{"text": "The case report by Nautiyal et al.  is an inActual measurement with exophthalmometry clearly demonstrates the lack of enophthalmos. As stated by Loewenfeld"}
{"text": "The first of these can be attributed to a melting phase transition arising from the rotation of two trifluoro\u00admethyl groups. In the crystal structure, both trifluoro\u00admethyl groups are disordered over two sites with occupancy factors of 0.660\u2005(17) and 0.340\u2005(17) for the major and minor orientations, respectively. The introduction of trifluoro\u00admethyl groups inhibits \u03c0-stacking between the diaza\u00adfluorene  units. Three short F\u22efO contacts between 2.80\u2005(3) and 2.95\u2005(1)\u2005\u00c5 are observed in the crystal structure.The title compound, C \u00c5                           b = 12.357 (3) \u00c5                           c = 26.941 (6) \u00c5                           V = 3090.4 (12) \u00c53                                                   Z = 4                           K\u03b1 radiationMo \u22121                        \u03bc = 0.11 mmT = 173 (1) K                           0.38 \u00d7 0.30 \u00d7 0.20 mm                                 Rigaku/MSC Mercury CCD diffractometerAbsorption correction: none20599 measured reflections3171 independent reflectionsI > 2\u03c3(I)2849 reflections with R                           int = 0.040                                                            R[F                           2 > 2\u03c3(F                           2)] = 0.056                           wR(F                           2) = 0.146                           S = 1.05                           3171 reflections498 parameters24 restraintsH-atom parameters constrainedmax = 0.28 e \u00c5\u22123                        \u0394\u03c1min = \u22120.25 e \u00c5\u22123                        \u0394\u03c1                                 CrystalClear  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008PLATON (Spek, 2003Mercury (Macrae et al., 2006WinGX (Farrugia, 1999Data collection: 10.1107/S160053680803420X/hb2809sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S160053680803420X/hb2809Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:"}
{"text": "Abstracttask clusters) that are common to efficient and effective practices in the digitization of biological specimen data and media. Examples of these clusters come from the observation of diverse digitization processes. The staff of iDigBio  visited active biological and paleontological collections digitization programs for the purpose of documenting and assessing current digitization practices and tools. These observations identified five task clusters that comprise the digitization process leading up to data publication: (1) pre-digitization curation and staging, (2) specimen image capture, (3) specimen image processing, (4) electronic data capture, and (5) georeferencing locality descriptions. While not all institutions are completing each of these task clusters for each specimen, these clusters describe a composite picture of digitization of biological and paleontological specimens across the programs that were observed. We describe these clusters, three workflow patterns that dominate the implemention of these clusters, and offer a set of workflow recommendations for digitization programs.This paper describes and illustrates five major clusters of related tasks (herein referred to as  These tasks typically include photography of specimens and labels, image processing, capture of label information as text, and locality georeferencing. The presentation of workflow characteristics in this paper provides the framework for analyzing the effectiveness and efficiency of workflows and for the development of new effective workflows. It should be noted that the workflows we observed represent a major departure from a historical practice of pulling a single specimen, creating a comprehensive database record, including researching localities, georeferences, collectors, taxon names, nomenclature, and other related details, then moving on to the next specimen  and the specimen itself (e.g. scientific name and identifying number). Digitization of label information includes capturing the text as characters, dividing the text into specific properties, and storing this information in a database. Digitization may also include capturing digital images and other media. References to media objects are added to the database records.http://www.gbif.org ), iDigBio (http://www.idigbio.org ) and the Thematic Collections Networks funded by the National Science Foundation\u2019s Advancing Digitization of Biological Collections (ADBC) program (http://www.nsf.gov/pubs/2011/nsf11567/nsf11567.htm), Atlas of Living Australia , http://vbrant.eu/ ), and VertNet (http://www.vertnet.org ). However, little has been published that characterizes modern existing and effective digitization workflows for a broad range of collections . We believe such characterizations are an early step in the process of building a common framework for sharing efficiencies across biological and paleontological research collections.The collections community has recognized that digitization processes need to be made more efficient to meet pressing scientific and societal needs (a topic broadly reviewed by (URLs provided for first mention only. Please see Appendix 2 for URLs of software and websites.)PageBreakgrounded theory research methodology . We asked that PageBreakIn the digitization workflows we observed, protocols for the digitization of biological and paleontological specimens were typically divided into clusters of related tasks. The order in which these task clusters were accomplished was based on a combination of staff availability, equipment, space, facilities, institutional goals, and the type of collection being digitized. Hence, though there was a general pattern to the components included within a particular task cluster, the order of accomplishment of the clusters and the tasks within each cluster varied by institution.These five task clusters were important components of digitization, but not all were essential to meeting the digitization goals of every organization or of every specimen for every organization. These clusters are presented here in a common order of operation:\u2022 pre-digitization curation and staging,\u2022 specimen image capture,\u2022 specimen image processing,\u2022 electronic data capture, and\u2022 georeferencing specimen data.It should be noted that quality control and data cleaning tasks were integral to each of these task clusters  to a specimen, container, or cabinet,\u2022 discover important but previously unknown, lost, or dislocated holdings ,\u2022 update nomenclature and taxonomic interpretation,\u2022 reorganize the contents of cabinets, cases, trays, and containers, especially when these are the units of digitization,\u2022 vet type specimens, and\u2022 select exemplars for digitization, when that approach is appropriate.The last five activities in this list may require the greatest knowledge of the organismal group of any during digitization. Many institutions use students, interns, dependable volunteers, or other full- or part-time technicians to accomplish the other pre-digitization curatorial tasks on this list, including the selection of exemplars for digitizing. However, some institutions also reported success with allowing technicians to take on more responsibility for at least some of the last 5 tasks in the above list .In addition, as collections data become more generally available online, updating nomenclature and taxonomic interpretations and vetting type specimens can occur after the publication of data and images on the internet, providing an opportunity for off-site experts to comment on the specimens. The latter approach will avoid what can become a bottleneck in the digitization workflow caused by the limited availability of in-house taxonomic experts or well-trained technicians.Although the application of specimen barcodes is treated here as part of pre-digitization curation, this placement in the digitization workflow is not universal. Some institutions applied barcodes at or just prior to the time of image or data capture, depending on the customized order of operations. In all cases where barcodes were used, they were applied prior to image capture to allow for the barcode value to be seen in the image, and prior to data capture to ensure that the physical specimen identifier is accurately included in the electronic data record.PageBreak of specimens from a single collecting event, barcodes were sometimes affixed to or inserted into the enclosing container. In most instances, when a container was barcoded, the number of specimens within the container was recorded, but individual specimens within a common container and not segregated by separate vials were neither barcoded nor otherwise individually identified. When individual vials containing single specimens were aggregated into larger jars, a replica of the label for the containing jar was sometimes inserted into each vial. In a few cases, the container was barcoded as were the individual specimens within that container (e.g. with Lepidoptera). In this latter case, the specimens were digitized individually, with both the individual specimen and container barcodes recorded in the database.Barcodes were used for two primary purposes. For individual specimens, barcodes were affixed or pinned to the single specimen or inserted into a wet container that held a single specimen. For specimen groups, such as taxon trays, wet containers, or a collectionLinear, one-dimensional barcodes are relatively large and are used in cases where sufficient space is available, for example on vascular plant specimens, bryophyte and lichen packets, and other dry, flat specimens. A smaller version of this type of barcode, printed the size of a standard insect label, was also used in entomology collections. Space is an important constraint in barcode selection.One-dimensional barcodes used for insect collections had two advantages. They mimicked the other labels in size, thus conserving space between specimens, and, if positioned near the bottom of the pin, were easily viewed and hand scanned without removal.Two-dimensional barcodes were also used, especially for small specimens. They were preferred by some entomology collections because they could be included on an insect pin with the coded end clearly visible and easily scanned.Determining what to image varied by institution and collection type. Most herbaria imaged entire specimen sheets. Close-up images of particular morphological features  were also sometimes captured. Certain entomological , paleontological, and ornithological collections captured several images of the same specimen with various views .http://www.jpeg.org/committee.html ) (jpeg or jpg) file format for distribution on the internet. Some institutions preferred camera raw formats for archiving images as these formats retain all data originally recorded when the image was made. Others preferred the well-documented and widely used http://partners.adobe.com/public/developer/tiff/index.html ) (tiff or tif), which retains all of the original image data and most of the Exchangeable Image File Format (EXIF) data (a topic reviewed by http://www.adobe.com/products/photoshop.html ) and Lightroom (http://www.adobe.com/products/photoshop-lightroom.html ))PageBreak. It should be noted that capturing and preserving high quality specimen label images offers opportunities to take advantages of future improvements in image analysis . A few camera manufacturers  have adopted the digital negative format as the native output for some of their cameras.Manufacturer-controlled raw formats are not openly documented and are subject to change without public notice. Hence, in 2004, Adobe, Inc. developed the publicly documented digital negative format (dng) as well as a freely accessible software application that converts many proprietary raw formats to digital negatives with little or no data loss  or a custom-designed replica), SatScan ,http://www.canon.com ), Nikon Capture NX2 (http://www.nikonusa.com ), Photoshop, and Lightroom), image stacking equipment and software, for example Helicon Focus (http://www.heliconsoft.com/heliconfocus.html ) or Auto-Montage (http://www.syncroscopy.com/syncroscopy/automontage.asp ) ,\u2022 copy stand and/or specimen holder,http://www.mkdigitaldirect.com/products/lighting-systems/mk-photo-ebox-plus-1419.html )),\u2022 studio lighting, flash units, or light/diffuser box .The most common brand of camera in use across collections was a Canon DSLR equipped with a medium-length macro lens, although Nikon DSLR cameras were also sometimes used. Megapixel ratings generally ranged from about 17 to 21.5, but were sometimes lower or higher, depending upon the expected use of the images.PageBreakstudy were usually captured at megapixel ratings of 17 and above. Macro lenses in the range of 50\u201360 mm were common, but a few institutions used macro lenses in the range of 100\u2013105 mm, which allowed for close focusing and performed well for smaller objects, such as small birds and mammals. Collections requiring macro images of very small specimens usually used a Leica microscope equipped with a Canon, Nikon, or Leica camera.It is instructive to note that generally, the larger the megapixel rating, the better the quality of the resulting images. Hence, images to be used for morphological To control for image quality, some institutions located the imaging station in a darkened or minimally lit windowless room. This prevented strong extraneous light, like that from a window, from contaminating or overpowering studio lighting or producing visible shadows on the resulting images. Light control was also sometimes accomplished by draping diffuser material across studio lights. A more elegant solution utilized a diffuser box with internal lighting that can be closed prior to image capture. Preferred for this was the MK Photo-eBox Plus Digital Lighting System, originally designed for photographing jewelry, coins, and collectibles. The box is slightly larger than a standard herbarium sheet, rests on a copy stand, includes halogen, fluorescent, and LED lighting, and is equipped with an oval port on the upper surface that allows an unobstructed camera view of the specimen. Herbaria using this system usually place the color bar and scale at the top of the sheet to preserve the aspect ratio of the resulting image, thus obviating the need for image cropping and reducing the number of steps required for image processing. Although the requirement to open and close the doors of the light box seemingly slowed the imaging rate, time lost was likely recaptured from a reduction in time spent on post-imaging batch cropping and light level adjustments.http://gpi.myspecies.info/ ). GPI specifications require that specimens be scanned at 600 ppi resolution, beyond the capacity of most DSLR cameras when used for whole sheet images of herbarium specimens. HerbScan uses a flatbed scanner  and a platform that raises the specimen sheet to the face of the inverted scanner. Scanning requires 4-6 minutes per scan for a maximum effective rate of about ten images per hour. Because the specimen sheet is pressed against the rigid glass face of the scanner, the acceptable depth of the specimen sheet is limited to about 1.5 cm, hence some specimens are too bulky for this equipment.HerbScan is the imaging system used for scanning type specimens for the Global Plants Initiative (GPI) project ,\u2022 adjusting camera height,\u2022 changing or attaching lenses, and\u2022 loading ancillary image management/processing software.In some institutions, especially those where all specimens are similarly sized (e.g. herbaria), camera settings and equipment mountings were usually not changed from session to session and required only a spot check prior to commencing a new imaging session. With collections of variously sized organisms , camera distance to subject was frequently adjusted, lighting re-arranged, camera settings altered, and custom or specialized specimen holders repositioned. In some instances, grouping like-size specimens alleviated the need for continuous camera adjustment and increased workflow efficiency. In these situations, the potential increase in imaging error due to increased demands for technician judgment were effectively offset by a higher level of detail in written protocols, elevated attention to specialized training, and diligent monitoring during the early phases of a new technician\u2019s tenure. Institutions that imaged only labels that required only moderate resolution sometimes dispensed with much of the equipment listed above in favor of a small digital camera and less elaborate copy stand that afforded more mobility .PageBreakProcuring and organizing the next batch of specimens for imaging was sometimes facilitated by ensuring proximity of the specimens to the imaging station. Institutions used mobile carts or cabinets to transport specimens from the pre-digitization curation or data entry areas to a location in close proximity to the imaging station. Moving specimens from station to station rather than returning them to storage cabinets and re-retrieving them reduced the amount of time devoted to travel and handling. From our observations, workflows that began with image capture, imaged every specimen, and extracted data directly from the image rather than the physical specimen effectively eliminated the need to handle or move specimens beyond the imaging stage, facilitating re-storage immediately following imaging . To ensuImage acquisition focuses on the process of camera operation for image capture. For collections with standard sized specimens (e.g. herbaria), the process involved repeating a rote procedure for each new specimen. Even for such collections, however, the technician was required to pay close attention to quality by periodically examining images to ensure that:\u2022 lighting, exposure, and focus remained constant,\u2022 file naming progressed according to plan,\u2022 exposure was correct,\u2022 focus remained sharp,\u2022 images lacked imperfections such as blemishes or streaking,\u2022 files were not corrupted, and\u2022 barcodes or identifiers were in place and readable.For wet collections, exemplar specimens were usually removed from the container before imaging. One successful technique we observed for imaging fish, reptiles, amphibians, and other organisms with a reflective epidermis submerged them in a shallow, ethanol-filled container, allowed the ripples to settle, and acquired the image through the ethanol. This method increased detail by reducing reflectance and increasing contrast. Coating fossil specimens with a thin layer of alcohol also increases contrast and provides for a sharper image .PageBreak clearly visible in the resulting image. One institution (Museum of Comparative Zoology) designed and constructed a custom specimen holder (http://www.imagemagick.org/ ).Protocols and workflows for efficiently imaging insects\u2014with the possible exceptions of bees, ants, and butterflies\u2014are under development and continue to pose special challenges. In nearly every case where we observed butterflies being imaged, specimens were removed from the pinning substrate, labels were carefully removed and placed on a custom-designed holder with the labels and barcodes (or other identifier)n holder  with sufn holder . Other in holder . Some inImaging productivity varied by collection. For herbaria, rates per imaging station ranged from as few as 10 sheets per hour using a single HerbScan, to 75\u2013120 sheets per hour using a camera (average rate slightly less than 100 sheets per hour). Imaging rates for insects are not well documented and their derivation is sometimes confounded by the inclusion of data entry and image acquisition in a single, linear workflow that makes it difficult to segregate strictly imaging tasks from data entry. For example, the imaging step might include removing the label from the pin, taking the photo, and putting the label(s) back on the specimen pin.Image processing involves all tasks performed on an image or group of images following image capture. Nine tasks are addressed here, reflecting common practices:\u2022 quality control,\u2022 barcode capture,\u2022 file conversion,\u2022 image cropping,\u2022 color balance or light level adjustments,\u2022 image stacking,\u2022 redaction,\u2022 file transfer, and\u2022 optical character recognition (OCR).Some institutions include one or more of these nine tasks  at other stages of the digitization process, as noted in the discussion below.http://www.snsb.info/SNSBInfoOpenWiki/attach/Attachments/JSTOR-Plants-Handbook.pdf ).Quality control was usually effected by selecting and examining sample images at regular intervals. In some institutions, all images were visually scanned for obvious deficiencies before individual images were selected for more thorough review. Selected images were evaluated for correct focus and exposure, blemishes, scan lines, mismatches between file names and barcode values (in situations where these are expected to match), and other obvious signs of imperfections or errors. Imperfections in camera images usually related to incorrect focus or exposure. Institutions using HerbScan, especially as part of the GPI, followed a more elaborate and rigorous process (not detailed here) that included converting images to high contrast in Photoshop and running scripts that track pixilation and banding, and that expose scanner-produced flaws such as minute streaks and lines caused by wear and tear on scanner parts. The standard for GPI images, coupled with mechanical parameters of the scanners, demanded these enhanced quality control procedures  software to scan the image file for a barcode value and rename the file to the barcode value detected. The benefits of the latter approach included reduction of potential naming errors and greater efficiency due to reduced camera manipulation.Based on our observations, collections that chose to use barcode values as filenames generally used one of several options. Most high-end DSLR cameras allow for cuhttp://finereader.abbyy.com/corporate/) at the herbarium of Valdosta State University, where barcodes were carefully affixed in precise horizontal or vertical orientation. A fourth approach used custom-designed software to intercept the filename generated by the camera, simultaneously creating an associated record in the database for later data entry from PageBreakthe image. Image filenames were unique for the collection, and the image files were usually stored in a repository and linked to database records through a software interface.However, OCR software sometimes failed at detecting barcodes within images due to image quality or other issues, resulting in files not being appropriately renamed. According to our observations, barcode extraction failure rates on bryophyte packets ranged from 0.2\u20133%, based on tests with ABBYY Finereader Corporate edition (http://code.google.com/p/zxing/ ), compared the value to the image\u2019s EXIF data, and created a database record containing the image filename and barcode value. This allowed database records to be created by software without regard to the image\u2019s filename. The key point of this process is that camera-generated filenames can be stored verbatim in a database if software is responsible for associating image files with specimen records .Cropping is used to trim excess image data in order to achieve an acceptable aspect ratio or to reduce unnecessary borders surrounding the specimen. Where cropping was utilized, it was accomplished in large batches that did not require monitoring once set into motion. However, cropping was not universal.In general practice, it is considered unwise to use photo manipulation software to alter color balance, saturation, sharpness, or other image features . Doing shttp://en.wikipedia.org/wiki/Focus_stacking ) involved recording several images of a stationary specimen at varying depths of field, processing them through a stacking algorithm that essentially merged the several layers into a single image while preserving properly focused pixels in each layer. The result was a sharply focused image throughout the specimen\u2019s depth. Software packages in common use included proprietary Auto-Montage . Stacking worked best with cameras that supported a live view of the specimen in conjunction with camera control software that allowed precise focus control targeted to small percentage regions of the specimen.Specimens with significant depth, such as fossils, some insects, birds, mammals, and even some herbarium sheets, make it difficult to achieve sharp focus throughout the depth of field. Institutions used one of several stacking software packages to rectify this problem. Focus stacking ), Microsoft Voice Recognition (a standard component of the Microsoft Windows\u00ae operating system), and Dragon Naturally Speaking  are three software packages being used or tested. We note that programmers at the Botanical Research Institute of Texas (BRIT) are testing the Application Programming Interface that is packaged with the Microsoft Windows\u00ae operating system.We believe that voice recognition shows great potential for data capture and that the comparatively small cost for appropriate commercial products will be offset by greater workflow efficiencies. Most modern operating systems include built-in voice recognition capabilities of various qualities that should be tested using a high quality microphone. From our experience, the potential drawback to this technology is that substantial training to particular voices is often required for the software to perform adequately, which may limit its use where several data entry technicians are involved or when the rate of technician turnover is high. In addition, PageBreakwe noted from our interviews that simultaneous data entry by several technicians in close proximity might lead to distortion and interference, or be distracting to workers.Advances in voice recognition technology are evident in computer, tablet, and smart phone applications. Nevertheless, we saw only a single use of this technology, and this only for capturing a limited set of data, but we note that some institutions are experimenting with this technology. IBM ViaVoice  at BRIT and the Symbiota Software Project (http://symbiota.org/tiki/tiki-index.php ) at Arizona State University. Each of these interfaces simultaneously displays a specimen image, an OCR-rendered version of label data extracted from the image, and a collection of database fields into which data can be transferred. Apiary allows users to demarcate OCR regions of interest within the image and highlight OCR-generated text that can be transferred to associated data fields by mouse click. Symbiota provides for moving data to fields manually, but additionally includes functionality for searching the databases of the Consortium of North American Byrophyte Herbaria (http://symbiota.org/bryophytes/ ) and Consortium of North American Lichen Herbaria  for previously digitized duplicates from which data can be imported.Optical character recognition (OCR) was also being used or considered by several institutions. Two of the most effective uses we observed included the Apiary Project (http://code.google.com/p/tesseract-ocr/ ), OCRopus (http://code.google.com/p/ocropus/ ), and JOCR (GOCR) (http://jocr.sourceforge.net/ ), all of which are open source, and the proprietary ABBYY Finereader corporate version (http://www.abbyy.com/ ) and Adobe Acrobat Professional version (http://www.adobe.com/products/acrobatpro.html ), both of which can batch process large numbers of images. There is significant interest in natural language processing (NLP), which is designed to parse OCR text into fields, as well as intelligent character recognition (ICR) or handwriting analysis, but effective systems for using these technologies to extract data from biological specimens were not observed.Other institutions routinely process all images through OCR and store the OCR-generated output in text files, or import it into a field within the database for subsequent editing, data cleaning, and searching. Popular OCR software packages included Tesseract (http://specifysoftware.org/ ) (via Workbench), Brahms (http://herbaria.plants.ox.ac.uk/bol/ ) (via Rapid DataPageBreak Entry http://herbaria.plants.ox.ac.uk/bol/BRAHMS/Documentation ), and KE EMu (http://www.kesoftware.com/ ) provide this capability. Issues to resolve when importing legacy or external data include data quality, mapping imported data fields to those in the preferred database, dealing with imported fields that do not have database correlates, and time required for post-import data cleanup. In many cases, importing and transforming legacy data can be efficiently managed, resulting in large dataset acquisitions for relatively small investment in time, especially when compared to keystroking.In some instances data entry is accomplished by electronic import from spreadsheets or other delimited lists. Some software interfaces, e.g. Specify  and Biogeomancer  are software applications designed to assist in assigning latitude/longitude coordinates to textually described localities. Both of these applications convert locality descriptions into coordinate pairs based on statements of state, county, orthogonal direction, distance, and place names of geographical features. Both also provide protocols for uploading datasets for processing and bulk georeferencing similar localities. Each returns a map of the estimated location of each described locality, including a point-radius estimate of precision. Map interfaces allow technicians to manipulate and refine the georeferenced locations of these points before recording a final determination of the point\u2019s coordinates. Technician manipulation was required for points to be reliable. Both Geolocate and Biogeomancer are free to use. The third method we observed was based on the use of standard and customized map layers in conjunction with GIS software (such as ArcMap http://webhelp.esri.com/arcgisdesktop/9.2/index.cfm?TopicName=An_overview_of_ArcMap ) and paper maps to pinpoint locations. For best results, all of these systems rely on a technician\u2019s knowledge of the region in which a collection is made, facility with desktop GIS or online mapping software, general understanding of maps and mapping, and ability to recognize habitat signatures on aerial photographs.We observed three georeferencing methodologies in use where coordinate values were not present on the specimen. Geolocate , which requires specimen handling only during image aquisition.The image to data to distribution pattern fits institutions that image all specimens (e.g. most herbaria) and captures data from these images. It reduces specimen handling and with it the likelihood of specimen damage, increases efficiency by eliminating the need for return trips to storage locations, and offers the capacity to incorporate Optical Character Recognition and similar technologies within the data capture workflow.The Based on our observations, interviews, discussions, and readings, we offer the following recommendations for establishing and improving biological and paleontogical collections digitization programs.1. With planning, the pre-digitization curation step is an opportunity for the goals of specimen digitization and collection curation to be merged into an efficient workflow. Curation tasks that cannot be efficiently addressed in the workflow can be identified so that adequate resources can be assigned to them in the future .PageBreakmore efficient (2. Biodiversity informatics managers and other digitization personnel should look for bottlenecks in digitization workflows and seek ways to make them fficient . We reco3. There should be clear institutional policies guiding which specimens to expose to public access, including policies governing whether to redact or not redact locality data for sensitive species  and ensu4. Barcodes should be used only as identifiers; encoded barcode data should not incorporate taxonomic or related information that might change with time.5. Where possible, the aspect ratio of specimen to camera should be synchronized to eliminate the need for image cropping.6. Image processing should not include color balancing or other adjustments that result in images inaccurately reflecting actual specimens .7. A color bar and scale should be visible in all images .8. Protocols for periodic quality control should be established for all stages in the digitization workflow to ensure data accuracy and the production of high quality digital images .9. For institutions in which imaging is paramount, acquiring images of labels prior to data entry reduces specimen handling by allowing for data extraction from images rather than from specimens.10. Attention to the digitization of gray and published literature related to specimen data is an important consideration and should be accomplished whenever possible cf. .11. Georeferencing should be treated as an essential part of digitization protocols .12. Quality control should be integral to all steps in the digitization workflow, including post-digitization review and targeted testing should be designed to expose data inconsistencies or suspected anomalies .13. Detailed written protocols should guide every step of the digitization workflow, be uniquely designed for a given institution, and be amended regularly to reflect emerging technologies and improved efficiencies. These protocols should be electronically stored in a common folder that allows technicians to insert comments and suggestions to be reviewed and potentially adopted by biodiversity informatics managers.14. Selection of data entry and imaging technicians should be guided by employability skill sets strongly associated with success in digitization tasks, with particular attention to potential technicians\u2019 attention to detail, orientation to increased efficiency, and commitment to high productivity.PageBreak15. Institution-wide digitization tasks should be periodically evaluated for overall progress, organizational collaboration and cooperation, and compatibility with new and emerging technology, with plans to use results of the evaluation to implement improvements .16. Digitization workflows should be coordinated by a designated biodiversity informatics manager with IT experience, preferably from a biological sciences and collections background, to bridge the potential knowledge gap between collections managers and information technology professionals .17. Biodiversity informatics managers should construct a frequently asked questions document that outlines common problems and offers instructions about how to address these problems, whom to contact with questions about specific categories of problems, and guidelines for which types of problems should be elevated to a higher administrative level.18. Institutions should utilize a digitization workflow strategy that captures problems, remedies, lessons learned, and technician input for use in improving digitization protocols, and remain open to investigating possible changes in current practice .19. Determining an appropriate storage format for archived images is an important decision that should precede image capture. Here we recommend capturing images in native camera raw and converting them from camera raw to dng or tif (a topic addressed by"}
{"text": "Nothing is known, however, about; thedevelopmental mechanisms governing butterfly oogenesis, how polarity in theoocyte is established, or which particular maternal effect genes regulateearly embryogenesis. To gain insights into these developmental mechanismsand to identify the conserved and divergent aspects of butterfly oogenesis,we analysed a Pararge aegeria females expressed74.5% of the genes that are known to be essential for D.melanogaster oogenesis. We discuss the genes involved in allaspects of oogenesis, including vitellogenesis and choriogenesis, plus thoseimplicated in hormonal control of oogenesis and transgenerational hormonaleffects in great detail. Compared to other insects, a number of significantdifferences were observed in; the genes involved in stem cell maintenanceand differentiation in the germarium, establishment of oocyte polarity, andin several aspects of maternal regulation of zygotic development.A total of 17306 contigs were annotated, with 30% possibly novel or highlydivergent sequences observed. This study provides valuable resources to investigate a number of divergentaspects of butterfly oogenesis requiring further research. In order to fullyunscramble butterfly oogenesis, we also now also have the resources toinvestigate expression patterns of oogenesis genes under a range ofenvironmental conditions, and to establish their function. Drosophila melanogaster, areinvolved in setting up; 1) the location of the germ plasm and subsequent germ cellline development in the offspring . Th. ThBomby by mex-3. Pararge-3 Table\u00a0. Furthernslation . All of anogaster,65, presDrosophila melanogaster includes maternal hunchback(hb) transcripts into the egg, the protein of which will form an APgradient during early embryogenesis and cooperate with Bcd to specify theanterior of the embryo, whilst being repressed at the posterior by Nos [hb RNA or protein is transferred to the egg, as well as in thesignificance of the maternal contribution to the Hb gradient for AP patterning,the transcription of hb during oogenesis appears conserved [Schistocerca americana embryo, maternal hbtranscripts appear to be involved in distinguishing embryonic fromextra-embryonic cells along the AP axis, whilst in D. melanogastermaternal and zygotic Hb are redundant for AP patterning of the embryo [B. mori, the hb transcripts detected appear tobe transcribed by the zygote, not the mother [Pararge aegeria also did not express hb duringoogenesis ),which indeed appear to have divided up these functions [B. mori does not have agerm plasm, the location of maternal B. mori nos-O transcripts in theembryo seems to correspond with where the PGCs will form [nos paralogs, with the exception of nos-P,are expressed during oogenesis in both B. mori and P. aegeria,with maternal transcripts detectable in P. aegeria eggs genome [nos sequences showsnos-P to be quite different from the other paralogs  genome  and phylD. melanogaster nos RNA is accomplishedduring oogenesis by proteins encoded by glorund (glo) and in the earlyembryo by smaug (smg) [D. melanogaster oocytes [P. aegeria ortholog of smg was found, which waspresent as RNA in the oocyte, but not of glo  that preventsrapid deadenylation at the posterior pole, establishing nos as aposterior defining gene [osk ortholog [nosparalogs may not being involved in AP axis formation during oogenesis. Indeed,P. aegeria also does not possess an ortholog of osk(Table\u00a0P. aegeriagenome).Translational repression of ug (smg) . Transcr oocytes . A P. aelo Table\u00a0. Furtheranogaster. Rapid d nos RNA . AlthougR4 Table\u00a0. In D.ming gene . DitrysiPectinophora gossypiella[osk, which has been argued to have been co-opted as theessential gene in germ plasm formation in holometabolous insects [Pararge aegeria may not possess an osk ortholog,but it does express two genes, which in D. melanogaster silenceosk translationally during oogenesis; bruno[cup[D.melanogaster , vasa (vas) and valois(vls) [valois (vls) could not be found in the P.aegeria transcriptome  . Only vae Tables\u00a0.tud, vas and vls , Brother ofYb (BoYb) and Sister of Yb (SoYb) [P.aegeria transcriptome , TDRD7 andspindle E/homeless (TDRD9) [P. aegeria  . Indeed, (TDRD9) ,110 and  with osk. It is l with osk.piwi,aubergine (aub) and argonaute 3 (AGO3) [P. aegeria pathway genes often disrupt the axes of the developingoocyte, through their effects on the microtubule cytoskeleton; for examplemaelstrom (mael), zucchini (zuc) andsquash (squ) affect DV polarity [aub in D.melanogaster in silencing osk translation during oogenesis [armitage (armi)affects axis formation and is involved in osk translational silencingin D. melanogaster[zuc nor squ was found in the P.aegeria transcriptome, but mael and armi were . All thraFigure\u00a0 and 10. em cells . Furtherpolarity ,115. Theanogaster. NeithereTables\u00a0 and 10.D. melanogaster these can befound in nurse cells, but also appear to be involved in compartmentalisation ofmRNA decay and translation repression, for example of osk[EDC4/Ge-1 and pacman(pcm), genes that encode the essential components of P-bodies wereexpressed in P. aegeria  are aggregates of translationallyinactive ribonucleoproteins (RNPs). In le of osk,117. Witogenesis . This waD.melanogaster, a number of (late-acting) maternal-effect genes areessential in germline formation during early embryogenesis  and imp[germ cell-less (gcl) and polar granulecomponent (pgc)  is involved in germ cell migration inD. melanogaster embryos  and imp. However) Tables\u00a0.These gte Table\u00a0.Drosophila melanogaster uses an elaborate network of genes to patternthe DV axis during embryogenesis on the basis of the oocyte polarity establishedduring oogenesis , nudel (ndl), gastrulationdefective (gd), snake (snk), easter(ea), spn27A, spz, tube (tub)and pelle (pll) (Tables\u00a0weckle (wek) have yet been found outsideDrosophila, and wek was also not found in P.aegeria , rather than toll (tl). A siminespace . This pria Table\u00a0. In D.m)Tables\u00a0 and 13. igration , and DV igration . While Ppathways . As discster toll. It thusPararge aegeria did express cactus (cact) anddorsal (dl)  is essential for subsequent DV patterning in the D. melanogasterembryo. Dorsal protein activates some genes, whilst repressing others along theDV axis [dpp ventrally and the proteinencoded by grainyhead (NTF-1/grh) acts asco-repressor [grh is deposited maternally into the oocyte to betranslated and used ventrally during embryogenesis [dpp by a Dorsal gradient does not, however,occur in T. casteneum[D. melanogaster embryo and its concentration isfurther restricted ventro-laterally by Short gastrulation (Sog), which in D.melanogaster may also be maternally provided [vrille (vri) gene encodes a Bzip transcriptionfactor that interacts in D. melanogaster with Dpp signalling, acting asdominant maternal enhancers of embryonic DV patterning defects caused byea and dpp mutations [eclair (eca) andbaiser (bai) are essential for the activity of maternalTkv, a type I Dpp receptor [Pararge aegeria females did transfer maternal transcriptsof grh, dpp, tkv, eca, bai and vri into the oocyte,but did not express sog maternally  Table\u00a0. DorsalDV axis ,124. Whiprovided . Rather provided . The vriutations . Two P24receptor . ParargeDrosophila melanogaster females express a group of genes called theyema genes  during oogenesis,with most of them displaying strict maternal expression. This may be ofimportance in the development of the central nervous system of the embryo [yema genes arenot known and there are no orthologs outside Drosophila[P. aegeriatranscriptome ,elav, brainiac (brn), Fmr1, braintumor (brat), mnb, and terribly reduced opticlobes (trol)  biogenesis [torso (tor) encodes areceptor whose ligand is most probably encoded for by trunk(trk). Furthermore, the protein encoded by torsolike(tsl) plays a role upstream of trk in activating the Torreceptor in a localised manner, and is thought to be essential for terminalspecification [tor and tsl are involved in terminalspecification in T. castaneum, different tissues are patterned andTorso signalling plays a role in defining the posterior growth zone duringembryogenesis in this short germband insect [Apis mellifera) has the genetsl, but not tor and trk in its genome [Pararge aegeria does not express clear orthologs of eithertor or trk during oogenesis, but does express tsl(Table\u00a0Bombyx mori does have a RTKin its genome (BGIBMGA003976), which shows similarity to torso, as wellas to tie-like and Cad96Ca. Pararge aegeria did notexpress tie-like , although it hadgreater similarity to the receptor tyrosine kinase Fps85D than totor. This transcript is present in both P. aegeria oocytesand ovarioles, but its role in oogenesis has not been described in theliterature. It is clear that P. aegeria uses RTK signalling duringoogenesis and that the sequences of its ligands and receptors have diverged fromthose of other insects. However, at present it is unclear in which functionalcontext RTK signalling takes place.The Torso receptor tyrosine kinase (RTK) pathway has been implicated in a numberof different processes during ogenesis  and in pogenesis . The matfication . Althougd insect . Torso ss genome . The hons genome . ParargeslTable\u00a0. Bombyx D. melanogaster , sex combs onmidleg (scm), and Arginine methyltransferase 1 and8  andsimilar to D. melanogaster polyhomeotic (ph-p) gene. Both playversatile and important roles in D. melanogaster oogenesis,particularly in ovarian follicle formation [Pararge aegeria females did express and transfer orthologsof other PcG genes into the oocyte. These include the polycomb repressivecomplex 1 (PRC1) genes sex combs extra (sce),polycomb (ph), posterior sex combs(psc), the PRC2 genes extra sex combs (esc),Enhancer of zeste (E(z)) and the polycomb related genesEnhancer of polycomb (E(ph)) and additional sexcombs (asx)  important in oocyte production ,matrimony (mtrm), microcephalin (MCPH1)and chiffon (chif) .A large number of genes that regulate mitosis have been studied in a reproductivecontext in ri Table\u00a0. Among talin MCPHand chifD. melanogaster germarium [bruno is not onlyessential in regulating the translation of a number of genes during oocytedifferentiation, but it also appears to be involved in regulating the silencingof Cdk1 activity in order to achieve primary meiotic arrest [bruno was expressed by P.aegeria females (Table\u00a0The cell cycle becomes arrested in meiotic prophase I in the majority ofMetazoans oocytes. This is initiated during the first stages of oogenesis inregion 2 of the ermarium . The intc arrest . It shouc arrest . As indies Table\u00a0.D. melanogaster germarium [D. melanogaster for SCformation and thus possibly chiasmata formation: crossover suppressor on 2of Manheim (c(2)M); crossover suppressor on 3 ofGowen (c(3)G); corona (cona) andnipped-B , corona (cona),orientation disrupter (ord) and mei-S332, for example, is bothessential in D. melanogaster for maternal provisioning of the eggduring vitellogenesis and to ensure secondary meiotic arrest by stage 14 ofoogenesis in metaphase I [P. aegeriafemales did express this gene during oogenesis  interacts with polo kinase(polo) in mitotic regulation particularly during earlyembryogenesis, and is maternally provided  and LpR [yl/VgR RNA into previtellogenicoocytes, thus preparing the oocyte for Vtg uptake [Pararge aegeria females expressed not only Vtg/Vg,apoLp-III, apoLp, their receptors yl/VgRand LpR, but also the genes described in D. melanogastervitellogenic endocytosis , sec5, sec6,garnet (G) and jagunal (jagn), such as Vitellogenin (Vtg/Vg) and Apolipophorins (ApoLPs) ,9. Predoanogaster. Vitelloanogaster. LLTPs a and LpR . Nurse cg uptake . Pararge)Figure\u00a0.B. mori and released as ecdysteroids during yolk uptake in theembryo as a result of dephosphorylation by ecdysteroid-phosphate phosphatase (EPPase)[Pararge aegeria did express EPPase and Samia cynthia (ESP) had not been found [G. mellonella yolk protein 2 have been discovered inD. plexippus and Plodia interpunctella, whilstESP does show significant sequence similarity with genes encodingthe KK-42 binding proteins in Antheraea moth species [D. melanogaster, in particular lipase-1 and 3(lip-1 and 3) [. Rather interestingly, although not functioningas a yolk protein, lip-1, but not lip-3, is expressed invitellogenic follicles in D. melanogaster[lip-1, and possibly lip-3 , was expressed by P. aegeria, whilst no clearortholog of a minor yolk protein was found . ParargeseTable\u00a0. Further protein . The genen found . More re species  (Additio1 and 3) . Lepidopanogaster. An orthnd Table\u00a0.P. aegeria ovarioles is anortholog of the slime mold Physarum polycephalum genespherulin-2A. No transcripts were found for this gene in eggs  Chico precludes vitellogenesis, whilst a sharp increasein 20-hydroxy-ecdysone (20E) relative to juvenile hormone (JH) results inapoptosis of the egg chamber before vitellogenesis is initiated or completed [D. melanogaster[bbx genes are expressed predominantly in the brain, butsome may also be expressed in ovaries [B. mori, possess a large number ofbbx-like genes in their genome [D. plexippus appears tohave only three such genes [bbxA1-like andbbxA3-like) were transcribed in P. aegeria ovarioles,whilst a third partial IRS transcript showed more sequence similarity tochico than to any bbx-like gene (Table\u00a0insulin-like receptor (InR) was also expressed by P.aegeria during oogenesis  and the lipid storagedroplet proteins 1 and 2 (Lsd1 and Lsd2). Please refer to Table\u00a0Reproductive output depends on female nutritional status which not only affectsthe rate and duration of oogenesis significantly, but also whetherprevitellogenic egg chambers will enter the vitellogenic stage or apoptose . Two sigompleted ,155. Altanogaster. The bbx ovaries . Moths, r genome , but thech genes . OrtholoApart from nutritional status, environmental factors such as temperature canaffect hormone concentrations, providing a possibility for environmental controlof reproductive output ,26. The methoprene-tolerant (met) [taiman (tai) in D.melanogaster[tai gene was originally discovered as a gene that wasexpressed in follicle cells in the functional context of border cell migrationand was described as an ecdysone co-receptor  can also been found in the genome of D. plexippus[There has been extensive investigation of JH signalling ,26, but nt (met) -160. It anogaster,160. Theplexippus.kruppel-homolog 1 (krh1) has beendescribed as a JH response gene, inhibiting 20E induced broad(br) expression in D. melanogaster, but not in thespecific context of oogenesis [khr1 and br were expressed by P.aegeria females , which regulates polyamine biosynthesisand appears to be essential for vitellogenesis [odc and its antagonist gutfeeling(oda), also a mitotic cell-cycle regulator, were expressed inP. aegeria. Maternal transcripts of odc and odawere found in eggs  and the JH esterases (JHEs) . JHEs fuB. mori and in aJHbp CDSs were identified in P.aegeria ovaries; JHbp, cytosolic JHbp(cJHbp), hemolymph JHbp (hJHbp) and a sequenceshowing strong orthology to takeout (to) identified in D.melanogaster as involved in JH binding  to enableimmobilisation, regulate degradation or enable transport . Four cong Table\u00a0. Transcr, like P. aegeria, have been argued tobest match the criteria for group 4 [B.mori and D. melanogaster[There is a significant amount of life-history variation among insects andconsequently in the relative importance of 20E and JH on oogenesis , even wi group 4  where JHanogaster,146.Bombyx mori appears to be capable of producing ecdysteroidsin the ovaries [D. melanogaster[Drosophila melanogaster expresses start1 duringoogenesis in significant amounts in nurse cells, most likely in response toecdysone signalling. The cholesterol transporter Start1 may in turn facilitateecdysteroid production from cholesterol-based precursors [D.melanogaster cholesterol conversion in the ovaries is defective inthe avoidance of repellents (dare), which encodes an Adrenodoxinreductase [D. melanogaster the SGT1 protein homologecdysoneless (ecd) and disembodied (dib)have been described as essential for ecdysone, both for functionality and itsproduction in the ovaries [D. melanogaster start1 arehypothesised to be deposited into the egg to facilitate ecdysteroid signallingin the developing embryo [P. aegeria females did not expressdib, but did express ecd, start1, and dare. Weobserved the transfer of transcripts of all three genes into the oocytes and its partner ultraspiracle  in B. mori) in theovaries , thetranscription factor gene E74 and the Broad-Complex gene Br-C[B. mori andtheir expression increases during vitellogenesis [E74, all of these genes wereexpressed in P. aegeria  during choriogenesis [P. aegeria females did not expressESP, but did express the related gene lip-3 is expressed in all follicle cells and is essential for DVpatterning of the embryo in D. melanogaster[D. melanogaster embryo [Pararge aegeria females expressed ndl and as inD. melanogaster, no transcripts were found in the oocyte is formedhalfway through vitellogenesis , for whie oocyte . This al [B. mori, althouganogaster. Ndl is anogaster. As suchr embryo . ParargeteTable\u00a0. It remaD. melanogaster VMPgenes outside the genus Drosophila. The best-characterised VMPgene in Lepidoptera is VMP30[P. aegeria ovarioles. Once again, notranscripts were found in the oocyte (Table\u00a0Insect vitelline membrane protein (VMP) genes show tremendous sequence diversity.For example, no clear orthologs can be found for  is VMP30, for white Table\u00a0.D. melanogaster and clusters of choriongenes are selectively amplified or expressed at very high levels [P. aegeria did not express anortholog of G1/S specific cycE, which in D. melanogaster isessential for chorion gene amplification and endocycling in general  in D. melanogaster[P. aegeria (Table\u00a0B. morihave multiple cis-regulatory binding sites for CCAAT/enhancer bindingprotein (C/EBP) transcription factors and their expression levels are C/EBPconcentration dependent [D. melanogaster ortholog of C/EBP is slbo,which is also expressed in follicle cells though predominantly involved inborder cell migration , its negative regulator tribbles (trbl) andHMGa  [B. mori[D. plexippus[cf1 and CbZ were transcribed by P.aegeria, with transcripts of the latter rather intriguingly found to bepresent in the oocyte  . Further [B. mori and orthplexippus. HoweverD. melanogaster cp geneswith those identified in Lepidoptera, including P. aegeria, is very lowindeed , which forms part of the eggshell and is produced by the follicle cells [P. aegeriaoogenesis (Tables\u00a0Chorion protein (cp) genes evolve possibly even faster than vitelline membraneprotein genes  and sequed Table\u00a0. The infstrength . Furtherfication . Expressfication ,179. As le cells . BmEP80 D.melanogaster and B. mori, with nurse and follicle cellsundergoing apoptosis as oogenesis progresses, while complete egg chambers mayapoptose in response to environmentally induced hormonal signals such asstarvation [D. melanogasterovaries, where the effector caspase Dcp-1 and the inhibitor of apoptosis proteinBIR-superfamily domain protein Bruce regulate both autophagy and starvation-induced cell death [B. mori, and the results of the study by Zhang and co-workersshowed that most of these genes are highly conserved [D. melanogaster , which is required for posterior follicle cell maturation [D. melanogaster oogenesis  and mob as tumor suppressor   . It is essential for regulating tissue size and growth . Hippo sturation . Ortholo1) Table\u00a0. Merlin/M1 Table\u00a0, which iM2Table\u00a0. In D. mlisation , but clei) Table\u00a0. The latD. melanogaster, for example, Hsp60C is essential inorganising and maintaining cytoskeletal and cell adhesion components and thusfor establishing AP and DV oocyte polarity [D. melanogaster oogenesis(Tables\u00a0Heat shock proteins (Hsps) provide a possible mechanism for environmental controlof development in ovaries and as maternal effects. The transcription of genesencoding Hsps, or molecular chaperones in general, is not only regulated inresponse to various environmental factors (e.g. temperature), but is alsoessential during many developmental processes, including oogenesis. It isthought that Hsps are important for both developmental buffering anddifferentiation ,186(furtpolarity . A large)Tables\u00a0.D. melanogaster oogenesisand early embryogenesis  are among the most highly transcribed genes duringMetazoan oogenesis, as large amounts of the translation machinery are neededboth during oogenesis and by the developing embryo . Just li are amonis Table\u00a0.P. aegeria and present as maternal transcripts in the oocytes, has been implicated in DV axisformation [Orthologs of the majority of the genes identified from the literature as beinginvolved in immune response during oogenesis were also found to be expressed byesTable\u00a0 and 2. AesTable\u00a0, these iesTable\u00a0.Drosophormation .Wolbachia sp. is anendocytosymbiont in many arthropod species affecting oogenesis in a multitude ofways and the Bacterium is maternally transmitted [D. mauritiana, Wolbachia increases egg production byaffecting the maintenance and division of germ-line stem cells [Asobara tabida, Wolbachiaconfers a reproductive advantage to the females by properly regulating apoptosisduring oogenesis via its regulation of iron metabolism and ferritinexpression [D. melanogaster highly infected females sufferfrom a range of oogenesis defects mediated via grk signalling [Pararge aegeria females were also found to be infected withWolbachia, but how this affects oogenesis in this species is atpresent not known. However, we did observe that the gene encoding an ortholog ofthe Ferritin 2 light chain protein (FER2-LCH) was amongst the most highlytranscribed genes during P. aegeria oogenesis M19/wisp)tail elongation of bcd, toll, and tor transcriptsupon egg activation. It is thus important for proper patterning of the embryo [Given that P. aegeria females did not expressbcd and tor, it remains to be investigated whether wisp isof any importance in patterning of the embryo.As discussed elsewhere in this paper, after vitellogenesis both the aphase I ,194. Unlaphase I , egg actme Table\u00a0,which mp)Table\u00a0. In D. mansition . The D. e embryo , but is e embryo . Given tD. melanogaster oogenesis)were transcribed by P. aegeria and transcripts were transferred to theoocytes. As this was an ovarian transcriptome study, the precise functional contextin which these genes were transcribed has not been identified. Differences in thefunctional context in which particular genes are expressed are to be expectedcompared to model organisms such as D. melanogaster and even B.mori. What is perhaps more revealing, however, is the absence of certaintranscripts in the database, in particular where these transcripts concern paradigmsof maternal regulation for various aspects of early insect embryogenesis [Pararge aegeria differed most significantly from D.melanogaster (and quite a number of other insect species), both in terms ofstem cell maintenance or differentiation in the germarium and in establishing (andmaintaining) polarity along AP, DV and at the termini of the oocyte. In particular,although Pararge aegeria females expressed an ortholog of aspi/krn-like EGF ligand and possibly its receptor, manycomponents of the EGF pathway involved in patterning of the axes in D.melanogaster embryos, as well as pipe andmirror, were not expressed. This may either suggest that there is not muchevidence for a significant role of EGF signalling in establishing P.aegeria oocyte polarity, or that its functional role and genes involved isdivergent from other insects. This requires further study, as well as the functionalrole and significance of Dpp and Notch signalling in this context.A large proportion of the genes currently described in the literature as beingessential during insect oogenesis . Although progress has beenmade in investigating B. mori embryonic patterning [P.aegeria may prove an ideal model these future studies.Although the more derived species such as and-like , it is mand-like ,54, as teristics . It is vtterning ,54, how Unfortunately, maternal effect gene expression and regulation have receivedsignificantly less research attention in Lepidoptera compared to vitellogenesis,choriogenesis and reproductive physiology . This isP. aegeria as an eco-evo-devo modelsystem for the study of butterfly oogenesis. In order to fully unscramble butterflyoogenesis, an investigation of the spatio-temporal expression patterns of the genesdiscussed in this study, as well as establishment of their function, is required.Further studies are also required to establish the function and expression patternsof the uncharacterised contigs identified in this study, which make up 30% of thetotal contigs found, and are undoubtedly composed of genes that are of highimportance in butterfly oogenesis.This study has taken a much-needed first step in determining the conserved anddivergent elements of the butterfly oogenesis GRN  and establishes P. aegeria. This population originatedfrom a woodland population from the south of Belgium  and by the time of the experiment, the butterflies had been rearedin the laboratory for 10 generations. Newly hatched larvae were placed on pottedhost plants (4 larvae per plant) of Poa trivialis L. with access toad libitum food and were reared until eclosion in a climate roomunder a regime  that promotes direct development(i.e. no diapause). On the day of eclosion  females from this laboratory stock placed individually in netted cages (0.5m3) along with a potted P. trivialis plant foroviposition and an artificial flower containing a 10% honey solution [As butterflies were used in this study, no ethical approval was required. Eggswere collected from a large outbred laboratory population of solution . Later tEggs from 50 mated 4-day old females were collected within 20 minutes of beinglaid, which is well before the onset of cleavage and thus early embryogenesis inbutterflies . The eggThe homogenate (both of eggs and ovarioles/ovary) was first centrifuged at 13000gfor 10min primarily to remove the yolk, after which the supernatant was vortexedwith 200\u03bcl of chloroform. Phases were separated at 13000g for 15min at roomtemperature. The aqueous phase was removed and precipitated in 0.5ml isopropanol . The RNAPararge aegeria egg and ovary RNA was sequenced by Source BioScience using Illumina short read RNA-Seq technology. Both total RNAsamples went through polyA selection, fragmentation and double stranded cDNAconversion to produce two separate libraries (300bp insert size) in accordancewith the Illumina mRNA-seq library preparation protocol . Sequencing was performed on the Illumina Genome Analyzer IIx platform withone flowcell lane allocated to each library. A total of 61,400,070 single-readsof 38 base pairs (bp) in length were obtained from the ovary and egg flowcelllanes which were pooled to produce a de novo assembly in CLC GenomicsWorkbench v4.0  using the default settings for shortread data (automatic word and bubble size) [le size) . The assde novo assembly usingTopHat on the Galaxy server [The RNA-seq data was analysed by FASTQC on the Galaxy platform ,199. Aday server . FPKM vay server  with quade novo assembly  was discarded from each rank, using the next best hit (which may bein a lower rank or group)  used for annotation, includingthose genes annotated manually, were used as input in the BLAST2GO software  and assi\u00b0C for 30 min after an initial 5 min denaturation step at70\u00b0C. Negative reverse transcription (NRT) controls were run inparallel without both Verso RT enzyme mix and primers. A final heat deactivationat 95\u00b0C for 2 min was also implemented to deactivate the RTenhancer. The resulting cDNA was stored at \u221220\u00b0C.For of a subset of 19 genes the expression in the ovarioles and the presence oftranscripts in the oocyte were confirmed further by means of RT-qPCR . Only those primers exhibiting the best stability were selected. Eachprimer pair was tested on a 3-step 5-fold dilution series of the ovary cDNA intriplicate, which enabled the primer pair efficiencies to be determined usingthe CFX Manager software . Primers withadequate efficiency (>65%) were then used for investigating the transcriptabundance in the egg and ovary cDNA  on white 96-well plates in ABsolute Blue qPCR SYBR Green Mastermix with the recommended amount of ROX reference dye was used to reveal whetherany individual transcript was used as a maternal effect gene transcript or wasmerely necessary for oocyte production. Relative transcript abundance in theovaries and eggs were obtained using the relative expression software tool RESTv2.0.13.0 software package , which uP. aegeria transcriptome investigatedby means of qPCR, transcript abundance calculated on the basis of Cq values bymeans of the methods described in [41 = 2.37, P = 0.011;Additional file The number of reads mapping to a transcript of a particular gene in RNA-seq datawas argued to be correlated linearly with the number of transcripts of that gene . Rather ribed in  showed sThe sequence read data reported in this manuscript have been deposited in theNCBI Sequence Read Archive and are available under the accession numbersSRR771147 (ovarian reads) and SRR772253 (oocyte reads). Additional file GRN: Gene Regulatory Network; eco-evo-devo: Ecological evolutionary development; AP:Anterior-posterior; DV: Dorso-ventral; RNA-seq: RNA-sequencing; RNP:Ribonucleoprotein; RTK: Receptor Tyrosine Kinase; CDK: Cyclin-dependent kinase; SC:Synaptonemal Complex; RN: Recombination Nodules; IRS: Insulin Receptor Substrate;20E: 20-hydroxy-ecdysone; JH: Juvenile Hormone; FPKM: Fragments Per Kilobase of exonper Million of fragments mapped; ORF: Open Reading Frame; SS: Similarity Score; GO:Gene Ontology; RT-qPCR: Real-time reverse transcription quantitative polymerasechain reaction; NRT: Negative reverse transcription; NTC: No template controlThe authors declare that they have no competing interests.nanos. JT assisted in manualannotation of the transcriptome. MG and CJB designed and supervised the study,performed the manual annotation of the transcriptome, and co-wrote the manuscript.All authors have provided comments on earlier drafts of the manuscript and approvedthe final version of the manuscript for publication.JMC collected and analysed RT-qPCR data, designed the automatic annotation pipeline,performed bioinformatic analyses, and co-wrote the manuscript. SCB assisted inRT-qPCR study design and data collection. RP and DRFC prepared RNA samples forRNA-seq. AC performed phylogenetic analyses of Oogenesis genes. Contains a tabulated and fully referenced list ofgenes identified from the literature, which have been studied in the contextof insect oogenesis and maternal regulation of early embryogenesis. The vastmajority of papers concern the fruitfly Drosophila melanogaster andthe silkmoth Bombyx mori. Many genes have multiple functions duringoogenesis, but to avoid repetition, and keep the size of the Tablemanageable, each gene has been listed only once in the functional contextfor which it is probably best known. Referencing has been kept to a minimum,highlighting key papers and databases. Hyperlinks have been provided foralmost all of the genes listed, which will provide full database informationon their myriad functions and further references. Presence (Y) or absence(N) of orthologs in the Pararge aegeria combined oocyte andovariole transcriptome are indicated.Click here for fileAnnotation summary of the combined transcriptome of the Pararge aegeria ovarioles and oocytes. Details the results of both automaticand manual annotation of 25266 contigs. Egg and ovary FPKM values aregiven for each contig. Each column contains a pop-up comment box with anexplanation of the column contents.Click here for fileOverview of the primer pair properties and performance in qPCRconditions. Gives an overview of the forward and reverse primersdesigned for qPCR of a set of 19 oogenesis and 3 housekeeping genes.Efficiency and R2 values are provided for each of theprimers.Click here for fileData generated by the CFX96 qPCR experiments. Details themeasurements from a total of 8 96-well white plates. Cq are given foreach gene of interest or reference gene.Click here for fileRelative Abundance Data generated by REST. Gives the results fromusing REST v2.0.13.0 to process Cq measurements and efficiencies inorder to estimate relative transcript abundance, and thus comparerelative transcript abundance between ovaries and eggs.Click here for fileTranscript abundance: Cq - FPKM correlation. Provides the resultsof the correlation analyses between two measures of transcriptabundance: Cq and FPKM-values.Click here for fileMapping of raw RNA-seq reads against egfr and wingless coding sequences as predicted from the draft Pararge aegeria genome. Provides the complete egfr andwingless (wg) CDS fasta information from ourunpublished P. aegeria genome. Furthermore, raw RNA-seq readswere mapped against these sequences and coverage determined.Click here for filePhylogenetic analysis of Nanos. Provides a phylogenetic analysisof insect Nanos protein sequences.Click here for filePhylogenetic analyses of both chorion and minor yolk proteins inLepidoptera. Provides the phylogenetic analyses of both chorionand minor yolk proteins in Lepidoptera.Click here for fileOocyte and ovarian RNA quality. Provides the Agilent BioAnalyzerElectropherograms detailing oocyte and ovarian RNA quality prior to cDNAsynthesis.Click here for fileFiltering of BLAST hits in the automated annotation. Provides avisualisation of the similarity score distribution and thresholdsapplied in the automated annotation of the P. aegeriatranscriptome.Click here for fileAutomated annotation based on different BLAST Strategies.Provides a summary of the automated annotation method, detailing thedifferent queries.Click here for fileDistribution of similarity classes across BLAST sources. Providesdetails regarding the number of Pararge aegeria contigs in eachof the similarity classes, according to the BLAST strategy used in theautomated annotation.Click here for fileThermocycler and qPCR reaction setup. Provides details regardingthe reaction conditions and thermocycler programming parameters forsuccessful qPCR amplification for each qPCR measurement reported in thisstudy.Click here for fileCombined annotated ovarian and oocyte transcriptome of Pararge aegeria. Provides the fasta format sequences of the contigs, which inAdditional file 2 had a YES in the SubmitFlag column (i.e. to besubmitted to NCBI TSA). Suggested annotated names are given on the basisof the BLAST results listed in Additional file 2, and as described inthe main text. The start and end of the open reading frames can be foundin the final two columns of Additional file 2.Click here for file"}
{"text": "N\u200a=\u200a138) were divided into three groups: patients with a history of suicide attempts (N\u200a=\u200a23); patients with current suicidal ideation (N\u200a=\u200a59); and patients without current suicidal ideation (N\u200a=\u200a56). After controlling for covariates, no significant differences were found among the three groups on any measure of temperament or character except self-directedness and self-transcendence. The self-transcendence scores of the lifetime suicide-attempt group were significantly higher compared with those of the suicidal-ideation group; post hoc analysis revealed that self-directedness was significantly lower in the suicide-attempt group compared with the non-suicidal group. The results from the present study suggest that remitted depression patients with a history of suicide attempts do not differ from non-attempters in temperament, but do differ in certain character traits.The aim of this study was to evaluate the Temperament and Character Inventory (TCI) scores of a sample of Korean patients with remitted depression who had attempted suicide and reported suicidal ideation and to compare their scores with those of remitted depressed patients without suicidal ideation. Adult depression patients who had completed 12 weeks of follow-up ( Suicide is a major public health problem worldwide, especially in patients with depression. The World Health Organization (WHO) estimates that 873,000 people commit suicide each year worldwide. This represents 1.4% of the total global burden of disease Although a variety of risk factors for suicidal behavior have been identified among persons with depression, including prior suicide attempts, hopelessness, co-morbid alcohol/substance abuse, further depressive episodes, and earlier age of onset of a major depressive disorder (MDD) In several previous studies, personality, as represented in Cloninger's temperament and character model and measured by the Temperament and Character Inventory (TCI), has been investigated in suicidal patients. TCI is a widely used inventory that evaluates four major dimensions of temperament: harm avoidance (HA), novelty seeking (NS), reward dependence (RD), and persistence (PE), together with three major character dimensions: self-directedness (SD), cooperativeness (CO), and self-transcendence (ST) Two temperament traits have been repeatedly associated with suicidal behavior: higher NS To date, no study has specifically compared remitted depression patients who have attempted suicide to patients with and without suicidal ideation in the context of Cloninger's temperament and character model as measured by the TCI. The aim of the present study was to compare the personality traits, indexed by TCI scores, of Korean patients with remitted major depressive disorder across three groups: patients with a history of suicide attempts (suicide-attempt group); patients with current suicidal ideation but without a history of suicide attempts ; and patients with neither current suicidal ideation nor a history of suicide attempts .The present study used data from the ongoing Clinical Research Center for Depression study (CRESCEND), whose design has been detailed elsewhere Between January 2006 and August 2008, CRESCEND enrolled 1,183 patients with depressive disorders  from 18 South Korean hospitals . Enrolment occurred in a naturalistic clinical environment from both outpatient and inpatient settings irrespective of depression subtype or physical co-morbidities. The study was approved by all of the relevant university and/or hospital institutional review boards and was conducted according to the Declaration of Helsinki and good clinical practices. All participants reviewed the consent form, and written informed consent was obtained by research staff prior to their participation.All patients with depressive disorders reviewed at the study hospitals were approached regarding participation. The clinicians assessing and diagnosing the patients applied the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria The CRESCEND research protocol recommended assessments at baseline, 1, 2, 4, 8, 12, 24, and 52 weeks and annually thereafter. The analysis described herein was restricted to data obtained at baseline and week 12 because the TCI was only completed at week 12. Socio-demographic and clinical characteristics were evaluated at baseline. Baseline and week 12 scores were used in this analysis.We categorized patients into three groups: patients without current suicidal ideation and without a history of suicide attempts ; patients with current suicidal ideation but without a history of suicide attempts ; and patients with a history of suicide attempts with or without current suicidal ideation (suicide-attempt group). To determine suicidality, we investigated patients' suicide attempt histories, and we administered the Beck Scale for Suicide Ideation (SSI-B) p<0.1), we excluded variables with low variance and/or significance. Finally, a post hoc analysis was performed to compare the three groups. Statistical significance was set at p<0.05 for all tests. All statistical analyses were conducted using the Statistical Analysis System software package .Descriptive statistics are provided for socio-demographic data and psychometric tests. The chi-square test, Fisher's exact test, and analysis of variance (ANOVA) were conducted to compare the groups across categorical and continuous variables. The three groups' TCI scores were compared via analysis of covariance (ANCOVA), with sociodemographic variables that were significant at baseline and clinical variables significant at week 12 used as covariates. If two or more significant variables exhibited strong correlations , and 23 patients (16.7%) had a history of suicide attempts. Included patients were more likely to be employed and had lower baseline scores on the CGI-S, HAMD, BDI, and SSI-B and higher scores on the SOFAS and WHOQOL compared with the excluded patients (data not shown).Of 1,183 total participants, 253 (21.4%) patients who had attempted suicide and 407 (34.4%) other patients completed follow-up to the week 12 visit. Information from the TCI was available for 325 patients (79.9%); 138 patients (42.5%) met the inclusion and exclusion criteria for the analysis described herein. The majority of the patients were female , age of onset , marital status , and religious status . Among the baseline psychiatric symptom scales, the HAMA , BDI , WHOQOL-BREF , and SSI-B  differed significantly across the three groups.The baseline socio-demographic and clinical characteristics of the non-suicidal, suicidal-ideation, and suicide-attempt groups are summarized in Post hoc analysis indicated that the suicide-attempt group was significantly younger compared with the suicidal-ideation (p\u200a=\u200a0.004) and non-suicidal (p\u200a=\u200a0.001) groups. Age of onset was higher in the suicidal-ideation group compared with the suicide-attempt group (p\u200a=\u200a0.042). Patients in the suicide-attempt group were more likely to be unmarried/divorced/widowed (p\u200a=\u200a0.005) and to have no religious affiliation (p\u200a=\u200a0.026) compared with the other groups. Compared with the non-suicidal group, the baseline HAMA total score was higher in the suicidal-ideation (p\u200a=\u200a0.015) and suicide-attempt (p\u200a=\u200a0.019) groups. Baseline total BDI scores were higher in the suicidal-ideation group compared with the non-suicidal group (p\u200a=\u200a0.004), and baseline WHOQOL scores were significantly lower in the suicide-attempt group compared with the non-suicidal group (p\u200a=\u200a0.005).p\u200a=\u200a0.009).At week 12, the total HAMD, BDI, CGI-S, WHOQOL-BREF, and SOFAS scores were not significantly different across groups. However, total HAMA scores were higher in the suicide-attempt group compared with the non-suicidal group , and the SD scores of the suicide-attempt group were significantly lower compared with the non-suicidal group (p\u200a=\u200a0.033).In the second phase of the analysis, ANCOVA was performed to compare group TCI scores at week 12, with age, gender, and week 12 HAMA total scores used as covariates. Age of onset, marital status and religion were excluded because they were correlated with age (data not shown), although they were significantly different across groups at baseline . There wT and SD . ST scorThe aim of present study was to evaluate patients who were currently in remission from MDD, with or without suicidal ideation and prior suicide attempts, to identify a distinct personality profile for suicide risk based on the TCI. We compared TCI scores in remitted MDD patients with and without suicidal ideation and in patients who had attempted suicide. The suicide-attempt group had higher ST scores compared with the suicidal-ideation group and had lower SD scores compared with the non-suicidal group. Interestingly, there were no significant group differences in HA, NS, RD, CO, and PE scores among groups.Several previous studies have reported that high HA, NS, and ST and low SD scores were associated with the risk for suicide In the present study, the suicide-attempt group had higher ST scores compared with the suicidal-ideation group and lower SD scores compared with the non-suicidal group. In a study with schizophrenic patients, high ST was associated with previous suicide attempts n\u200a=\u200a59) in remitted depressive patients in the present study. However, the widely accepted relationship between impulsivity and suicidality It is unclear why suicidal ideation was observed so frequently (42.8%, The present study had a number of strengths. First, the study used data from the CRESCEND study, which is the largest-scale depression cohort study ever conducted in Asia and includes a wide range of data pertaining to the socio-demographic and clinical characteristics of depressed Korean patients. Second, we excluded depressed patients with psychiatric comorbidities to control for the confounding effects of personality disorders or other Axis I disorders, such as anxiety disorders, eating disorders, and substance misuse-related disorders. Third, we included only patients who were in remission from depressive episodes, and assessed TCI at week 12 to control for the possible effects of depressive symptom severity.The present study also has several limitations. First, we included only Korean patients because cultural issues may lead to variation when assessing dimensions of temperament or character In conclusion, we did not observe differences among patients who attempted suicide and those with and without suicidal ideation in any of the dimensions of the TCI, with the exception of ST and SD. Longitudinal studies that include younger age groups are required to disentangle the personality profile of individuals with high suicidality from underlying psychopathology. The findings of the present study may further the delineation of this complex phenotype."}
{"text": "The use of sulfur mustard (SM) as a chemical weapon for warfare has once again assumed center stage, endangering civilian and the military safety. SM causes rapid local skin vesication and late-onset systemic toxicity. Most studies on SM rely on obtaining tissue and blood for characterizing burn pathogenesis and assessment of systemic pathology, respectively. However the present study focuses on developing a non-invasive method to predict mortality from high dose skin SM exposure. We demonstrate that exposure to SM leads to a dose dependent increase in wound area size on the dorsal surface of mice that is accompanied by a progressive loss in body weight loss, blood cytopenia, bone marrow destruction, and death. Thus our model utilizes local skin destruction and systemic outcome measures as variables to predict mortality in a novel skin-based model of tissue injury. Based on our recent work using vitamin D (25(OH)D) as an intervention to treat toxicity from SM-related compounds, we explored the use of 25 (OH)D in mitigating the toxic effects of SM. Here we show that 25(OH)D offers protection against SM and is the first known demonstration of an intervention that prevents SM-induced mortality. Furthermore, 25 (OH)D represents a safe, novel, and readily translatable potential countermeasure following mass toxic exposure. Sulfur mustard (SM) has been used as a chemical weapon in World War I and during inter-war periods causing life-threatening injury for which there is currently no specific treatment. Indeed the recent past has once again witnessed the horrors of SM re-emerging as an arsenal for combat so the pressure is on, more than ever, to intensify the search for effective intervention. In this study we use SM as the inducer of chemical injury which possesses a range of toxicities with exposure to varying intensities. Low dose cutaneous exposure to SM results in erythema and burning , while hPathogen-free, six week old female C57/BL6J mice were purchased from Jackson Laboratories . Animals were quarantined according to Battelle standard operating procedure (SOP). All animals receive standard laboratory diet. Anesthetic calculations were based on weights obtained during quarantine. An anesthesia cocktail of a 10 ml/kg mixture of ketamine hydrochloride  and xylazine hydrochloride  was administered by intraperitoneal (IP) injection. Hair on the dorsal back of mice was removed by clippers and depilation as previously described . Mice weSM was supplied by U.S. Army Edgewood Chemical Biological Center . The SM purity value was >96 percent as determined by gas chromatography. As part of the dose finding study, SM dissolved in DMSO , was applied at 8 different concentrations  over an area approximately 8 mm in diameter on the dorsal surface of each mouse. To determine a stable LD70 model, doses of 50, 55 and 60 mg/kg were applied on mice. Control mice were applied DMSO only.25(OH)D (Sigma-Aldrich) was reconstituted in ethanol and further diluted in mineral oil Sigma-Aldrich Chemical Company, Inc. , for use. 5 ng of 25(OH)D was injected intraperitoneally using a 27 gauge needle  1 h following SM exposure. Untreated mice receiving SM only were injected with mineral oil.Wounds were measured with a digital caliper daily for 5 days following SM exposure. All mice in the study were weighed daily till day 5 or till drop in body weight met euthanasia criteria.A drop of blood was collected from SM exposed and control mice and transferred onto a glass slide by tail snip, smeared using a microscope slide (Fisher Scientific), fixed in 100% methanol (Fisher Scientific), and stained with Wright-Giemsa to observe cell types and enumerate red blood cells per high power field (HPF).Blood was collected from mice undergoing sacrifice on day 5 and analyzed for complete blood count using a hematology analyzer at Battelle R&D facility Sternums and skin of euthanized mice were removed and fixed overnight in 10% formalin diluted in PBS (Fisher Scientific). Sternums were embedded in paraffin, sectioned (8 \u00b5m thickness), stained with H&E and observed under the microscope to evaluate SM-induced cellular depletion.Freshly harvested skin and sternum designated for immunofluorescence studies were OCT-embedded and analyzed as previously described . PrimaryTibiae and femurs of mice were isolated to harvest bone marrow cells as described before . Viable A 2 sided unpaired t-test was used to compare mean values. The Kaplan-Meier method was used to plot the survival distributions of all groups (SM and SM + 25(OH)D) which were then compared using the log-rank test. A Cox regression analysis was used to assess mortality hazard as a function of lesion size and body weight loss. Data are presented as mean \u00b1 s.e.m, and p values \u22640.05 were considered significant.2 circular area (8 mm diameter). The challenge dose range (4.8\u2013116 mg/kg SM) was selected in order to reduce confidence limits around LD90 and obtain a statistically significant probit slope. Skin lesions were evident in all SM challenged groups with representative images shown in We developed a SM skin exposure mouse model in which dorsally shaved mice received 8 different concentrations of SM topically applied (in DMSO) onto a 50 mm2  and 68 mg/kg  (data not shown). Interestingly, we observe no mortality with exposure to SM at doses \u226440 mg/kg, although doses \u2265 45 mg/kg appeared to be a critical threshold for death resulting in a stable LD70 at 50 mg/kg D in rescuing mice from NM-induced pathology (70 model in mice that is attenuated by 25(OH)D.The data presented defines 50 mg/kg SM in DMSO as the minimum dose that elicits a sufficient local (skin lesion) and systemic  response to generate an LDathology , 25(OH)Dathology . These dOur findings suggest that skin exposure to SM precipitates in an acute phenotype in mice characterized by skin ulceration, body weight loss and death, all of which progress in a dose dependent manner. We demonstrate that the early effects following challenge with SM include skin destruction that penetrates deep into the dermis through massive infiltration of activated macrophages producing elevated levels of iNOS. In most experimental animal models studied cutaneous wounds from SM burn progress with an inflammatory burst which critically influences the kinetics of wound healing . While mVitamin D is widely available and has been shown to be beneficial in a NM skin vesication model and thus could have a broad public health impact in the event of chemical weapon exposure. A prospective study on patients with leg ulcers are associated with acute vitamin D deficiency that upon treatment with vitamin D shows a trend towards accelerated healing  suggestiIn conclusion this study establishes that SM exposure to skin sustains skin vesication including formation of inflammatory foci at the site of exposure. Local skin destruction is accompanied by progressive weight loss that contributes to mortality. Our results provide the first evidence that early intervention with 25(OH)D offers protection from SM induced injury. Utilizing progressive increase in wound area and loss in body weight as the two key variables, our model presents a stable platform for advancing research to test the efficacy of potential countermeasures like 25(OH)D that would offer treatment options to exposed victims."}
{"text": "Roofers are at increased risk for various malignancies and their occupational exposures to polycyclic aromatic hydrocarbons (PAHs) have been considered as important risk factors. The overall goal of this project was to investigate the usefulness of phosphorylated histone H2AX (\u03b3H2AX) as a short-term biomarker of DNA damage among roofers.Blood, urine, and dermal wipe samples were collected from 20 roofers who work with hot asphalt before and after 6\u00a0h of work on Monday and Thursday of the same week (4 sampling periods). Particle-bound and gas-phase PAHs were collected using personal monitors during work hours. \u03b3H2AX was quantified in peripheral lymphocytes using flow cytometry and 8-hydroxy-2-deoxyguanosine (8-OHdG) was assessed in urine using ELISA. General linear mixed models were used to evaluate associations between DNA damage and possible predictors . Differences in mean biomarker and DNA damage levels were tested via ANOVA contrasts.Exposure measurements did not show an association with any of the urinary biomarkers or the measures of DNA damage. Naphthalene was the most abundant PAH in gas-phase, while benzo(e)pyrene was the most abundant particle-bound PAH. Post-shift levels of \u03b3H2AX and 8-OHdG were higher on both study days, when compared to pre-shift levels. Cigarette smoking was a predictor of \u03b3H2AX and urinary creatinine was a predictor of urinary 8-OHdG. Between-subject variance to total variance ratio was 35.3\u00a0% for \u03b3H2ax and 4.8\u00a0% for 8-OHdG.\u03b3H2AX is a promising biomarker of DNA damage in occupational epidemiology studies. It has a lower within-subject variation than urinary 8-OHdG and can easily be detected in large scale groups. Future studies that explore the kinetics of H2AX phosphorylation in relation to chemical exposures may reveal the transient and persistent nature of this sensitive biomarker of early DNA damage.The online version of this article (doi:10.1186/s12940-016-0182-4) contains supplementary material, which is available to authorized users. Workers around the world experience daily exposures to potentially carcinogenic chemicals. Identifying the role of these exposures in cancer development later in life has been a major challenge in occupational epidemiology. Estimating the exposure-cancer association becomes more complicated by simultaneous exposures to other environmental and lifestyle factors. A further challenge is the long latency period between carcinogenic exposure and cancer diagnosis. Many times occupational studies rely on estimates of current exposures and their association to short-term markers of health effects. Among these markers, measures of DNA damage are viewed as reliable indicators of increased cancer risk , as theyRoofers are at increased risk for different malignancies such as lung, bladder, stomach, skin and buccal cavity cancers, and leukemia \u201310. ExpoExposure to PAHs can increase reactive oxygen species (ROS) formation in the body. When the cellular antioxidant defense system is disturbed, the increased amount of ROS can cause oxidative damage to biomolecules such as DNA, proteins and lipids. Recent studies have supported the link between insufficient cellular defense towards oxidative DNA damage and increased susceptibility to cancer development \u201333. UrinDouble-strand DNA breaks have also been linked to PAH exposures; this form of DNA damage can be measured using phosphorylated histone H2AX (\u03b3H2AX) in individual cells . IncreasBefore a biomarker can be comfortably used in epidemiology studies, it needs to be validated based on the following criteria: 1) The relationship between the biomarker and exposure in question, 2) The formation, distribution and elimination of the biomarker in humans, 3) Variation of the biomarker between- and within- study participants, 4) Baseline values of the biomarker in the general population, and finally 5) Cost and difficulty of analytical techniques . Here, wTwenty roofers employed by one roofing company were recruited. The study site was a roof replacement project located in Colorado Springs, Colorado and was visited by the field study team over four weeks between July and September of 2013. Potential participants were informed about the study at the site and those who signed the informed consent under University of Colorado\u2019s IRB (COMIRB) approved protocol (COMIRB Protocol # 12\u20130443) were recruited. Each week, a new group of workers participated in the study over two workdays: Monday and Thursday. Study questionnaires were administered before and after the work-shift in either English or Spanish; the latter applied by a Spanish speaking interviewer. Biological samples (urine and blood) were collected before and after 6\u00a0h of work. Study participants also provided hand wipes (with 3\u00a0ml sun flower oil) at each sampling period. Information on personal characteristics , life-style factors , use of protective equipment during the study day  and specific work tasks performed during the day  were collected via questionnaires. The before-work questionnaire focused on non-occupational sources of PAH exposures and the after-work questionnaires contained more detailed questions on work practices. Both of these questionnaires are provided in Additional file After the completion of morning questionnaires and collection of biological samples, the participants were given lightweight vests with air monitors to collect air samples from within each worker\u2019s breathing zone air during the shift. The participants were then asked to return to their work. After 6\u00a0h of work participants returned the vests with the air monitors. The total number of samples collected from 20 participants is as follows: 79 urine, 79 blood, 40 air, and 79 dermal wipes. One of the participants had to leave the site on the second study day due to a family emergency and could not provide samples that afternoon (period 4).2.5 sampling inlets  and 37\u00a0mm Teflon filters. Gas-phase PAHs were collected immediately downstream of the filters using standard adsorbent tubes . Method details are provided in Additional file Polycyclic aromatic hydrocarbons (PAHs) in ambient air were measured within the breathing zone of workers via personal sampling. Particle-bound PAHs, 4-ring and above, were collected using personal sampling pumps (SKC XR5000) fitted with PMPAH metabolites were analyzed using an automated solid-phase extraction based on a method developed by Romanoff . DetailsDermal exposure samples were collected using a previously published hand washing method with sunflower oil . Detailsn\u2009=\u200980) were tested to evaluate levels of \u03b3H2AX. This method was optimized by treating fresh lymphocytes, in triplicate from one volunteer who was not a roofer, with various amounts of H2O2 (0.02-0.24\u00a0mM) and freezing via the same method described above. Frozen samples were thawed in a 37\u00a0\u00b0C water bath  and 500,000 cells were added to wells in a round-bottom 96 well plate . Freezing media was removed and cells were washed 3 times with PBS. All washes and buffer removals involved a 600\u00a0g spin for five minutes at room temperature. Cells were then fixed with 200\u00a0\u03bcL BD Cytofix fixation buffer (BD Biosciences) and incubated for 15\u00a0min at room temperature. Next, the fixative was removed, and cells were washed twice with 200\u00a0\u03bcL of PBS. Cells were then permeabilized with 200\u00a0\u03bcl \u221220\u00a0\u00b0C Perm Buffer III (BD Biosciences) for 5\u00a0min at RT. After one wash in 200\u00a0\u03bcL of 1x perm/wash buffer (BD Biosciences), 200\u00a0\u03bcl of 1x stain buffer (BD Biosciences) was added to each well to block non-specific binding. After 20\u00a0min at room temperature the cells were washed two times with 200\u00a0\u03bcl of Perm/Wash buffer. Next, 100\u00a0\u03bcl stain buffer and 5\u00a0\u03bcl BD antibody  were added and cells were incubated for 60\u00a0min at room temperature in the dark. After antibody removal, wells were washed three times with 200\u00a0\u03bcl of 1x perm/wash buffer. Finally, the cells were resuspended in 300\u00a0\u03bcl FACS fix  and read with CFlow Plus software on a C6 flow cytometer . All samples were run in triplicate and results are given in mean fluorescence intensity (MFI) of the lymphocyte gated FL1 channel . Plots for publication were made using the FCS Express4 Flow Research Edition software. Figure\u00a0Methodological details on processing of peripheral blood samples are described in Additional file All statistical analyses were conducted using SAS system software  at a significance level of 0.05. All tests were performed after  logarithmic transformation of urinary analytes , \u03b3H2AX and PAH levels  to satisfy the normality assumption, and data were summarized as geometric means (GMs) and geometric standard deviations (GSDs). Average levels of naphthalene and pyrene in personal breathing zone samples or on dermal wipes were compared between the two study days or between smokers and nonsmokers for each day, using Student\u2019s t-tests. Specific contrasts  were applied to test for differences in log-transformed mean levels of urinary biomarkers and DNA damage measures by sampling period (before and after the work-shift on first and second day) and cigarette smoking status. For this purpose, a two-way analysis of variance (ANOVA) procedure was applied for each day separately. For each sampling day, Pearson\u2019s correlation coefficients (with 95\u00a0% CI) were used to measure the strength of association between different pairs of exposure and biomarker measurements.In personal air samples using filters (FLT) 42.5\u00a0% of naphthalene measurements and 35\u00a0% of benzo(e)pyrene measurements were below limit of detection (LOD). For personal air XAD samples 42.5\u00a0% of pyrene was below LOD, while naphthalene was detected in all of the XAD samples. For dermal wipes, 3.5\u00a0% for naphthalene samplers were below LOD and pyrene was detected in all of the samples. Measurements of \u03b3H2AX (lymphocytes) and 8-OHdG (urine) were above detection limit for all of the samples. For urinary biomarkers: 1-OHPyr was below LOD in 24\u00a0% of the samples while 1-OHNap was below LOD in one urine sample (1.2\u00a0%). 2-OHNap and creatinine were both detected in all of the urine samples. For samples that were below the LOD, a proxy measurement was assigned using the value of LOD/\u221a2 before statistical analyses .Information for a number of general and work-related variables was collected via questionnaires  were retained. Backward selection of all retained independent variables and their plausible two-way interactions were used to achieve final models (using a significance level of p\u2009<\u20090.05). Multivariable models had the general form:Repeated-measures general linear mixed modeling (PROC MIXED) was used to examine associations between DNA damage measures (\u03b3H2AX and 8-OHdG) and sampling period (before/after work on two separate sampling days) adjusting for confounders. For the general linear mixed models only confounders with values at the four different sampling periods were considered. Candidate variables that were considered amounted to eight variables for the model of \u03b3H2AX , and nine variables for the model of urinary 8-OHdG , plus the two-way interactions between urinary biomarkers and cigarette smoking in both models. The most likely candidate variables were screened as follows. First, DNA damage measures were regressed on each covariate separately and variables that suggested significant contributions (Yi represents the subject-specific mean of log-transformed levels of \u03b3H2AX (or 8-OHdG) for the ith subject at sampling period jh, \u03b1 is the intercept representing the average level of Yi when all independent variables are zero for an average worker, bi is the random intercept for subject i that captures the heterogeneity between individuals, \u03b2k is the regression coefficient for the kth independent variable Xik for the ith subject, and \u03b5ij is the error term. Given the sample size of 80 measurements from 20 individuals, a linear mixed model with about 4\u20136 predictors will likely be a stable model. Estimates of the percentage of variance explained by each of the significant covariates in the linear mixed models were calculated using the conditional and marginal formulas of R2 [where as of R2 . The intp-value\u2009<\u20090.0001 for difference). When divided by smoking status, post-shift levels of 8-OHdG remained to be higher than those observed pre-shift in nonsmokers  and in smokers , difference was statistically significant for both groups (p\u2009<\u20090.05). The overall difference between post-shift and pre-shift levels was smaller for \u03b3H2AX  and remained small in nonsmokers  and in smokers .Levels of DNA damage markers were at higher concentrations in samples collected after work when compared to those observed before work. The overall difference between post-shift and pre-shift levels was 1.7-fold for urinary 8-OHdG , naphthalene was the most abundant PAH, quantified in all of the samples. Pyrene on the other hand, was only quantified in 57.5\u00a0% of the XAD samples. For PAH measurements obtained from filters (representing particulate phase) the most abundant compound was benzo(e)pyrene quantified in 65\u00a0% of all samples, followed by naphthalene quantified in 57.5\u00a0%. Only 35\u00a0% of filter samples had quantifiable levels of pyrene. Table\u00a0Levels of naphthalene on dermal wipes did not differ significantly before or after the work on both study days Table\u00a0. Levels Table\u00a0p\u2009=\u20090.005, Additional file All urinary biomarkers were higher after work on Monday in both smokers and nonsmokers, but the post-shift to pre-shift difference was statistically significant only for urinary 1-OHPyr and 8-OHdG in smokers .For \u03b3H2AX, according to the conditional RIn models of urinary 8-OHdG measurements, cigarette smoking was not a significant predictor, but urine creatinine had a great impact on the levels and was kept in final models. Sampling period also remained a significant predictor. When compared to Monday morning, levels collected on Monday afternoon corresponded to 55\u00a0% higher levels of urinary 8-OHdG, and levels collected at the end of the week (period 4) were increased by 50\u00a0%. The percentage of the between-workers variation of 8-OHdG in the log scale was 4.8\u00a0% of the total variance Table\u00a0.Based on our model .For the analysis of logged 8-OHdG, according to the conditional RWe have recently shown that urinary 8-OHdG is a promising biomarker reflecting early effects of occupational exposures to PAHs during a single work day . Here weIn this study, sampling period, reflecting four different time points within one workweek, was an important predictor of both DNA damage markers. Other important predictors were cigarette smoking for \u03b3H2AX and urinary creatinine for urinary 8-OHdG. Our results once again support that urinary 8-OHdG is highly affected by urine dilution. This is an important concern since many times roofers are exposed to heat and can be dehydrated during the course of a single work day.We also observed that about 35.3\u00a0% of the unexplained variance of \u03b3H2AX was between subjects, while this number was only 4.8\u00a0% for urinary 8-OHdG. The proportion of within-subject variance appears larger for urinary 8-OHdG. These two measures cannot be directly compared as they reflect different types of DNA damage  and are measured in different biological media (\u03b3H2AX from lymphocytes and 8-OHdG in urine). However, a low ICC value reflects high within-individual variation of a biomarker and is a sign of poor reproducibility . Other sLevels of pyrene on dermal wipes were higher after work when compared to before work measures. This increase was not observed for naphthalene. Consistently, urinary 1-OHPyr levels significantly increased over work hours on Monday, while the increase in urinary naphthalene metabolites was small. Naphthalene is the most abundant PAH in many environments and naphthalene based biomarkers can potentially increase sensitivity of assays. However, our results in this population suggest that environmental influences and cigarette smoking can overwhelm those of occupational exposures to naphthalene. Results of this study are consistent with our previous findings that urinary 1-OHPyr is a promising biomarker of occupational exposures in roofers and that dermal contact can be an important exposure route .Urinary metabolites of PAHs, particularly urinary 1-OHPyr, are established biomarkers of occupational exposures. Overall, levels of PAH exposures in this study were lower than previous reports in asphalt exposed workers , 72. WhiDespite the increase in urinary biomarkers during work hours, we did not observe consistent correlations between measures of exposure, urinary metabolites, and DNA damage, making it difficult to reach a final conclusion on the association between exposure and biomarkers. However, this is possibly related to the overall low occupational exposures observed during the study period. Consistently, Monday morning levels of urinary 8-OHdG were are approximately 36-fold lower than those observed in our previous study in roofers .Cigarette smoking is an important factor to consider when analyzing \u03b3H2AX. The correlation between \u03b3H2AX and urinary metabolites of naphthalene on the second study day was possibly due to their common association with cigarette smoking. This is supported by the fact that levels of urinary 1- and 2-OHNap among smokers were much higher before the work shift on Thursday when compared to levels after the work. It is tempting to limit future evaluations to nonsmokers. However, considering the high proportion of smokers  and the There are some limitations of this study that constrain the interpretation of results. The most important limitations are the low levels of exposure observed during the study period and the small number of participants. Repeated sampling at four different time points, however, provided us with a larger effective sample size. We also used ANOVA to test for differences between measurements conducted before and after the work shift without adjustment for repeated measures. While before and after-work samples do not reflect identical conditions, they are also not independent. However, this was only a preliminary approach that has been addressed by the use of linear mixed models where the specific contrasts of interest have been tested. We also recognize that we are conducting some hypothesis tests without adjustment for multiple comparisons, which in turn might produce a few false positive associations.Another limitation is the widespread environmental exposure to PAHs and other toxicants that cause DNA damage. 1-OHPyr has been widely viewed as the gold standard biomarker of PAH exposures \u201377. NaphThe PAH biomarkers studied here reflect short-term exposures. The estimated elimination half-lives for urinary 1- & 2-OHNap and 1-OHPyr are around 4\u00a0h  and 13\u00a0hWhile \u03b3H2AX is an early response to genotoxic insults, a number of non-occupational factors can contribute to the DNA double strand breaks (DSBs), such as ultraviolet light (UV), environmental chemicals, and even endogenous triggers of DNA damage. Tobacco smoke, a common source of PAHs, is also a potent inducer of DSBs. With the high number of factors influencing \u03b3H2AX response, it is important to distinguish between baseline levels in the general population and \u03b3H2AX kinetics following specific exposures. Two types of \u03b3H2AX foci have been reported previously: the fast transient \u03b3H2AX foci associated with rapid repair which takes place within minutes or hours, and the residual foci that persist for several days or months . The majOur overall goal was to explore the usefulness of \u03b3H2AX as a possible marker of DNA damage in workers exposed to PAHs using a high throughput flow cytometry assay. One of our evaluation criteria was its association with exposure data, which we could not observe for \u03b3H2AX. Urinary 8-OHdG, a commonly used marker of DNA damage, was also not associated with exposure levels in this group. It is possible that the relatively low levels of exposures may have impacted our analyses. As a second criterion, we evaluated the between- and within-subject variation of \u03b3H2AX. Here we observed that \u03b3H2AX has a smaller within-worker variation when compared to urinary 8-OHdG. Our analyses also confirmed that baseline values of \u03b3H2AX are easily detectable in this population, using an inexpensive method such as flow cytometry. Despite the lack of association with exposure data, we propose that \u03b3H2AX is a sensitive biomarker of early DNA damage related to occupational exposures. The low within-subject variation, easy and high throughput methodology makes \u03b3H2AX a feasible alternative in epidemiology studies. We perceive the need for additional studies to understand baseline values of \u03b3H2AX, between- and within-individual variation in different study populations, and the impact of developmental and degenerative diseases, as well as dietary, environmental, and life-style factors on this promising biomarker . This wi"}
{"text": "This paper focuses on nano-morphology-controlled small-molecule organic solar cells without solvent treatment for high power-conversion efficiencies (PCEs). The maximum high PCE reaches up to 7.22% with a bulk-heterojunction (BHJ) thickness of 320 nm. This high efficiency was obtained by eliminating solvent additives such as 1,8-diiodooctane (DIO) to find an alternative way to control the domain sizes in the BHJ layer. Furthermore, the generalized transfer matrix method (GTMM) analysis has been applied to confirm the effects of applying a different thickness of BHJs for organic solar cells from 100 to 320 nm, respectively. Finally, the study showed an alternative way to achieve high PCE organic solar cells without additive solvent treatments to control the morphology of the bulk-heterojunction. During the last decades, bulk-heterojunction (BHJ) small-molecule organic solar cells (OSCs) have received more research attention ,6,7,8,9.i.e., chlorobenzene, 1,2-dichlorobenzene) -phenyl-C71-butyric-acid-methyl-ester (PC70BM) to form a BHJ photoactive layer. Chemical structures for OSC showed in p-DTS(FBTTh2)2 and PC70BM were dissolved in chlorobenzene (CB) and stirred over 24 h with a total concentration of 50 mg/mL. Indium tin oxide (ITO)-coated glass substrates were cleaned sequentially by ultrasonic treatment in Alconox detergent, deionized water, acetone and isopropyl alcohol. A polymeric conducting thin layer of PEDOT:PSS (40 nm) was spun-cast on top of the ITO-coated glass substrate. Before spin-coating of BHJ thin film, co-dissolved donor-acceptor blend was heated at 100 \u00b0C for 30 min. Then the p-DTS(FBTTh2)2:PC70BM BHJ layer was spin-cast from the heated and blended solution with different spin speed to form a different thickness of BHJs from 300 to 2000 rpm. During the solution preparation and film formation processes, 1,8-diiodooctane (DIO) additive solvent treatment was avoided. Finally, Ca (5 nm) and Al (100 nm) electrodes were deposited on top of BHJ photoactive layer  characterization system was supplied by PV Measurement Inc.  to obtain the data. A GTMM analysis was accomplished to calculate and analyse the multi-layered interface OSCs. For transfer matrix analysis, optical constants of all layers have been obtained by the ellipsometry method. Finally, the nano-morphology of BHJ film was investigated by atomic force microscopy (AFM).To address the particular issues mentioned above, OSCs fabricated through the incorporation of a donor-acceptor blend of ve layer b. Post-a2 Air Mass (AM) 1.5G illumination. The optimized ratio of the small molecule donor (p-DTS(FBTTh2)2) to PC70BM was chosen to be the same as the reported ratio (60:40 w/w) [J-V) characteristics of SM BHJ OSCs, in terms of BHJ without DIO treatment, with different photoactive thicknesses are shown in SM BHJ OSCs have been tested under simulated 100 mW/cmVoc as high as 0.74 V was observed in all devices. Combined with its high Jsc and fill factor (FF), a high power conversion efficiency (PCE) of 6.83% on average was measured with 320-nm-thick devices and the highest PCE was measured at 7.22% . The measured PCE of our device (2.78%) is much greater than the reported PCE value (1.8%) with the same BHJ thickness of 100 nm [p-DTS(FBTTh2)2 and small molecule donor materials.A J-V result. The results are shown in Jsc value by integrating the EQE data. The calculated Jsc values are in good agreement with the directly measured values. Furthermore, these EQE spectra confirm Beer-Lambert law by increasing the photoactive layer thicknesses. It is good to note that EQE spectra results obtained much better results to compare with the literature [The EQE spectra were obtained to confirm the accuracy of the SM BHJ OSC\u2019s photo-generated terature ,3,4,8. A\u22121\u2219cm\u22123) of SM BHJ OSCs obtained by the generalized transfer matrix method (GTMM). These calculated charge generation rate results provide a further explanation as to why a thicker junction of OSC has a higher Jsc value than a thinner junction of OSCs. From Jsc value in thin junction OSCs. By increasing the photoactive layer thickness containing BHJ, the OSC follows Beer-Lambert law. Therefore, the photo-generation rate is exponentially decreased by increasing the photoactive layer thickness.p-DTS(FBTTh2)2:PC70BM BHJ film (thickness = 320 nm) is shown in An AFM two-dimensional 2D) topography image of the D topograIn conclusion, we studied the high performance of small-molecule organic solar cells without additive solvent treatment on the control of the photoactive layer in nano-morphology. Significantly, we have obtained small-molecule organic solar cells with a comparable PCE of 7.22% by eliminating the DIO additional solvent treatment. Also, GTMM analysis was accomplished to confirm the effect of the thickness variations of SM BHJ OSCs. Lastly, AFM measurement also confirms that smaller domains have achieved efficient charge generation. These results provide significant progress in showing that solution-processed small-molecule organic solar cells without solvent treatment can be an alternative method to having a high-PCE device with polymeric and/or small-molecule counterparts."}
{"text": "The Uncoordinated 5A (UNC5A) protein is part of a family of receptors that play roles in axonal pathfinding and cell migration. We previously showed that the Fanconi anemia C protein (FANCC) interacts with UNC5A and delays UNC5A-mediated apoptosis. FANCC is a predominantly cytoplasmic protein that has multiple functions including DNA damage signaling, oxygen radical metabolism, signal transduction, transcriptional regulation and apoptosis. Given the direct interaction between FANCC and UNC5A and that FANCC interferes with UNC5A-mediated apoptosis, we explored the possibility that FANCC might play a role in axonal-like growth processes.Here we show that FANCC and UNC5A are localized to regions of neurite outgrowth during neuronal cell differentiation. We also show that absence of FANCC is required for neurite outgrowth. In addition, FANCC seems required for UNC5A expression. Results from this study combined with our previous report suggest that FANCC plays a role in tissue development through the regulation of UNC5A-mediated functions. FancA and FancC are highly expressed in the developing brain, specifically in the intermediate zone, which contains migrating neurons . Thase (AD) .UNC5A were found in the posterior cingulate brain region of AD patients, while mutations in UNC5C seemed to predispose to late-onset Alzheimer\u2019s disease [Furthermore, significant expression changes in  disease \u201331. ThesIt is unclear whether interaction between FANCC and UNC5A is required for neurite outgrowth. Further work is needed to determine whether FANCC regulates UNC5A apoptosis during cellular development or axon guidance in vivo."}
{"text": "The adverse drug reactions (ADRs) related to clonazepam are mild, and only two cases of myotoxicity induced by clonazepam have been reported, with both patients recovering well. We present a unique case of a serious ADR outcome after taking clonazepam.A 24-year-old woman with a long-standing history of polio and a 2-year history of epilepsy developed a serious ADR after repeated exposure to oral clonazepam combined with sodium valproate that manifested as myotoxicity and elevated levels of creatine phosphokinase. The patient is currently bedridden and unable to take care of herself.Clinicians should be vigilant of the possibility of myotoxicity induced by clonazepam, especially in specific populations such as polio patients or when clonazepam is used in combination therapies. Epilepsy is one of the most common serious disorders of the brain, affecting about 50 million people worldwide. Nearly a quarter of these patients have drug-refractory epilepsy . ClonazeThere has been no previous report on serious adverse drug reactions (ADR) associated with clonazepam. Here we describe a patient who developed myotoxicity and became bedridden after repeated exposure to clonazepam combined with sodium valproate.A 24-year-old woman had experienced non-progressive polio from 1\u2009year of age despite having been inoculated with polio vaccine on time. She could walk with a limp but could not control her right hand sufficiently well to write, and so had not received the usual schooling. However, she could take care of herself and help her parents with a small amount of housework. About 2\u2009years previously she had suffered from epilepsy in the form of bilateral tonic-clonic seizures, but she had not received regular treatment due to financial reasons. The epilepsy had not resulted in any deterioration of her polio symptoms.The patient had been taken to the hospital to receive regular treatment for epilepsy for the first time 6\u2009months previously, at which time her antiepileptic regimen was sustained-release sodium valproate (500\u2009mg p.o.) plus clonazepam (2\u2009mg p.o.) every 12\u2009h. The patient developed drowsiness after the first combined dose of sodium valproate and clonazepam, and slept from 3\u2009p.m. on the first day to 10\u2009a.m. on the following day. She could not walk unaided and felt very tired, but she did not contact her doctor, instead continuing on this antiepileptic regimen regularly for 21\u2009days until she ran out of clonazepam tablets. Her epilepsy symptoms were well controlled during this 21-day treatment period, but she developed muscle weakness and muscle pain, and remained in bed since she could not take care of herself.The patient continued taking the sustained-release sodium valproate tablets (500\u2009mg p.o. every 12\u2009h), during which her epilepsy symptoms remained well controlled. Her muscle weakness symptoms started to improve gradually over the following 2\u2009weeks, allowing her to stand but not walk. At this time she was taken to hospital for the second time. The patient and her family unfortunately refused electromyography, a muscle biopsy and other tests with the exception of some simple blood tests due to financial reasons. The findings of preliminary blood examinations including the complete blood count, urine test, liver and kidney function test, electrolytes, and plasma ammonia were within clinically acceptable limits, but her serum CPK was markedly raised at 4261\u2009U/L (38\u2013174\u2009U/L), while her serum sodium valproate concentration was 101.89\u2009\u03bcg/mL (50\u2013100\u2009\u03bcg/mL) and her serum globulin concentration was 36.4\u2009g/L (25\u201335\u2009g/L). There had been no preceding illness, infection or trauma, the patient did not have a history of statins or other drugs, and there was no family history of any neuromuscular disorder. Based on the relationship between medication times and symptoms, we attributed the myotoxicity to clonazepam, and assigned the patient to an antiepileptic regimen of sodium valproate monotherapy, with clonazepam remaining discontinued.A 2-month telephone-based follow-up revealed that the patient had started taking clonazepam irregularly because of insomnia, and suffered from muscle weakness and muscle pain again. Her clinical condition had deteriorated to the point that she was unable to stand or walk, and was unable to take care of herself. The patient and her family refused further physical examinations and treatment because they had lost confidence in curing the disease and also for financial reasons. We recommended that the patient discontinued clonazepam immediately and never take it again. At another follow-up 3\u2009months later, the frequency and severity of epileptic seizures were significantly reduced in the patient, but her myotoxicity condition had not improved, she still could not stand or walk, and she was now bedridden.Our patient developed muscle weakness after taking sodium valproate and clonazepam at the same time, and the myopathy caused by sodium valproate and by clonazepam had been reported \u20139. HowevThe patient and her family declared that her polio and epilepsy were non-progressive. However, the dearth of neurological examinations meant that the woman might have actually had a slowly progressive early-onset type of muscle-wasting disease of neurological or myopathic origin, whose acceleration by the epilepsy treatment could have caused her clinical manifestations. Moreover, the probable myopathy varied with her clonazepam intake, representing further evidence that clonazepam was related to the myotoxicity. Numerous agents exhibit well-documented myocytoxicity, including anticholesterol statins, antirheumatic/inflammatory/immunosuppressive drugs, antinucleoside analogues, contaminated products, and dietary agents, and they result in symptomatologies ranging from mild discomfort and inconvenience to permanent damage and disability , 12. TheBecause our patient was taking sodium valproate at the same time as clonazepam, drug interactions might have been present. Jeavons et al.  reportedThe reported myocytoxicity symptoms including ataxia, fatigue and weakness induced by clonazepam were resolved and the CPK level reduced to the normal reference range after stopping clonazepam in our patient , 6. The We have described a case of the serious ADR of myotoxicity that was probably induced by clonazepam. Our findings suggest that clinicians should be vigilant of the possibility of myotoxicity induced by clonazepam, especially in specific populations such as polio patients or when clonazepam is used in combination therapies."}
{"text": "Subsequently, the synthesized compounds have been evaluated for their antimalarial activity against the drug-sensitive P. falciparum NF54 strain. All of them were inactive. In addition, they did not show any toxicity against L6 cells . These results contribute to a better understanding of artemisinins mechanism of action.Herein, we describe a biomimetic entry to (+)-3-hydroxymethylartemisinin ( Gratify9 and 10 (12 and 13 after treatment with Martin sulfurane [12 using NiCl2/NaBH4 in methanol as solvent yielded derivative 14 with excellent diastereomeric ratio (dr = 1:0.03). On the other hand, derivative 13 was reduced under Birch conditions (Li/NH3) to afford ester 15 in 61% yield (dr = 1:0.4). Gratifyingly, both artemisinin precursors 14 and 15 possess the desired stereoconfiguration at carbon center 8a.The structure of all these isomers was confirmed by NOE experiments  in 24% and 16% yield, respectively. Protection of the free hydroxy group of 16 as a silyl ether and methylation of the obtained lactone in \u03b1-position (LDA/MeI/HMPA) afforded derivative 17. After removal of the TES protecting group (+)-3-hydroxymethyl-9-epi-artemisinin  was produced from 17 in two steps including epimerization and cleavage of the TES group in excellent yield  as well as of the derivatives 16 and 18 against the drug-sensitive P. falciparum NF54 strain as described previously [2 did not show any toxicity against L6 cells . In both assays the highest concentration used was 100 \u03bcg/mL.Finally, we evaluated the antimalarial activity of (+)-3-hydroxymethylartemisinin  [2+ in vitro (Fenton reaction), it is difficult for us to explain their inactivity against Plasmodium. Furthermore, in cellular systems free ferrous iron [Plasmodium parasite. Derivative 2 will be used to attach further substituents at position 14 of artemisinin to prove this hypothesis.In the past it has been postulated that artemisinins kill intraerythrocytic parasites such as es (ROS) . As arteous iron  as well ous iron \u201321 have ous iron ,22. Our Experimental details, NMR spectra and other physical data are shown in File 1Experimental part."}
{"text": "Giant cell tumor of bone (GCTB) is a biologically benign and locally aggressive tumor that most often affects the epiphyseal and metaphyseal sites of long bones in the young adult population. Overexpression of receptor activator of nuclear factor kappa B ligand (RANKL) by cancerous mesenchymal stromal cells stimulates a signal transduction cascade that recruits and activates multinucleated osteoclast-like giant cells, resulting in pathologic bone resorption. Denosumab, an RANKL inhibitor that blocks the RANKL-mediated osteoclast activation, has been recently approved by the United States Food and Drug Administration (FDA) for the treatment of aggressive GCTB. Although uncommon, several studies reported drug-related malignant morphological transformation of benign GCTB following treatment with denosumab therapy. The aim of the article was to review the clinicopathological characteristics of all the reported cases of malignant sarcomatous transformation of GCTB after treatment with denosumab therapy in patients without any history of prior exposure to radiotherapy. Giant cell tumor of bone (GCTB) is a benign osteolytic neoplasm that primarily involves the epiphyseal and metaphyseal regions of long bones . AlthougThe optimal management of GCTB continues to be controversial . NonetheFrom a histological point of view, GCTB has a distinctive bi-phenotypic cell pathology. GCTB comprises osteoclast-like multinucleated giant cells that express receptor activator of nuclear factor kappa B (RANK)\u00a0and neoplastic mesenchymal stromal cells that express RANK ligand (RANKL). In patients with GCTB, the RANK\u2013RANKL interaction mediates the differentiation and activation of osteoclasts\u00a0and thus results in the osteolytic phenotype of bone destruction ,6-8.Denosumab is a fully humanized monoclonal antibody that targets RANKL. Thus, denosumab inhibits the RANK\u2013RANKL interaction and prevents bone destruction . In mid-Several studies including clinical trials ,10-12, cPrior exposure to radiation therapy has been proposed as a predisposing factor for the development of malignant transformation of GCTB . AlthougPubMed\u00ae database was searched until 15th December 2018 using the following keywords: \u201cgiant cell tumor of bone\u201d, \u201cdenosumab\u201d, and \u201cmalignant transformation\u201d. Only English-published literature was included. Further references from published articles were also manually screened for potential additional studies.\u00a0The study inclusion criteria included patients who were (i) initially diagnosed with benign GCTB, (ii) received denosumab therapy, (iii) developed malignant sarcomatous transformation during or post-therapy, and (iv) did not have any history of previous exposure to radiation therapy. For each reported case of sarcomatous transformation of GCTB during or after denosumab therapy, the following details were reported (whenever available): authors, year of publication, patient age, patient gender, tumor site, sarcoma histology, pulmonary metastases, causal relationship with denosumab therapy, time of sarcomatous transformation from the time of denosumab therapy, and duration of denosumab therapy.In 2010, Thomas et al. \u00a0reportedIn 2013, Chawla and colleagues  reportedIn 2015, Rutkowski and associates  reportedIn 2015, Aponte-Tinao and partners \u00a0reportedIn 2015, Broehm and friends  reportedIn 2016, Park and peers  reportedIn 2017, Tsukamoto and co-workers\u00a0 reportedTable The exact mechanism by which the sarcomatous transformation of GCTB occurs during or after denosumab therapy is poorly defined . HoweverFurthermore, from a technical point of view, a potential hypothesis has been proposed to account for the sarcomatous transformation of GCTB that takes place during or after denosumab therapy. This hypothesis takes into account that osteosarcoma arising in a histologically benign GCTB may encompass some scattered foci of malignant tumor. These foci of malignant tumor (osteosarcoma) may have been missed by means of sampling error at the time of initial core biopsy ,40. MoreIn literature, apart from the\u00a0sarcomatous transformation, a wide variety of morphological transformation of GCTB has been reported during and after denosumab therapy. For instance, some authors described growth patterns resembling osteoblastoma , fibrousAt the present time, the optimal duration of denosumab therapy is not yet established. In addition, whether the duration of denosumab therapy is directly associated with the increased potential of developing sarcomatous transformation of GCTB remains poorly defined. Nevertheless, long-term treatment with denosumab therapy should be limited to the minimum possible in order to avoid dose-dependent toxicity side effects, for example, osteonecrosis of the jaw .n\u00a0= 11)\u00a0may not represent the actual figure.Lastly, it should be noted that\u00a0this review article may not be reporting all the cases of malignant sarcomatous transformation of GCTB, as under-reporting of such cases by healthcare centers remains a possibility. Thus, the number of related cases reported in this review (n\u00a0= 11) related cases have been reported so far in the English literature. The exact mechanisms or triggering factors by which sarcomatous transformation of GCTB occurs during or after denosumab therapy are poorly defined. Additional isolated case reports and large-sized cohort studies are needed to thoroughly explore a causal association between the\u00a0malignant sarcomatous transformation of GCTB and denosumab therapy.In conclusion, healthcare providers should be aware that malignant sarcomatous transformation of GCTB during or after denosumab therapy is a possible consequence. To the best of knowledge, 11 ("}
{"text": "In both groups, the non-treated side served as a control. A numerical rating scale (NRS) of pain, pressure pain thresholds (PPTs), cold detection thresholds (CDTs), warmth detection thresholds (WDTs), cold pain thresholds (CPTs), and heat pain thresholds (HPTs) were tested on both sides at the gingiva and canine tooth and on the hand. The data were analysed by a repeated measures analysis of variance (ANOVA). The NRS pain scores were significantly lower in the LG group (P\u2009=\u20090.01). The CDTs, CPTs, WDTs, HPTs, and PPTs at the gingiva and the PPTs at the canine tooth were significantly less sensitive on the treatment side of the LG compared with that of the PG (P\u2009<\u20090.033). The parameters tested also showed significantly less sensitivity on the non-treatment side of the LG compared to that of the PG (P\u2009<\u20090.043). There were no differences between the groups for any quantitative sensory testing (QST) measures of the hand. The application of LLLT appears to reduce the pain and sensitivity of the tooth and gingiva associated with orthodontic treatment and may have contralateral effects within the trigeminal system but no generalized QST effects. Thus, the present study indicated a significant analgesia effect of LLLT application during orthodontic treatment. Further clinical applications are suggested.Low-level laser therapy (LLLT) may have an effect on the pain associated with orthodontic treatment. The aim of this study was to evaluate the effect of LLLT on pain and somatosensory sensitization induced by orthodontic treatment. Forty individuals  scheduled to receive orthodontic treatment were randomly divided into a laser group (LG) or a placebo group (PG) (1:1). The LG received LLLT  can reduce pain and sensitivity in teeth and surrounding tissues. They divided 40 patients into two groups; one was given repeated LLLT in the hours and days following procedures, while the other group received a placebo course. They tested both groups for sensitivity to stimuli including heat and pressure, in the mouth and on the hands. LLLT significantly reduced pain in the mouth relative to the placebo group. No differences were found in tests on the hands, suggesting LLLT works as a targeted analgesia. The loading and forces may lead to an inflammatory process in the periodontal ligament, which can cause pain.10 The pain usually begins within 4\u2009h after the force is applied, reaches a peak after ~24\u2009h, and then dissipates by day 7.12 The intensity of the pain during orthodontic treatment is sometimes reported to be even stronger than the pain related to dental extractions.13 Thereby, it could be one of the reasons for non-compliance with and even withdrawal from orthodontic treatment.The most common problems for patients during orthodontic treatment are pain and discomfort evoked by the appliances and mechanical loading.15 Overall, studies on clinical efficacy have shown equivocal results.16 The efficacy of LLLT in alleviating orthodontic pain has also been studied in recent years.22 It has been reported that LLLT has analgesic properties and anti-inflammatory effects25 through increases in the local blood flow, reduction of prostaglandin levels E2 and inhibition of cycloxygenase-2.24 Various studies have been designed to investigate the pain during orthodontic treatment, including telephone interviews and questionnaires.27 However, because of small sample sizes, controversial results, and different methodological issues in previous studies, good evidence for a positive treatment effect of LLLT is still lacking. Randomized, placebo-controlled and double-blinded studies are needed.28Low-level laser therapy (LLLT) has attracted attention for decades because of its obvious advantage in pain management as a non-invasive and inexpensive technique without significant adverse effects.29 QST in the trigeminal region has been characterized in terms of specificity, sensitivity, repeatability, and reliability.31 The application of QST could help to study the underlying neurobiological mechanisms of orthodontic pain and could serve as a valuable measure to determine the effects of LLLT on somatosensory function. One of our recent studies has shown that pressure pain thresholds (PPTs) applied to the teeth have excellent intra-examiner and inter-examiner agreements in healthy participants.32 A modified intra-oral QST has been applied in several of our previous studies34 and has been shown to be an easy and reliable technique for assessing mechanical pain sensitivity  in the periodontal ligament, which is associated with endodontic or periodontal conditions.Quantitative sensory testing (QST) is a psychophysical method in which different standardized stimulus modalities  are applied to different tissues , and the test person\u2019s response, in terms of a sensory or pain threshold or a report of the magnitude of the perceived intensity, is assessed.As a measure of self-reported levels of pain, the numerical rating scale (NRS) score, in which 0 represents \u201cno pain\u201d and 10 indicates \u201cthe most pain imaginable\u201d, can be recorded from the participants. The aim of the present study was to evaluate the effect of LLLT on the self-reported pain and somatosensory function induced by orthodontic treatment using our recently established intra-oral QST techniques in a randomized, placebo-controlled and double-blinded study design.P\u2009>\u20090.916). There were no significant differences between the LG and PG at baseline for cold detection threshold (CDT) , cold pain threshold (CPT) , warmth detection threshold (WDT) , heat pain threshold (HPT) , pressure pain threshold (PPT) at the gingiva , or pressure pain threshold (PPT) at the canine tooth .There were no significant age or gender differences between the laser group (LG) and placebo group (PG) at baseline . There was also a significant effect of time , with the highest NRS scores being recorded at 24\u2009h (P\u2009=\u20090.018). However, there was no significant interaction between the groups and time  , side  and time  and a significant interaction between group, side, and time . Moreover, the interaction between group and time was also significant ; whereas, the interaction between side and time was not significant . The relative PPT changes were significantly larger in the LG compared with the PG at 2\u2009h , 24\u2009h , 4 d , and 7 d  at the treatment side and non-treatment side. When the treatment side in the LG was compared to the non-treatment side in the same group, there were significant effects of side  and time  but no significant interaction between side and time . The relative PPT changes on the treatment side were significantly higher than those on the non-treatment side from 2\u2009h to 7 days in the LG , 24\u2009h , 4d , and 7d . Finally, there was a significant interaction between group and time .Interestingly, the relative PPT changes at the non-treatment side in the LG were significantly higher compared with those at the non-treatment side in the PG at 2\u2009h  and side (P\u2009=\u20090.243) but a significant effect of time (P\u2009<\u20090.001). However, when the non-treatment sides in the LG and PG were compared, there were significant effects of group  and time . The interaction between group and time was not significant   Fig.\u00a0.The mean thermal QST values are shown in Table\u00a0F\u2009=\u20099.2, df\u2009=\u20091, P\u2009=\u20090.003) and time  but no significant effect of side . The interaction between group, side and time was not significant , but the interaction between group and time was significant . The relative CDT changes were significantly smaller in the LG than in the PG at 2\u2009h , 24\u2009h , 4 d , and 7 d   Fig.\u00a0. The relF\u2009=\u20097.2, df\u2009=\u20091, P\u2009=\u20090.009) and time  but no significant effect of side . The interaction between group, side and time was not significant , but the interaction between group and time was significant . The relative WDT changes were significantly smaller in the LG than in the PG at 2\u2009h , 24\u2009h , and 4 d   Fig.\u00a0. The relF\u2009=\u200928, df\u2009=\u20091, P\u2009<\u20090.001) and time  but no significant effect of side . The interaction between group, side, and time was not significant , but the interaction between group and time was significant . The relative CPT changes were significantly smaller in the LG than in the PG at 2\u2009h , 24\u2009h , 4 d , and 7 d  . The relative CPT changes were also significantly lower on the non-treatment side than on the treatment side in the PG at 2\u2009h, 24\u2009h, 4 d and 7 d   Fig.\u00a0. The rel01) Fig.\u00a0.F\u2009=\u20097.1, df\u2009=\u20091, P\u2009=\u20090.009) and time  but no significant effect of side . The interaction between group, side and time was not significant . The relative HPT changes were significantly larger in the LG than in the PG from 24\u2009h to 7 d , WDT , CPT , HPT  and PPT . There were significant time effects for CDT , WDT , CPT , and HPT , but there was no time effect for PPT  , ATP production, and prostaglandin reduction.41 In addition, LLLT might alter nerve conduction by influencing the synthesis, release, and metabolism of encephalin, endorphins and many other neurochemicals.42 This study was not designed to elucidate the neurobiological mechanisms potentially underlying an analgesic effect of LLLT on orthodontic pain, but the present findings so far indicate that the observed reduction in the NRS pain scores cannot be attributed to placebo-based mechanisms. Although placebo effects cannot be completely ruled out based on the self-reported NRS pain scores, the observation of consistent and significant effects on somatosensory function further supports the notion of a neurobiological effect of LLLT.The exact mechanisms responsible for the apparent analgesic effect of LLLT are still unclear.33 To our knowledge, it was the first time that QST was applied to evaluate the effect of LLLT on orthodontic pain in a systematic manner. The QST parameters are considered to offer a diagnostic sensitivity of 67%\u2013100% for small-fiber neuropathy and to be potentially valuable for evaluating small-fiber function.44 The CDT is considered to represent small myelinated A\u03b4 nerve fiber conduction, the WDT is considered to represent unmyelinated C nerve fiber conduction, and the CPT and HPT may represent conduction by both A\u03b4 and C nerve fibers.46 The PPT was measured to test deep pain sensitivity, which is probably mediated through both C and A\u03b4 fibers.47 The PPT can also be used to assess mechanical pain sensitivity in the periodontal ligament and gingiva.32As a special feature of the present study, intra-oral QST was applied in addition to traditional self-reports of pain intensity evoked by the orthodontic treatment. QST is believed to contribute to a better understanding and profiling of the underlying neurobiological mechanisms related to orthodontic pain.The present QST results demonstrated that not only tooth-related pain but also sensitization of thermal and mechanical somatosensory channels were evoked by the orthodontic treatment. In fact, both mechanical (PPT) sensitization and thermal  sensitization were observed during the orthodontic treatment until day 7. In the present study, all of the QST parameters were normalized to their baseline values (0\u2009h), and the relative changes at the 5 time points in the two treatment groups were calculated and presented Figs.\u00a0 and 3. I32 The force from the pressure algometer was applied directly to the crown of the canine tooth, and the recorded PPT may partially reflect the innervation patterns and receptor density in the periodontal ligament.48 Interestingly, in the present study, significantly higher PPTs  on the LLLT-treated side compared to the non-treated side were observed in the LG. Thus, it can be speculated that LLLT may decrease the sensitization of the periodontal ligament evoked by orthodontic treatment. Surprisingly, the analgesic effect induced by LLLT was not restricted to the treatment side and was also observed on the contralateral side, indicating an extended effect in the trigeminal area. Studies on animals have shown the existence of branched nerves innervating both intrapulpal and periodontal tissues.49 It has also been shown in animal studies that LLLT may promote neural regeneration,37 but until now, only few animal studies have been available that allow for the study of the activation of the trigeminal-vascular system. Within the meninges, the activation of the trigeminal-vascular system  leads to an ipsilateral inflammatory response characterized by vasodilation.50 Animal studies have also indicated that noxious stimulation may produce marked blood flow changes in various orofacial structures.51 Noxious stimulation of the human teeth is indeed associated with bilateral increases in blood flow in both the maxillary and mandibular nerve innervation territories.52 In the trigeminal-vascular system in rats, prostaglandin E2 (PGE2) is considered to be a key mediator for pain and nocifensive responses.53 PGE2 causes vasoconstriction and is increased in both blood and saliva during migraine attacks, which is a mechanism related to pain. Interestingly, PGE2 may be reduced by LLLT,13 which is related to the activation of the trigeminal-vascular system, which, again, might be associated with the bilateral effects of LLLT.Mechanical allodynia in the periodontal ligament around the canine tooth was investigated with the same method used in our previous study.54 and lead to an increase in the intrapulpal pressure.55 Thus, increased fluid flow from the root canal system through the apical foramen and dentinal tubules may cause the delivery of SP and MMP-8 to the periodontal ligament space.56 These data support the possibility for the local neurogenic spread of inflammation from intrapulpal tissues to surrounding periodontal tissues, which may further explain the bilateral effect of LLLT.Tooth pain caused by orthodontic tooth movement is associated with elevated gingival crevice fluid contents of matrix metalloproteinase (MMP)-8 and neuropeptide substance P (SP) levels, which are more abundant in pulp tissues from painful human teeth33Pain only evoked local elevations in the gingival crevice fluid content of SP and MMP-8 levels and caused no marked modulations in systemic cardiovascular parameters. Thus, systemic stress mechanisms probably did not significantly contribute to the present results, which may explain the lack of significant QST effects on the hand. Time effects of the QST parameters at control sites, such as the hand, have also been observed in a previous study and may be attributed to minor drifts in psychophysical performance, e.g., an adaptation or a habituation of the test procedures.57 The evidence suggests that LLLT can be effective in enhancing the rate of orthodontic tooth movement since LLLT is found to increase the rate of bone remodeling without imposing any adverse effects.59 However, other studies have reported conflicting results60 and have failed to observe any significant improvement in the rate of orthodontic tooth movement associated with LLLT.61 The rate of tooth movement was not recorded in the present study, and further studies will be needed to clarify the effect that LLLT has on tooth movements.The process of bone remodeling in periodontal tissues is a major determinant of orthodontic tooth movement.62 Nevertheless, the strength of the mixed study group is that the present results may be more generalizable. Another limitation of the present study could be argued to be the subjective nature of the self-reported pain. However, in addition to self-reports of pain, we also included psychophysical responses (QST), which also indicated significant effects on the treatment side in the LG. In future studies, objective measures of somatosensory function  and nociceptive activity  could be incorporated into the study design to further support the potential analgesic effect of LLLT in relation to orthodontic pain. A particular strength of the present study was its double-blinded and placebo-controlled design. However, a limitation may still be that no information was collected to demonstrate that participants were indeed successfully blinded and that participants in the PG had the same degree of anticipation and expectancy of a positive outcome. Previous studies have clearly demonstrated that expectancy reports are crucial for the understanding of placebo effects.63 Thus, it may not be possible to completely rule out that the present findings were not contaminated by placebo effects. However, the QST findings may nevertheless suggest that there could be physiological responses related only to LLLT. Further studies are obviously needed to address these questions in more detail.One of the limitations of the present study could be argued to be the mixed group of participants, which includes both children and adults and includes participants of both genders. A total of 10 individuals were under 18 years old, but so far, there is no systematic information about age-related differences in acute pain responses evoked by orthodontic treatment, although gender differences in pain sensitivity have been documented.The volunteers were recruited from the orthodontic clinic at the Hospital of Stomatology of Nanjing Medical University . The inclusion criteria were individuals who were going to start comprehensive orthodontic treatment for slight crowding without tooth extraction, and all of the participants were to be healthy without signs or symptoms of pain or any kinds of on-going therapy and no other systemic disease. The exclusion criteria were active caries, periodontal diseases, visible lesions of the oral mucosa, and any kind of chronic use of analgesics or drugs affecting the function of the central nervous system.20 All of the participants gave informed consent to the procedures, which were approved by the local Ethical Committee  in accordance with the Helsinki Declaration II. In addition, all of the participants understood that they were free to withdraw from the experiment at any time.Fifty participants requiring orthodontic treatment were initially recruited in this study: six participants declined enrollment after receiving study information, and an additional four individuals were excluded according to the exclusion criteria. A total of 40 participants  were included in the study  by Examiner 2. The LG received the low-level laser therapy (LLLT) on one randomized side from Examiner 2 immediately after (0\u2009h) the arch wire (0.014 super plastic nickel-titanium arch wire) was placed in self-ligating brackets  and then again at 2\u2009h, 24\u2009h, 4 d, and 7 d. The PG received inactive treatment with the LLLT device, which was applied in a similar way at the same test sites and at the same time points as for the LG. The treatment sides in the LG and PG groups were also randomly determined using the same computer program by Examiner 2. QST was performed after the application of active LLLT or placebo LLLT in both of the groups. A 0\u201310 numerical rating scale (NRS) score, in which 0 represented \u201cno pain\u201d and 10 indicated \u201cthe most pain imaginable\u201d, was recorded from the participants, and the modified intra-oral QST was performed at the canine tooth and the surrounding gingiva on both sides at 0\u2009h, 2\u2009h, 24\u2009h, 4 d, and 7 d after the orthodontic forces were applied. The participants were requested to close their eyes during the LLLT application. The study was performed in a quiet and temperature-controlled room. The contralateral, non-treatment side served as an additional control in both groups. Examiner 1 was blinded to the information about the allocation to the LG or PG, as well as to which side had been treated or not.The experiment was performed as a randomized, placebo-controlled and double-blinded trial. All of the participants were tested by the same examiner (Examiner 1: Song Wu). The laser treatment and randomization were performed by another examiner (Examiner 2: Huijie Shen). The forty participants were randomly divided into two experimental groups: a LG and a PG. The randomization code of the two treatment groups (20 in each group) for the 40 participants was obtained using a computer program  or when the temperature reached a sensation of painful hot or cold (CPT and HPT). The temperature of the thermode returned to the baseline after the mouse button had been pushed. The range of the stimulation temperatures was between 0\u2009\u00b0C and 50\u2009\u00b0C. The CDT and the WDT were always measured first, followed by the CPT and the HPT. The mean of three repeated trials was used to determine the threshold values.Thermal quantitative sensory tests were performed using a computerized thermal stimulator . The contact area of the intra-oral thermode was 6\u2009mm\u00d7\u20096\u2009mm. The cold detection threshold (CDT), warmth detection threshold (WDT), cold pain threshold (CPT) and heat pain threshold (HPT) were tested at the area of the gingiva around the upper-left and right canines and on the left hand  at the five time points. The thermode started from a baseline temperature of 37\u2009\u00b0C. The temperature decreased or increased by 1\u2009\u00b0C\u00b7s33 The pressure was increased with a constant application rate of 30\u2009kPa\u00b7s-1. The participants were instructed to concentrate on the test stimulus and to press the switch button as soon as they felt that the pressure changed to the slightest sensation of pain. The amount of pressure (kPa) at this point was defined as the PPT. Three measurements per site were made at 1-min intervals to obtain a mean value.The mechanical PPT was measured by an algometer . The diameter of the intra-oral probe was 8\u2009mm. The algometer was applied perpendicular to the surface of the test sites. The measuring sequence was the left hand, upper-left canine gingiva, upper-right canine gingiva, upper-left canine, and upper-right canine.-2 .LLLT was applied buccally and lingually to an upper canine at 6 points: mesial, distal, and at a site corresponding to the middle of the root of the canine tooth for 20\u2009s each in the LG. The laser tip diameter was 1\u2009mm, and the laser tip was kept 10\u2009mm away from the surface of the gingiva during the stimulation. From the cementoenamel junction of the tooth towards the apex of the tooth, the laser tip was directed perpendicular to the long axis of the tooth. In the PG, the LLLT device was held at the same 6 points for the same duration with the light on for the operation indicator. The same procedure was repeated for the LG but without any active laser output. Both the examiner and the participant wore protective laser glasses. LLLT was applied using an 810-nm gallium-aluminium-arsenic diode laser in continuous mode with the power set at 400\u2009mW, 2\u2009J\u00b7cmThe sample size was calculated with risks for type I and type II errors of 5% and 20%, respectively, an estimate of the inter-individual variation of 25% and a minimal relevant difference for detection of 20%. A total of 40 patients were recruited in the present study.Descriptive statistics were used to summarize the data. Baseline comparisons between the two groups in regards to age, gender and the QST parameters at baseline were performed. The necessary logarithmic transformation was performed when the data were not normally distributed. The mean values and SD of the NRS score, CDT, WDT, CPT, HPT, and PPT at each time point and test site were calculated. All of the data were normalized to the baseline, and the relative changes of each parameter were compared between the treatment groups. A three-way mixed model analysis of variance (ANOVA) with repeated measures was used to analyse the different outcome parameters of the CDT, WDT, CPT, HPT, and PPT at the test sites at different time points. The between group factor in the ANOVA was group (LG and PG), while the within-group factors were side (treatment and non-treatment side), and time point . Post hoc tests were performed with the LSD honestly significant difference test with corrections for multiple comparisons. The significance level was set at 0.05. Blinding of the data was maintained in the statistical analysis.The QST data that support the findings of this study are available in figshare with the identifier 10.6084/m9.figshare.5532226.v1.A repeated application of LLLT using a gallium-aluminium-arsenic diode laser with an 810-nm wavelength was able to significantly reduce self-reported pain scores and sensitization of the periodontal and gingival tissues evoked by orthodontic treatment.The effect also extended to the contralateral side in the trigeminal region but not in the extra-trigeminal region, indicating that LLLT treatment may have some degree of bilateral effects within the orofacial region. This interesting finding calls for future studies on the clinical application of LLLT with larger cohorts, as well as for those including additional measures of nociceptive function and sensitization."}
{"text": "FN1, FLNA, FLNB, VCL, GSN, MYH10, ACTN4, KDR and EREG, and they were all down-regulated after 6-TG treatment. The coexpression network consisted of 18 microRNAs (miRNAs), 9 long noncoding RNAs (lncRNAs) and 20 mRNAs. Hsa-mir-16-5p and Hsa-mir-335-5p targeted the greatest number of mRNAs in the network. These molecules could bind to PAX8-AS1 and eliminate the inhibition of target mRNA expression. We showed that PAX8-AS1 is the main lncRNA affected by 6-TG and that PAX8-AS1 regulates the hub genes in tumor pathways by competitively binding with miR-16-5p and miR-335-5p.Breast cancer is one of the most prevalent and recurring cancer types that leads to deaths in women. Triple-negative breast cancer (TNBC) is difficult to treat due to the lack of therapeutic targets. Many studies have focused on identifying drugs for use as alternative treatments for breast cancer. Thioguanine (6-TG) exerts antitumor effects in cancer. Increasing evidence has demonstrated that competitive endogenous ribonucleic acids (ceRNAs) are involved in cancer processes. However, the mechanism by which 6-TG regulates lncRNA\u2013miRNA\u2013mRNAs has not been elucidated. We evaluated the antitumor effect of 6-TG in MDA-MB-231 cells and comprehensively analyzed the RNA-Seq data of MDA-MB-231 cells treated with 6-TG. Our results showed that most tumor pathways were blocked by 6-TG. The hub genes were  Breast cancer is one of the common causes of deaths in women. Among all diagnosed cases of invasive breast cancer, approximately 15\u201320% are triple-negative breast cancer (TNBC) .DNMT1 [2-M arrest and cell death by DNA mismatch repair mediated in human colorectal carcinoma. Our previous studies have shown that 6-TG can induce FAS-mediated exogenous apoptosis and p21-dependent G2/M arrest by restoring TP53 activity in MCF-7 breast cancer cells [Thioguanine 6-TG) is a classic leukemia therapy drug with potential for cancer therapy . 6-TG acDNMT1 . Inki et-TG is a DNMT1  suggesteer cells .BCRT1 is overexpressed in breast cancer and promotes breast cancer metastasis by targeting miR-1303 [NKILA suppresses TGF-\u03b2-induced epithelial\u2013mesenchymal transition by blocking NF-KB signaling in breast cancer. Therefore, the theory of competitive endogenous ribonucleic acid (ceRNA) has emerged, which states that lncRNAs can competitively combine with the miRNA response element (MRE) and inhibit the negative regulation of target mRNAs by miRNAs. The ceRNA network has been confirmed in many diseases. However, the role of ceRNA in the regulation of breast cancer remains to be further studied.Intensive research in the past two decades has uncovered the presence and importance of noncoding RNAs (ncRNAs), which include microRNAs (miRNAs) and long ncRNAs (lncRNAs) . LncRNAsmiR-1303 . Wu et amiR-1303  reportedIn our study, we found that 6-TG plays an antitumor role in TNBC cells (MDA-MB-231 cells). To elucidate the interactive mechanism of lncRNAs and the lncRNA-mediated regulatory network, we performed an integrative analysis to identify differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) of TNBC cells after treatment with 6-TG and identified a ceRNA network that can down-regulate core genes that dominate the tumorigenesis process. The present study contributes to the exploration of the ceRNA regulatory mechanism of 6-TG and provides valuable insight for further functional research.2 at 37\u00b0C. The MDA-MB-231 cell line has been authenticated using STR profiling within the last 3 years. All experiments were performed with mycoplasma-free cells.The human breast cancer cell line MDA-MB-231 was purchased from the Cell Bank of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Cells were cultured in RPMI 1640 medium  supplemented with 10% fetal calf serum, 100 IU/ml penicillin and 100 \u03bcg/ml streptomycin . The cells were cultured in a humid environment with 5% CO6-TG was purchased from Selleck Chemicals . Dimethyl sulfoxide (DMSO) was purchased from Sigma\u2013Aldrich .3 cells were seeded per well in a 96-well plate and treated with 6-TG at 0.5, 1, 2, 4 and 8 \u03bcM and DMSO. Thereafter, CCK-8 solution was added, and the absorbance was measured using a microplate reader  at 450 nm.Cells were treated with different concentrations of 6-TG and then subjected to the Cell Counting Kit-8 (CCK-8) assay according to the manufacturer\u2019s protocol . Briefly, 6.5 \u00d7 10Total RNA was extracted with TRIzol  reagent following the manufacturer\u2019s instructions. The insert size was assessed using the Agilent Bioanalyzer 2100 system, and qualified insert sizes were accurately quantified using the StepOnePlus\u2122 Real-Time PCR System . Sequencing libraries were generated using the NEBNext\u00ae Ultra\u2122 RNA Library Prep Kit for Illumina\u00ae  following the manufacturer\u2019s recommendations, and index codes were added to attribute sequences to each sample (GEO number: GSE137418).http://bioinformatics.sdstate.edu/idep/). Genes with false discovery rates (FDRs) \u2264 0.1, |log2FC| \u2265 1.5 and P-value \u2264 0.01 were selected as candidate genes. Heatmaps were utilized to identify the gene expression differences between the control group and the 6-TG group. DEmRNAs and DElncRNAs are shown in Supplementary Tables S1 and S2, respectively.DEmRNAs and DElncRNAs were identified by the IDEP website (http://www.genome.jp/kegg/) database. The volcano plot of GSEA was structured on the website . Hierarchical clustering and heatmap plot analyses were performed for the ten main down-regulated pathways via the METASCAPE website (https://metascape.org/gp/index.html#/main/step1). The genes in the top ten pathways and seven pathways are shown in Supplementary Tables S3 and S4, respectively.Pathway analysis was used to investigate the DEmRNAs according to the Kyoto Encyclopedia of Genes and Genomes  network of genes involved in the main pathways was constructed to understand the relationships of different genes. The PPI network was constructed using Cytoscape3.7.2. The submodule of the PPI network was analyzed by using the MCODE plugin of Cytoscape. GO and KEGG pathway enrichment analyses for the DEGs involved in the module were performed.http://starbase.sysu.edu.cn/) and miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/). Correlation analysis was performed to evaluate the interaction between DEmRNAs and DElncRNAs. Pairs with absolute values of Pearson correlation coefficients not less than 0.95 were selected. The miRNAs of these lncRNAs were predicted by DIANA tools (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php).DEmRNAs in the MCODEs were collected for further analysis. Then, miRNAs of mRNAs were predicted from StarBase (https://echarts.apache.org/examples/zh/editor.html?c=sankey-simple).According to the ceRNA hypothesis, there were potential interactions among lncRNAs, mRNAs and miRNAs. We constructed the ceRNA coregulated network of DElncRNAs, DEmiRNAs and DEmRNAs using echarts. In brief, we used TarBase and miRTarBase to predict 18 miRNAs of 21 hub DEmRNAs. We also predicted coexpressed lncRNAs of the 21 hub genes according to Pearson correlation coefficients \u2265 0.95. Then, we utilized DIANA tools to predict the coexpressed miRNAs of the abovementioned DElncRNAs. Finally, we constructed a ceRNA network according to Supplementary Table S5 on the echarts website  reagent following the manufacturer\u2019s instructions. Complementary DNA (cDNA) was synthesized using TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR  . qPCR was performed with FastStart Essential DNA Green Master  via a StepOnePlus Real-Time PCR system. Primers sequences are listed in Supplementary Table S6. The results were analyzed using the 2https://www.oncolnc.org) and Kaplan\u2013Meier plotter  to perform Kaplan\u2013Meier survival analyses of the association between the expression of DElncRNAs, DEmiRNAs and DEmRNAs in the ceRNA network and the prognosis of patients. We only selected the TNBC sample set, which is composed of 255 patients with ER negative, PR negative, and HER-2 negative.To evaluate the potential roles of genes in the ceRNA network in the patients, we used Cox regression analysis of survival packages and Kaplan\u2013Meier curves on OncoLnc . **50 of 6-TG in 48 h was 2.5 \u03bcM. Moreover, the cell morphology was changed after 6-TG treatment , Hsa-miR-1 (connection degree = 7) and Hsa-miR-335 (connection degree = 6) were the top three miRNAs that targeted most mRNAs, suggesting that they were major miRNAs in the coexpression network. Only six lncRNAs  were identified in the network according to the Lnc2Cancer 3.0 database. Therefore, PAX8-AS1 targeted the top two miRNAs and interacted with the majority of mRNAs ITGA2, LAMC1, ITGB4, KDR, FLNA, ACTN4 and EREG. To verify our analysis, the expression levels of the majority of mRNAs were assessed using qPCR. As shown in KDR, ITGA6, ACTN4 and EREG were significantly lower in the 6-TG group than in the control group. These data suggested that ceRNA played a potential role in regulating gene expression in MDA-MB-231 cells treated with 6-TG.To better understand the pivotal roles of DEmRNAs and DElncRNAs in DMA-MB-231 cells under 6-TG treatment, a ceRNA network was constructed. We used TarBase and miRTarBase to predict 18 miRNAs according to the abovementioned 21 hub DEmRNAs. We also predicted coexpressed lncRNAs of the 21 hub genes according to Pearson correlation coefficients \u2265 0.95. Then, we utilized DIANA tools to predict the coexpressed miRNAs of the abovementioned DElncRNAs. Echarts was used to construct the coexpression network. As shown in Hsa-miR-16 and Hsa-miR-335 seemed to exhibit protective functions, as the prognosis of patients with higher expression levels was longer than that of patients with lower expression levels. However, the ACTN4, EREG, FLNA and FLNB mRNAs  was reported as a regulator of cell proliferation, and its reduced expression could inhibit cell growth [Vash2 inhibited tumor growth by down-regulating EREG and IL11, suggesting the antitumor effects of EREG in tumors. Fortunately, our results showed that the tumor-related pathways, Regulation of actin cytoskeleton, MAPK signaling pathway, ECM\u2013receptor interaction, Proteoglycans in cancer, Focal adhesion, PI3K-Akt signaling pathway and Pathway in cancer, were inhibited by 6-TG. In addition, we identified the hub genes in these pathways, such as FN1, FLNA, FLNB, ACTN4, VCL, GSN, MYH10, ACTG1, EREG and KDR, suggesting that they were the dominant genes in these pathways under 6-TG treatment.To the best of our knowledge, changes in a variety of cancer signaling pathways are involved in the development and progression of breast cancer. Previous studies have shown that MAPK signaling, the PI3K/AKT pathway and focal adhesion pathways play important roles in malignant transformation in TNBC ,18. KDR l growth . Yasuhirl growth  reportedCCND1, CCND3, CCNE1 and CDK [MiR-335 suppresses metastasis and migration by targeting the progenitor cell transcription factor SOX4 and has been identified as a metastasis suppressor miRNA in human breast cancer [miR-335-5p to regulate cell proliferation, apoptosis and invasion in pancreatic cancer. However, we did not observe any studies reporting lncRNAs with potential competing abilities. Increasing evidence indicates the crucial role of lncRNAs in the ceRNA network in terms of modulating other RNA transcripts [PAX8\u2010AS1 expression levels had shorter RFS times. While the isoform of PAX8-AS1 is named PAX8-AS1-N, it can bind to miR-17-5p and upregulate miR-17-5p targets, such as PTEN, CDKN1A, and ZBTB4. The reduced expression of PAX8-AS1-N indicated poor survival of breast cancer patients [PAX8-AS1 in ceRNAs is unknown. In our results, we found decreased lncRNA PAX8-AS1 expression under 6-TG treatment in MDA-MB-231 cells. The lncRNA PAX8-AS1 targeted the top two miRNAs and interacted with the majority of mRNAs in the ceRNA network. Moreover, the mRNAs with reduced expression after 6-TG treatment were consistent with those predicted by PAX8-AS1. Therefore, we predicted that PAX8-AS1 may sponge miR-16 and miR-335. This lncRNA can mediate ACTN4, EREG, KDR, FLNA, and FLNB expression by competing with miR- 16 and miR-335 to participate in the progression of MDA-MB-231 cells. Therefore, lncRNA PAX8-AS1 may inhibit MDA-MB-231 cells by sponging miR-16 and miR-335 to exert ceRNA regulation. Meanwhile, the high expression levels of miR-16 and miR-335 and the low expression levels of ACTN4, ERGE, FLNA, and FLNB could prolong patient survival, suggesting that 6-TG influenced the gene expression pattern and played a positive role in TNBC treatment.ceRNAs are transcripts that can regulate each other at the post-transcriptional level by competing for shared miRNAs . Amelia  and CDK . MiR-335t cancer . An et at cancer  found thnscripts . Wei et nscripts  reportedpatients . NeverthPAX8-AS1 is the main lncRNA influenced by 6-TG and that PAX8-AS1 regulates hub genes in tumor pathways by competitively binding with miR-16-5p and miR-335-5p. The present study contributes to the exploration of the ceRNA regulatory mechanism of 6-TG and provides valuable insight for further functional research.In summary, we showed that Click here for additional data file."}
{"text": "In February 2020, a novel coronavirus (SARS-COV2) broke out in Wuhan city of China. The Chinese government decisively imposed nationwide confinement. This study comprised a structured, online questionnaire, based on 40 items inquiring about socio-demographic information and anthropometric data (reporting weight and height), as well as changes in food intake, physical activity, and sleep during the COVID-19 outbreak. Questionnaires were distributed to residents of Jiangsu and other provinces from 29 March to 5 April. A total of 889 respondents were included, aged between 16 and 70 years . There was a significant increase in total food intake by 9.8% and a slight increase by 29.2% of respondents, and a significant decrease in physical activity by 31.5% and a slight decrease by 23.4% of respondents, especially in snacks and drinks, and outdoor activities. The rate of weight gain in the total population was 30.6% and the average weight gain was 0.5 \u00b1 2.8 kg. The main factors contributing to weight gain were increased food intake and reduced physical activity. Additionally, normal-weight people were more likely to gain weight than people with overweight/obesity during the COVID-19 confinement. This study provided a good warning and educational reference value on lifestyle changes during the COVID-19 confinement. A novel coronavirus disease, later abbreviated to COVID-19 by World Health Organization (WHO) . It was As definitive vaccines and cures for COVID-19 are unlikely to be identified soon , social Nevertheless, the prolonged home life may unintentionally lead to increased sedentary behaviors, such as spending excessive amounts of time sitting, reclining, or lying down for screen activities , reducing regular physical activity (thus lowering energy expenditure), or indulging in excessive food intake that, consequently, causes an increased risk for and potential worsening of chronic health conditions . Some ofn = 1.097) found that almost 30% of subjects experienced weight gain (3.0 \u00b1 1.6 kg), and overweight, obese, and older subjects tended to gain weight more frequently during COVID-19 lockdown [During the global pandemic COVID-19 lockdown, studies reported weight gain in different countries. For instance, the perception of weight gain was observed in 48.6% of the population in an Italian survey . A studylockdown .To comprehensively understand and analyze the impact of the domestic lifestyle due to the COVID-19 outbreak on the diet, exercise, and sleep of the Chinese people, we conducted this survey. It is worth mentioning that during the period of this investigation the epidemic in China was controlled. However, at the same time, the rest of the world was in the midst of a rapid outbreak, people were living in dire conditions, and the global hazards caused by the COVID-19 pandemic were far from over. Therefore, this survey will have a good reference value for countries that adopt \u2018limiting to go out\u2019 measures and also provide a basis for the active intervention of residents\u2019 quality of life in similar public health emergencies in the future.In order to cope with the outbreak of the COVID-19 epidemic, this survey needed to evaluate the lifestyle of the population in a short period of time. So, a rapid epidemiological assessment was adopted. Due to the \u201cstay at home\u201d regulation of the Chinese people, the on-site survey could not be carried out and an online questionnaire could only be chosen. Accordingly, a cross-sectional sample of 1040 Chinese residents aged 16\u201370 years was recruited online via our research group from 29 March to 5 April. The gender, age, urban and rural areas, family income, educational level, and so on of the collected population were taken into account. These people were all voluntary participants, which greatly reduced the non-response bias that often exists in the on-site survey. However, as our research group is located in the Jiangsu Province, according to the survey results, residents in the Jiangsu region accounted for the largest proportion (68.46%), followed by nearby places such as Shanghai City (8.17%) and Zhejiang Province (4.23%), and the remaining 26 provinces together accounted for 19.14%. Therefore, this survey may be not a good representation of all Chinese residents. However, under such urgent conditions at that time, it more or less reflected the lifestyle of the residents in the most economically developed and ideologically active regions of China for the first time.All the participants were informed of the purpose and procedures of the study and participated voluntarily and anonymously. This study was approved by the ethical review committee of Nantong University (Ethics number: 202062).A very popular professional questionnaire platform, named \u2018Wen Juan Xing\u2019 in Chinese or \u2018Sojump\u2019 in English, was used to develop the questionnaire survey system. Through several simulation tests, the content of the questionnaire was continuously improved. Key content areas were developed by applying the socio-ecological model, including responders\u2019 demographics, current diet, physical activity, and sleep behaviors, as well as changes before and during the outbreak. It should be noted that this study was a cross-sectional survey that collected the data at the time of the outbreak, while the data before the COVID-19 outbreak were recorded by the responders through their recollections. For those subjective qualitative questions, it was reported using a five-point Likert type scale, ranging from \u201ca lot less\u201d (score = 5) to \u201cno change\u201d (score = 3) to \u201ca lot more\u201d (score = 1). This self-designed questionnaire consisted of four parts, as follows.Gender, date of birth, living in the urban or rural areas during the epidemic, family per capita monthly income, resident population, educational background, body height, and body weight were included.(1)Change of dietary intake before and during the epidemic, including 10 categories: total intake, staple foods, vegetables, dairies, legumes or products, livestock/poultry meats, fished or fishery products, eggs, fruits, snacks/beverages;(2)Six psychological emotions on dietary behavior before and during the epidemic: dysphoria, anxiety, sadness, nervous, loneliness, depression; and(3)Dietary restriction, overeating, and storing food behavior during the epidemic.(1)Physical activity time, sedentary time, outdoor exercise time, and average daily steps before and during the epidemic;(2)Time spent using mobile phones and computers and doing housework during the epidemic; and(3)Weekly frequency of vigorous exercise and moderate exercise, as well as going out and walking during the epidemic.(1)The time to go to bed, fall asleep, and get up; the length of sleep; the quality of sleep before and during the epidemic;(2)Factors affecting sleep during the epidemic: disturbance in respiration, cough or snore, feeling cold or hot, having a nightmare; pain or ache; and(3)Feeling sleepy and undynamic during the epidemic.n = 100) and correlation coefficient range = 0.76\u20130.98 (strong reliability).Moreover, for every certain number of questions in the questionnaire, a quality control question was inserted to ask the respondents to choose the specified answer, to find the respondents who give a random answer. Test-retest (one week) reliability was assessed via bivariate Pearson correlation  before and after the COVID-19 outbreak. Analysis of variance (ANOVA) was used for comparison of weight gain between groups . The confounders  were adjusted for by analysis of covariance (ANCOVA). The count data were expressed by frequency (n) and percentage (%), and Pearson\u2019s \u03c72 test was used for comparison between groups. The linear regression equation between the total score of the three main factors  and weight gain was calculated. Multiple linear regression modeling was used to analyze the interactions between the changes in total food intake, changes in physical activity, and the pre-outbreak overweight/obesity effect on weight gain. A p-value < 0.05 was considered statistically significant.In this study, the quantitative data were expressed as mean value \u00b1 standard deviation (mean \u00b1 SD). A paired Excluding the invalid and illogical data, the final number of respondents was 889. Among them, 347 (39%) were males and 542 (61%) were females. The ages ranged from 16\u201370 years old and the average was 31.8 \u00b1 11.4 years. For residence type, 661 (74.4%) people lived in urban areas and 228 (25.6%) people lived in rural areas. During the COVID-19 outbreak, 607 (68.3%) people lived in Jiangsu province, 71 (8.0%) people lived in Shanghai city, 36 (4.0%) people lived in Zhejiang province, and 175 (19.7%) people lived in the other 31 provinces except Hainan, Ningxia, Hong Kong, Macao, and Taiwan. The education background was 78 (8.8%) completed junior high school, 116 (13.0%) completed senior high school, 618 (69.5%) completed bachelor\u2019s degree, 57 (6.4%) completed master\u2019s degree, and 20 (2.2%) completed doctoral degree. Per capita monthly household income was 211 (23.7%) people with less than 3000 yuan ($462), 252 (28.3%) people with 3000\u20136000 yuan ($462~924), 211 (23.7%) people with 6000\u201310,000 yuan ($924~1540), 143 (16.1%) people with 10,000\u201320,000 yuan ($1540~3080), and 72 (8.1%) people with more than 20,000 yuan ($3080). Permanent resident population was 1 population, 15 (1.7%); 2 population, 73 (8.2%); 3 population, 355 (39.9%); 4 population, 228 (25.6%); 5 population, 155 (17.4%); 6 population, 40 (4.5%); and 6 population and above, 23 (2.6%).Changes in food intake, physical activity, sleep duration, and sleep quality were rated on a scale of five compared to before the outbreak. The detailed results are shown in p = 0.074), with an average weight gain of 0.5 \u00b1 2.8 kg. Firstly, through the one-way analysis of variance (ANOVA), it was found that changes in total food intake (p = 0.000) and physical activity (p = 0.020), as well as whether overweight/obesity before the COVID-19 outbreak (p = 0.004), had a significant effect on weight gain due to the \u201cstay at home\u201d lifestyle. After further adjusting for potential confounding factors by using the analysis of covariance (ANCOVA), the above three variables still had significant effects on weight gain, as shown in The proportion of respondents who gained weight (>1 kg) during the COVID-19 outbreak was 30.6% , age (p = 0.302), urban or rural residents (p = 0.312), per capita monthly household income (p = 0.408), permanent resident population (p = 0.384), and educational background (p = 0.390), as well as changes in sleep duration (p = 0.416) and sleep quality (p = 0.062) had no significant effect on weight gain. Other demographic indicators including gender  on weight gain, each individual was assigned a score, and then the sum was added to calculate the total score, so that the linear regression equation between the total score of the three main factors and weight gain was calculated .(1)p-interaction < 0.001)  A.(2)p-interaction = 0.001)  B.(3)p-interaction < 0.001)  C.Furthermore, the interaction analysis of the three main factors that significantly affect weight gain during the \u201cstay-at-home\u201d lifestyle was carried out, and it was found that:Lastly, all variables correlated with weight gain during the COVID-19 outbreak are listed in The purpose of this study was to assess the immediate changes in food intake, physical activity, and sleep quality in Chinese residents during the initial period of the COVID-19 outbreak. We found that Chinese residents had more food intake, were less active, were more sedentary, and gained weight during the initial period of the COVID-19 outbreak compared with before the restrictions. These changes did not differ in demographic indicators such as gender, age, urban or rural areas, household income, educational background, and so on. The largest changes reported were the increased intake of snacks/beverages and decreased outdoor activity. These findings confirm speculations that pandemic-related confinements were unfavorably related to lifestyle. This observation had triangulated support from quantitative  and qualitative  evidence.The COVID-19 outbreak is a classic public health emergency. A review reported negative psychological effects after isolation, including post-traumatic stress symptoms, confusion, and anger . AccordiSimilarly, in this study, we found that about 40% of the respondents had different degrees of increase in total food intake, and the increase in food intake was probably associated with psychological factors. The rapid outbreak of COVID-19, its high infectivity, and long epidemic period, together with the various lockdown measures adopted, constitute a stressor for the general public, making some especially vulnerable groups more anxious and depressed than before the outbreak. Either acute mild stress or prolonged chronic stress can influence our appetite, including our desire to eat and the types of food we are likely to choose . StudiesIn terms of energy consumption, due to the suspension of work and production, as well as the closure of fitness venues and public playgrounds, people\u2019s exercise during the epidemic period was significantly reduced, replaced by more screen time. This survey found that more than half of the respondents had reduced physical activity during the COVID-19 outbreak compared with before (with a \u201csignificant decrease\u201d accounting for 31.5%), including decreased outdoor activity, decreased step number, and increased sedentary time, which were also significantly associated with weight gain. Studies have shown that the abrupt interruption of physical exercise and prolonged inactivity may promote many adverse health changes, including the development of insulin resistance, muscle atrophy, bone loss, decreased aerobic capacity, increased blood pressure and heart frequency, fatty liver disease, nonalcoholic steatohepatitis, and dyslipidemia, as well as a higher risk of collapsing upon resuming exercise . TherefoContrary to previous studies that people with overweight and obesity were more likely to gain weight ,35, thisMoreover, the three factors most significantly associated with weight gain, namely, increased food intake, reduced physical activity, and non-overweight and obesity before the outbreak were analyzed through interaction analysis and multiple regression analysis. It was found that when these three main factors were combined, respondents had a higher risk of weight gain. Additionally, while this study found a slight increase in \u201csleep time\u201d and \u201csleep quality\u201d, there was no correlation with weight gain. But another domestic study found that after the outbreak of COVID-19, residents\u2019 sleep quality declined, with an obvious sleep disorder . Certainly, in addition to the weight gain, there were also some harmful health problems that were not involved in this study, such as behavioral addiction disorders, inadequate sunshine exposure, and social isolation . In partThis study provided data on lifestyle changes during the COVID-19 lockdown about Chinese residents for the first time. There was an increase in total food intake by 39% of respondents, especially in snacks and drinks. The major psychological factors leading to the increase in food intake were loneliness and anxiety. There was a decrease in physical activity by 54.9% of respondents, especially in outdoor activities. The rate of weight gain in the total population was 30.6% and the average weight gain was 0.5 \u00b1 2.8 kg. The main factors contributing to weight gain were increased food intake and reduced physical activity, and normal-weight people were more likely to gain weight than overweight or obese people during the \u201cstay at home\u201d lifestyle caused by the COVID-19 outbreak. Furthermore, the three main factors associated with weight gain interact with each other and can have a synergistic effect on weight gain."}
{"text": "Innate antiviral immunity is the first line of host defense against invading viral pathogens. Immunity activation primarily relies on the recognition of pathogen\u2010associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Viral proteins or nucleic acids mainly engage three classes of PRRs: Toll\u2010like receptors (TLRs), retinoic acid\u2010inducible gene I (RIG\u2010I)\u2010like receptors (RLRs), and DNA sensor cyclic GMP\u2010AMP (cGAMP) synthase (cGAS). These receptors initiate a series of signaling cascades that lead to the production of proinflammatory cytokines and type I interferon (IFN\u2010I) in response to viral infection. This system requires precise regulation to avoid aberrant activation. Emerging evidence has unveiled the crucial roles that the ubiquitin system, especially deubiquitinating enzymes (DUBs), play in controlling immune responses. In this review, an overview of the most current findings on the function of DUBs in the innate antiviral immune pathways is provided. Insights into the role of viral DUBs in counteracting host immune responses are also provided. Furthermore, the prospects and challenges of utilizing DUBs as therapeutic targets for infectious diseases are discussed. This review systematically summarizes the role and mechanisms of deubiquitinating enzymes (DUBs) in regulating innate antiviral signaling via the Toll\u2010like receptors (TLRs), RIG\u2010I like receptors (RLRs), and cyclic GMP\u2010AMP synthase (cGAS). The role of viral DUBs counteracting host immune responses is also concluded. Moreover, the prospects and challenges of utilizing DUBs as therapeutic targets for treatment of infectious diseases like COVID\u201019 are also discussed. Upon recognition, PRR initiates a series of signaling cascades that ultimately lead to the synthesis and secretion of type I interferons (IFN\u2010I) and proinflammatory cytokines, thereby exhibiting antiviral immune responses.Our living environment is surrounded by various infectious viruses. To counteract these pathogens, hosts have evolved diverse innate immune systems. This is achieved by pattern recognition receptors (PRRs) expressed in cells that recognize molecules specific to the pathogens, namely pathogen\u2010associated molecular patterns (PAMPs).1, 2 This raises the question of how host cells limit innate immune signals to extremely low levels and meanwhile ensure its timely activation to deal with the challenge of pathogens. The rapid switch on of innate antiviral signaling requires certain posttranslational modifications (PTMs) of crucial sensors for viral RNA, such as ubiquitination.As insufficient interferon production results in chronic infection whereas excessive interferon leads to autoimmune and/or inflammatory disease, innate immunity must be tightly regulated to efficiently respond to invading pathogen and meanwhile avoid excessive harmful immune pathology.5 This To date, three classes of E3s have been identified: really interesting new gene (RING), homologous to E6AP C\u2010terminus (HECT), and RING\u2010between\u2010RING (RBR). RING E3s directly transfer the ubiquitin from E2s to the substrate, whereas HECT and RBR E3s first receive ubiquitin from E2s through a catalytic cysteine, after which the ubiquitin is transferred to the substrate. An isopeptide bond is then formed between the carboxyl group of the ubiquitin's glycine and the epsilon\u2010amino group of the substrate's lysine  and an N\u2010terminus methionine (M1) serving as points of ubiquitination, thereby resulting in complex linkage types. A substrate protein can be attached with a mono\u2010 or poly\u2010ubiquitin molecule. The resulting ubiquitinated proteins vary based on the linkage type of the ubiquitin chains. The most common linkage types are K48\u2010 and K63\u2010linked polyubiquitin chains, with the former recognized by the proteasome, leading to the degradation of the substrate, whereas the latter is mainly involved in nondegradative roles, including cellular signaling, intracellular trafficking, and DNA damage response. Those not linked via canonical K48 linkages or K63 linkages are known as atypical ubiquitin chains, whose physiological roles have only started to accumulate. For example, linear ubiquitin chains are linked through M1 and have been established to play crucial roles in inflammatory signaling and apoptotic cell death. Accumulating evidence has uncovered the role of both typical and atypical ubiquitin chains in the initiation, maintenance, and termination of antiviral immune responses.Ubiquitination is an enzymatic posttranslational modification in which a ubiquitin protein with 76 amino acids is conjugated to a target molecule. Ubiquitin is so named because it occurs ubiquitously in cellular processes. Ubiquitination requires a cascade consisting of three enzymes: ubiquitin\u2010activating enzymes (E1), ubiquitin\u2010conjugating enzymes (E2), and ubiquitin ligases (E3). In this cascade, E1 can bind with many E2s, which further bind with hundreds of E3s in a hierarchical way.7 To d DUBs cleave ubiquitin molecules from substrates to generate a free ubiquitin pool, thereby ensuring the ubiquitin cycle .18 DUB22.1 To date, DUBs have been divided into seven families, based on their structural features: six families of cysteine proteases, namely ubiquitin\u2010specific proteases (USPs), ovarian tumor proteases (OTUs), ubiquitin C\u2010terminal hydrolases (UCHs), the Josephin family, the motif interacting with ubiquitin (MIU)\u2010containing novel DUB family (MINDYs), zinc finger with UFM1\u2010specific peptidase domain protein/C6orf113/ZUP1 (ZUFSP), and the JAB1/MPN/MOV34 metalloenzyme family , which belongs to the zinc\u2010metalloprotease group. DUBs generally contain a catalytic domain surrounded by one or more accessory domains, some of which contribute to target recognition. These additional domains include zinc finger (ZnF) domain, ubiquitin\u2010like domain (UBL), coiled\u2010coil (CC) domain, and motif interacting with ubiquitin (MIU) domain  was the first identified DUB to exhibit linkage specificity, and many JAMMs are reported to be K63 specific. MINDY DUBs are K48 specific. Most OTU family DUBs can be linkage specific, whereas most USPs show little or no linkage preference. The underlying mechanisms of linkage specificity have been reviewed elsewhere and will therefore not be covered herein.DUBs cleave ubiquitin from ubiquitinated substrates in different ways, either by binding to a protein substrate that they deubiquitinate (target\u2010specific) or by directly binding to the ubiquitin chain that they cleave (linkage\u2010specific).25 In Ubiquitin chains are cleaved by DUBs either through endo\u2010 or exocleavage activity. While endo\u2010cleavage activity efficiently removes ubiquitin chains from a substrate, the released chain undergoing further processing to regenerate monoubiquitin, exo\u2010cleavage directly generates monoubiquitin, but requires multiple steps for the DUB to remove the entire polyubiquitin chain. Although it seems complicated to determine whether a DUB cleaves with endo\u2010 or exocleavage activity, several studies have elucidated that the activity depends on both the DUB structure and the ubiquitin linkage type.252.3 inflammatory diseases, and neurological diseases. DUBs can be regulated by a multifaceted approach, including the control of their abundance, catalytic activity, as well as their localization.Dysregulation of DUBs may lead to aberrant functions that can cause a variety of diseases, such as cancer,32 inf\u03baB (NF\u2010\u03baB) activation. Once expressed, DUBs can also be negatively regulated, as exemplified by the stimulation of the expression of OTUD\u20106B in B lymphocytes by IL\u20103, IL\u20104, IL\u201013, or granulocyte\u2010macrophage colony\u2010stimulating factor (GM\u2010CSF); however, an opposing effect occurs with sustained induction, leading to a decrease in OTUD\u20106B expression.The abundance of DUBs is modulated at the transcriptional level and the expression level of DUBs can be induced in a stimulation\u2010dependent manner. For example, the abundance of A20 is low in unstimulated cells, yet is upregulated in response to TLR4\u2010mediated nuclear factor tivation.38 Onc\u03baB essential modulator (NEMO). Second, DUB activity is commonly adjusted in an allosteric fashion. This occurs especially when DUBs function in complexes with other molecules. For instance, USP1 by itself is an ineffective enzyme, but its activity is boosted when bound to USP1\u2010associated factor 1 (UAF\u20101) and it undergoes conformational changes. Additionally, substrate binding may promote DUB activation, thereby enabling fine\u2010tuning of linkage specificity. For example, USP7 alters its inactive configuration toward an active one upon ubiquitin binding, which enables full activation of the USP7 catalytic domain. Third, DUB catalytic activity can be modulated by posttranslational modifications, such as phosphorylation, ubiquitination, and SUMOylation. For instance, our previous studies demonstrated that poly\u2010SUMOylation is required for OTUB2 to interact with and deubiquitinate Yes\u2010associated protein (YAP) and transcriptional coactivator with PDZ\u2010binding motif (TAZ). Intriguingly, these modifications can play dual roles in DUB activity, either inhibitory or activating, thereby revealing the fascinating intricacy of posttranslational regulation. Moreover, the cellular microenvironment can also influence DUB activity. DUBs that are cysteine proteases harbor a reactive cysteine residue that can be oxidized by reactive oxygen species (ROS), thus attenuating DUB activity.DUB catalytic activity can be regulated in a number of ways: binding to regulatory proteins, allosteric regulation, and posttranslational modification. First, DUBs can target a specific substrate by recruiting other factors, as demonstrated by USP10 recruiting and interacting with the monocyte chemotactic protein induced protein 1 (MCPIP1) to deubiquitinate its substrate, nuclear factor r (NEMO).40 Sec DUB localization can be regulated by various approaches. Posttranslational modifications, apart from affecting DUB catalytic activity, have also been reported to affect the nuclear localization of DUBs. To illustrate, phosphorylation of USP4 by protein kinase B  results in its redistribution from the nucleus to the cytoplasm, which enables USP4 to reach the cell membrane and deubiquitinate the transforming growth factor\u2010\u03b2 (TGF\u2010\u03b2) receptor I (T\u03b2R\u2010I). Furthermore, localization can also be indirectly modulated by changing the protein interactions that a DUB engages in ref. . For example, hydroxylation of OTUB1 by factor inhibiting HIF (FIH) leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. This alteration of OTUB1 interactome affects OTUB1 localization and substrate accessibility.DUB regulation is also exerted through subcellular localization, which is necessary for substrate accessibility. DUBs are often found to form complexes with other proteins, including adaptors and scaffolds. Interactions with these proteins can assure proper localization of the DUB so that the specific substrate is delivered to the DUB in the right place for its catalytic action.48 DUBThe diversity of the mechanisms for DUB regulation enables their function to be finely tuned, which ensures appropriate responses in different contexts, including innate immune response against viruses.33.1 TLRs have evolved to recognize a variety of PAMPs. To date, at least ten human TLRs have been identified, with TLRs 1, 2, 4, 5, 6, and 10 localizing at the cell surface, whereas TLRs 3, 7, 8, and 9 are anchored to endolysosomes. Of the identified TLRs, several have been linked to innate antiviral immunity. Among them, TLR2 and TLR4 sense both structural and nonstructural viral proteins, whereas TLR3, TLR7/8, and TLR9 detect virus\u2010derived dsRNA, ssRNA, and unmethylated CpG DNA, respectively, which together is responsible for the effectiveness of the antiviral immune surveillance. TLRs share similar domain structures, including an ectodomain comprising leucine\u2010rich repeats that recognize PAMPs, a transmembrane domain, and a C\u2010terminal cytoplasmic domain, known as Toll\u2010interleukin\u20101 receptor (IL\u20101R) homology (TIR) domain. The TIR domain in the cytoplasm is crucial for signal transduction.As the most widely studied PRRs, TLRs are transmembrane glycoproteins that are mainly expressed in immune cells. TLRs were first acknowledged to be critical for defending fungal infections.55 TLRFigure\u00a0\u03b2 (TRIF), all TLRs involved in antiviral immunity recruit myeloid differentiation primary response 88 (MyD88).TLRs initiate downstream signaling cascades by recruiting their respective adaptor proteins upon ligand binding Figure\u00a03. Apart TRAF6, cIAP1, and cIAP2 can also be recruited to Myddosome, functioning as ubiquitin ligases that mediate K48\u2010linked polyubiquitination of the tumor necrosis factor receptor\u2010associated factor 3 (TRAF3) and consequent degradation by the proteasome. In response to stimuli, Myddosome triggers IRAK4 autophosphorylation and activation, followed by IRAK4\u2010mediated phosphorylation and activation of IRAK1/2. IRAKs are then dissociated from MyD88, resulting in the activation of TRAF6. TRAF6 forms a complex with two E2 enzymes, Ubc13 and Uev1A, which promotes the synthesis of K63\u2010linked polyubiquitin chains, thereby acting as a scaffold to recruit the downstream signaling molecules, TGF\u2010\u03b2\u2010activated kinase 1 (TAK1) and \u03baB kinase (IKK) complexes, through their ubiquitin\u2010binding subunits, TGF\u2010\u03b2\u2010activated kinase 1 and MAP3K7\u2010binding protein 2/3 (TAB2/3) and NEMO, respectively. As a homologous binding partner of both IKK\u03b1/IKK\u03b2 complex and TANK\u2010binding kinase 1 (TBK1)/IKK\u03b5 complex, NEMO can be catalyzed with K27\u2010, K29\u2010, and M1\u2010linked ubiquitin chains, thereby regulating the phosphorylation of NF\u2010\u03baB and interferon regulatory factor 3 (IRF3) and subsequent translocation into the nucleus. Notably, linear ubiquitin chain assembly complex (LUBAC), which consists of heme\u2010oxidized IRP2 ubiquitin ligase 1L (HOIL\u20101L), HOIL\u20101\u2010interacting protein (HOIP), and Sharpin, catalyzes the M1\u2010linked ubiquitination  of NEMO, which is a prerequisite for recruiting the IKK complex (IKK\u03b1 and IKK\u03b2). TAK1 not only induces the phosphorylation of IKK\u03b2, leading to the activation of IKK, but also activates the mitogen\u2010activated protein kinase (MAPK) kinase family. These kinases lead to the activation of transcription factors NF\u2010\u03baB and activator protein 1 (AP1) and induction of proinflammatory cytokines. Of note, in the TLR7/8/9\u2010mediated signaling pathway, IRF7 can be directly recruited by both MyD88 and IRAK1, followed by IRAK1\u2010mediated phosphorylation and activation. TRAF6, TRAF3, and IKK\u03b1 are proved to be necessary for this activation. Besides, IRF5 downstream of MyD88 and TRAF6 is indispensable for TLR7/8/9\u2010mediated production of proinflammatory cytokines.TLR2/4, as well as TLR7/8/9 activation, induces the recruitment of MyD88 and the assembly of the MyD88 signaling complex, termed Myddosome, which consists of MyD88 itself and the IL\u20101 receptor\u2010associated kinase (IRAK) family kinases IRAK4 and IRAK1/2.64 TRA Activated TRIF binds TRAF3, a ubiquitin ligase crucial for the recruitment of TBK1 and the inhibitor of the IKK\u03b5 complex, following modification of TRAF3 with K63\u2010linked polyubiquitin. The TBK1/IKK\u03b5 complex recruits and phosphorylates IRF3. The activated IRF3 dimerizes and enters the nucleus and induces type I IFN production. Additionally, TRIF also activates the NF\u2010\u03baB pathway. TRIF recruits and binds to receptor\u2010interacting serine/threonine\u2010protein kinase 1 , which activates TAK1. The activation of NF\u2010\u03baB is then mediated by a similar mechanism downstream of TRAF6. Therefore, TAK1 results in a variety of signaling cascades and ultimately initiate the transcription of proinflammatory cytokines.In contrast, TLR3 activation leads to the recruitment and subsequent activation of TRIF through TIR\u2013TIR interaction.78 Act The E3 ubiquitin ligase Parkin negatively regulates the antiviral signaling pathway by targeting TRAF3 for degradation; however, the process is inhibited by the mitochondrial protein PTEN\u2010induced kinase 1 (PINK1), which associates with TRAF3 via the kinase domain. Smurf1/2 interacts with MyD88, leading to its K48\u2010linked ubiquitination and degradation, thus limiting the inflammatory response. Neuregulin receptor degradation protein 1 (Nrdp1) is another E3 ubiquitin ligase that leads to MyD88 degradation through K48\u2010linked ubiquitination. Intriguingly, Nrdp1 can also conjugate K63\u2010linked polyubiquitin on TBK1, thereby causing effects that are opposite. Similarly, TRIF can be targeted for K48\u2010linked polyubiquitination and subsequent degradation by the E3 ligase WW domain\u2010containing protein 2 (WWP2) and tripartite motif\u2010containing 38 (TRIM38). BICP0, a viral E3 ubiquitin ligase encoded by bovine herpes virus\u20101, is another negative regulator that promotes the K48\u2010linked ubiquitination and degradation of TRAF6. TRAF6 itself is the E3 ligase that can target IRF7 for K63\u2010linked polyubiquitination. This process is negatively regulated by TARBP2 by its participation in the interaction between IRF7 and TRAF6.Ubiquitination plays a critical role in TLR\u2010triggered MyD88 and TRIF signaling. Multiple E3 ligases have been identified to negatively regulate TLR signaling through K48\u2010linked polyubiquitination. Notably, Triad3A has been reported to target not only certain TLRs, including TLR3, 4, 5, and TLR9 but also RIP1 and TRAF3, ultimately leading to their degradation.81 The Nevertheless, the role of A20 is not that simple due to its structural features, including the presence of an OTU domain as well as seven zinc\u2010finger (ZnF) motifs, which confer both DUB activity and E3 ubiquitin ligase activity on A20, respectively. Whereas A20 removes K63\u2010linked polyubiquitin chains from its substrates, such as RIPK1, A20 also introduces K48\u2010linked polyubiquitin chains in the same substrate, thereby tagging it for proteasomal degradation. Additionally, A20 can bind polyubiquitin chains through its zinc\u2010finger motif, which leads to its specific binding to ubiquitinated NEMO. This allows phosphorylation of IKK to be sufficiently blocked by its upstream kinase TAK1, which inhibits NF\u2010\u03baB activation. Another study has shown that A20, with the help of the regulatory molecule Tax1 Binding Protein 1 (TAX1BP1), interacts with E2 enzymes Ubc13 and UbcH5c and triggers their ubiquitination and subsequent degradation, which antagonizes their interactions with the E3 ligases TRAF6, TRAF2, and cIAP1. The activities of these E3 ligases are consequently dampened. These findings together reveal the diverse and intricate roles of A20 in the inhibition of TLR signaling pathways. CYLD is another well\u2010studied DUB. In TLR\u2010mediated signaling, CYLD negatively regulates NF\u2010\u03baB signaling by cleaving K63\u2010linked polyubiquitin chains and M1\u2010linked polyubiquitin chains from RIPK1, TRAF2, and NEMO.Deubiquitination precisely controls the abundance and activity of signaling molecules in TLR signaling, thereby avoiding dysregulation Figure\u00a0. Over th91 Nev OTUD4 was previously shown as a K48\u2010specific DUB that is important for maintaining the stability of alkylation repair enzymes. In another study, OTUD4 has been reported to harbor K63\u2010specific DUB activity following phosphorylation at Serine202 and Serine204. Furthermore, this activity requires an adjacent ubiquitin\u2010interacting motif that increases the affinity of OTUD4 for K63\u2010linked chains. OTUD4 can target MyD88, thereby negatively regulating the TLR\u2010mediated NF\u2010\u03baB pathway. Indeed, OTUD4\u2010deficient mice have been shown to exhibit increased inflammatory signaling in response to TLR stimulation. TRAF6 and TRAF3 are essential for mediating downstream signaling, and their activities are tightly regulated by several DUBs. Myb\u2010like, SWIRM, and MPN domains 1 (MYSM1) targets both TRAF3 and TRAF6 for K63\u2010linked deubiquitination and subsequent inactivation, subsequently terminating TLR\u2010mediated pathways for antiviral responses, which is evidenced genetically. Specifically, the SWIRM and metalloproteinase domains have been identified as responsible for the interaction with both TRAF3 and TRAF6, and the removal of K63\u2010linked polyubiquitin chains, respectively. USP4 has been identified as a potent negative regulator of TLR/IL\u20101R signaling. USP4 interacts with and deubiquitinates TRAF6, thereby preventing the activation of NF\u2010\u03baB and AP\u20101 transcription factors and subsequent immune responses. Moreover, usp4\u2010depleted zebrafish (Danio rerio) larvae have been shown to produce more proinflammatory cytokines and were more susceptible to endotoxic challenge following LPS treatment. Ubiquitin carboxyl\u2010terminal hydrolase L1 (UCHL1) cleaves K63\u2010linked polyubiquitin chains on TRAF3, which ultimately leads to reduced production of type I IFN, proinflammatory cytokines, and chemokines in response to high\u2010risk human papillomavirus (hrHPV) infection. TRAF family member\u2010associated NF\u2010\u03baB activator (TANK) interacts with both monocyte chemotactic protein\u20101\u2010induced protein\u20101 (MCPIP1) and USP10, leading to the cleavage of ubiquitin chains on TRAF6 and termination of NF\u2010\u03baB activation in response to TLR activation. Of note, USP10 also interacts with NEMO via MCPIP1 and leads to the removal of NEMO\u2010attached M1\u2010linked polyubiquitin chains, thus inhibiting the genotoxic NF\u2010\u03baB signaling cascade. In addition to interacting with USP10, MCPIP1 itself regulates cellular inflammation pathways with distinct mechanisms. A study showed that MCPIP1 negatively regulates c\u2010Jun N\u2010terminal kinase (JNK) and NF\u2010\u03baB activities, induced by LPS, IL\u20101\u03b2, and tumor necrosis factor\u2010\u03b1 (TNF\u2010\u03b1), by removing ubiquitin moieties from TRAF2, TRAF3, and TRAF6. Consistently, the fatal inflammatory syndrome was observed in MCPIP1\u2010deficient mice. In addition to its DUB activity, MCPIP1 restricts cellular inflammation via its RNase activity. Interestingly, several studies have shown that MCPIP1 RNase activity renders specific mRNA degradation of IL\u20106, IL\u20101\u03b2, and IL\u20102. Conversely, MCPIP1 can also be a positive regulator of antiviral immunity. MCPIP1 ribonuclease exhibits broad\u2010spectrum antiviral effects via the binding and degradation of viral RNA. Studies have found that MCPIP1 not only inhibits RNA viruses, such as Japanese encephalitis (JEV), dengue virus (DEN), sindbis virus, encephalomyocarditis, influenza A, hepatitis C virus (HCV), and human immunodeficiency virus (HIV), but also DNA viruses, such as the hepatitis B virus (HBV). Moreover, while MCPIP1 can also promote IFN\u2010I\u2010mediated antiviral efficacy in a manner independent of its RNase and DUB activity, the underlying mechanism is not clear. Similarly, USP18 acts as a negative regulator of IFN responses by counteracting ISG15 conjugation upon viral infection. Additionally, it has been reported that USP18 also regulates TLR\u2010induced NF\u2010\u03baB activation by removing K63\u2010linked polyubiquitin chains of TAK1 and NEMO.Several DUBs in TLR signaling perform nondegradative roles. For example, USP19 was identified to play a role in downregulating TLR3/4\u2010mediated signaling. USP19 interacts with TRIF and removes the K27\u2010linked polyubiquitin moieties of TRIF, which inhibits the recruitment of TRIF to TLR3/4. In vitro and in vivo assays further demonstrated that USP19 negatively regulates TLR3/4\u2010mediated innate immune and inflammatory responses.98 OTU In response to noncanonical NF\u2010\u03baB stimuli, OTUD7B displays K48\u2010specific DUB function, which inhibits TRAF3 proteolysis and prevents aberrant non\u2010canonical NF\u2010\u03baB activation through binding and deubiquitination of TRAF3.It has been shown that some DUBs are related to the stability of signaling molecules in the TLR pathway. In response to LPS, USP25 prevents the K48\u2010linked ubiquitination of TRAF3 that is mediated by cIAP2. Deficiency in USP25 accelerates its degradation following TLR4 activation. Indeed, USP25 knockout mice exhibited increased susceptibility to LPS\u2010induced septic shock.117 InAlthough a large number of host DUBs that control TLR signaling have been characterized over the years, much of the data attempting to elucidate whether DUBs function in the context of defending viral infections or other pathogen infections are inconclusive. Hence, in this Review, we present a summary of the host DUBs that target key molecules in the TLR signaling pathway. It should be noted that the specific function of the host DUBs differs, depending on the PAMPs, cell type, and infection model examined. Further studies are needed to confirm the antiviral function of DUBs in TLR signaling.3.2 melanoma differentiation\u2010associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), all of which are distributed in the cytoplasm. All RLRs contain a C\u2010terminal domain (CTD) and an intermediate RNA helicase domain. At the N\u2010termini, all RLRs, except for LGP2, possess tandem caspase recruitment domains (CARDs), which are necessary for downstream signal transduction. Although sharing similar domain architectures, they each have different ligand preferences: RIG\u2010I recognizes ssRNAs bearing a 5\u2032\u2010triphosphate moiety, as well as short dsRNA (\u223c1kb)\u00a0bearing 5\u2032\u2010triphosphate or with a lower affinity 5\u2032\u2010diphosphate, whereas MDA5 prefers to sense longer dsRNA. LGP2 has been shown to engage in the recognition of and interaction between RNA and RIG\u2010I/MDA5, thus exerting a regulatory role in downstream signaling.While TLR\u2010mediated signaling exerts extensive antiviral immune responses, this system has its limitations due to a lack of TLR expression in most nonimmune cells. Therefore, alternative PRRs, such as RLRs, are needed. RLRs are cytosolic RNA sensors that consist of three members: RIG\u2010I,119 me MAVS further activates TBK1 and IKK by recruiting TRAF proteins. TBK1 and IKK complexes activate and phosphorylate IRF3 and NF\u2010\u03baB, respectively, leading to the induction of interferon and proinflammatory cytokines  through CARD\u2013CARD interaction.133 MA Moreover, the E3 ligases TRIM25 and Riplet/RING finger protein 135 (RNF135) have been reported to conjugate K63\u2010linked polyubiquitin chains to RIG\u2010I CARDs, thus stabilizing this complex and boosting its activation. RIG\u2010I can also be conjugated with K63\u2010linked ubiquitin chains by E3 ligase TRIM4 and Mex\u20103 RNA binding family member C (MEX3C), thus enhancing virus\u2010mediated type I IFN production. Similarly, unanchored K63 polyubiquitin chains are indispensable in the oligomerization of MDA5, the resulting oligomer complex being stabilized by the covalent binding of polyubiquitin chains. The E3 ligase TRIM31 interacts and catalyzes the K63\u2010linked polyubiquitination on MAVS, which promotes the formation of prion\u2010like aggregates of MAVS after viral infection. Furthermore, the E3 ligase TRIM21 positively regulates innate antiviral immunity through K27\u2010linked polyubiquitination of MAVS. LUBAC negatively regulates RIG\u2010I and TRIM25\u2010mediated type I IFN induction by catalyzing M1\u2010linked ubiquitination and subsequent degradation of RIG\u2010I, as well as competing with TRIM25 for its association with RIG\u2010I, thus inhibiting RLR\u2010mediated signaling.Ubiquitination is also crucially involved in regulating RLR signaling. Structural remodeling of RIG\u2010I into active conformations occurs upon RNA binding, which allows RLRs undergoing oligomerization to be active. RIG\u2010I forms tetramers with the help of E3 ligase TRIM25, which can catalyze and form free K63\u2010linked polyubiquitin chains that bind to its CARDs.136 Mo RNF122, carboxyl terminus of Hsc70\u2010interacting protein (CHIP), and STIP1 homology and U\u2010box containing protein 1 (STUB1) have been reported to attach K48\u2010linked ubiquitin chains on RIG\u2010I, and TRIM13 works on MDA5. TRIM40 has been reported to interact with both RIG\u2010I and MDA5, catalyzing K27\u2010 and K48\u2010linked polyubiquitination, respectively, thereby promoting their degradation. A few E3 ligases, such as RNF5, SMAD specific E3 ubiquitin protein ligase 1(Smurf1), Smurf2, TAX1BP1, membrane\u2010associated RING finger (C3HC4) 5 (MARCH5), poly(rC)\u2010binding protein 1 (PCBP1), PCBP2, TRIM25, and receptor for activated C kinase 1 (RACK1), are responsible for the K48\u2010linked ubiquitination on MAVS.K48\u2010linked polyubiquitination is needed for proteasomal degradation, thus negatively regulating the MAVS signaling cascade. The E3 ligases RNF125,147 RN Upon viral infection, or in the presence of a tumor necrosis factor, the CYLD expression level is downregulated to enhance IFN production. Notably, it has been shown that CYLD targets not only RIG\u2010I but also downstream TBK1, the kinase that phosphorylates and activates IRF3. In this case, Optineurin (Optn) was shown to dampen the response via targeting CYLD to TBK1 to impair its enzymatic activity. Similarly, USP3 also acts as a negative regulator of RIG\u2010I\u2010mediated signaling by removing K63\u2010linked ubiquitination on RIG\u2010I. Upon viral infection or ligand stimulation, USP3 binds to the CARD domain of RIG\u2010I and then removes its polyubiquitin chains through both the zinc\u2010finger Ub\u2010binding domain and USP catalytic domains. However, whether USP3 regulates RIG\u2010I in vivo remains unknown. Moreover, USP21 has been reported to remove K63\u2010linked ubiquitination of RIG\u2010I, resulting in impaired antiviral responses. Interestingly, USP21 presumably removes polyubiquitin chains from more than one lysine sites in either CARD or CTD domain, since USP21 can reverse the polyubiquitination mediated by both RNF135 and TRIM25. USP21\u2010deficient mice are more resistant to vesicular stomatitis virus (VSV) infection, concomitated with elevated IFN production. USP14, in another study, has also been shown to remove K63\u2010linked ubiquitin chains on RIG\u2010I. USP14\u2010specific inhibitor, IU1, was demonstrated to increase RIG\u2010I\u2010mediated type I IFN production and antiviral responses in vitro and in vivo. Recently, siRNA library screening identified USP27X as a negative regulator of type I IFN signaling by cleaving K63\u2010linked polyubiquitin chains from RIG\u2010I. While USP15 also plays a negative role in RIG\u2010I signaling, its activities are more difficult to decipher as it exhibits an opposite function in other contexts (see below).Since K63\u2010linked ubiquitination of RIG\u2010I is crucial for its activation and signaling, it is not surprising that several DUBs are involved in downregulating RLR signaling to avoid overactivation Figure\u00a0. As the 162 Up\u03b2 production. It is worth mentioning that both YOD1 and TRIM31 interact with the proline\u2010rich domain of MAVS, indicating competition between these two enzymes with opposing roles. Recently, our previous research identified OTUD3 as an acetylation\u2010dependent DUB that directly deconjugates the K63\u2010linked polyubiquitin chains of MAVS and terminates downstream antiviral response. Intriguingly, the catalytic activity of OTUD3 is dependent on the acetylation of Lysine129. To confirm this, acetyl\u2010lysine is incorporated into a recombinant OTUD3 to generate a fully Lys129\u2010acetylated OTUD3 (OTUD3Lys129\u2010Ac) with chemical biology approaches. In vitro Ubiquitin\u2010AMC assay and deubiquitination assay demonstrated that OTUD3Lys129\u2010Ac exhibits robust deubiquitinase activity toward K63\u2010linked ubiquitin chains and is more effective in targeting MAVS. Besides, this acetylated residue can be removed by sirtuin 1 (SIRT1) upon virus infection, which promptly inactivates OTUD3 and triggers the innate antiviral immune response promptly. Consistently, intensive antiviral immune response, diminished viral load, and morbidity were observed in OTUD3 knockout mice, and acetyl\u2010OTUD3 levels are negatively correlated with IFN\u2010\u03b2 expression in influenza patients. These findings collectively establish OTUD3 as a new repressor of MAVS and unveil a novel regulatory mechanism through which the catalytic activity of OTUD3 is tightly tuned in the control of the timely antiviral defense.YOD1 , a DUB of the OTU family, was the first identified DUB responsible for downregulating the K63\u2010linked polyubiquitination of MAVS. Following a Sendai virus infection, YOD1 was recruited to mitochondria and was shown to interact with MAVS. Subsequently, YOD1 removes the K63\u2010linked ubiquitin chain and abrogates the aggregation of MAVS, thus inhibiting not only IRF3 and p65 activation, but also IFN\u2010oduction.170 It Because various components are involved in virus\u2010induced signaling, biochemical assays have demonstrated that OTUB1 and OTUB2 inhibit RIG\u2010I\u2010, MDA5\u2010, and MAVS\u2010mediated but not TBK1\u2010 and IRF3\u2010mediated signaling. Notably, the negative regulation of OTUB1 in TRAF3\u2010elicited antiviral innate immunity is enhanced by HSCARG , which fosters the recruitment of OTUB1 to TRAF3 and abolishes its ubiquitination.Negative regulation of RLR signaling is also accomplished by targeting downstream molecules of MAVS. Coimmunoprecipitation analyses indicate that OTUB1 and OTUB2 associate with TRAF3 and TRAF6, negatively regulating antiviral responses by deubiquitinating their respective K63\u2010linked polyubiquitin chains.172 Be OTUD1 was also reported to inhibit IFN production during RNA virus infection. Mechanistic studies demonstrated that OTUD1 stabilizes Smurf1 by removing its ubiquitin chains, which allows the binding of Smurf1 to MAVS, TRAF3, and TRAF6 with subsequent degradation of each component of the MAVS/TRAF3/TRAF6 signalosome. Notably, OTUD1\u2010deficient mice were consistently shown to be more resistant to RNA virus infections when more antiviral cytokines were produced.Modulating the stability of key signaling molecules is another approach to inhibit RLR\u2010mediated signal transduction. A systematic functional screening revealed that USP5 can interact with and recruit STUB1 to mediate the degradation of RIG\u2010I, thus enhancing VSV replication.174 OT USP4 interacts with and cleaves K48\u2010linked polyubiquitin chains from RIG\u2010I, thereby promoting RIG\u2010I\u2010mediated IFN\u2010\u03b2 signaling and inhibiting VSV replication. Following virus\u2010induced RIG\u2010I activation, USP4 expression was shown to attenuate. Additionally, overexpression of USP4 dramatically enhanced the RIG\u2010I protein level, while knockdown of USP4 had an opposite effect. Although multiple E3 ligases responsible for the K48\u2010linked ubiquitination of MAVS have been identified over the past years, the corresponding DUBs remained elusive until the identification of OTUD4. It has been shown by DUB screening and biochemical analyses that OTUD4 removes K48\u2010linked polyubiquitin chains on MAVS in response to viral infection, thereby inhibiting the proteasomal degradation of MAVS and triggering downstream signaling. This is supported by the observation that knockout or knockdown of OTUD4 impairs the RNA virus\u2010triggered expression of type I interferon and enhances VSV replication, both in vitro and in vivo. Interestingly, as mentioned before, OTUD4 cleaves K63\u2010linked polyubiquitin chains after it is phosphorylated. Therefore, it might be the in vivo protein modification that strongly altered the chain specificity of OTUD4. TRAF6 and TRAF3 play crucial roles in the RLR\u2010mediated antiviral signaling pathway by recruiting other related proteins to form a complex that facilitate NF\u2010\u03baB signaling. A study has demonstrated that USP4 is capable of targeting TRAF6 for K48\u2010linked deubiquitination and inhibiting enterovirus 71 (EV71) replication. USP25 interacts with TRAF3 and TRAF6 following RNA virus or DNA virus infection and prevents TRAF3 and TRAF6 from proteasomal degradation via deubiquitinating K48\u2010linked polyubiquitin chains. Furthermore, USP25 deficiency results in enhanced ubiquitination and turnover of TRAF6 and TRAF3. Consistently, USP25 deficient mice were shown to be more susceptible to H5N1 or HSV\u20101 infection compared to their wild\u2010type counterparts.Over the years, a growing number of DUBs have been identified to upregulate the RLR\u2010mediated signaling pathway. Since K48\u2010linked polyubiquitination may bring signaling molecules to proteasomal degradation, several DUBs were identified to reverse the process. Among them, USP17 and USP4 are the only two identified DUBs that are responsible for upregulating the stability of RIG\u2010I. USP17 facilitates RIG\u2010I\u2010 and MDA5\u2010mediated induction of type I IFN via the deubiquitination of RIG\u2010I, as well as MDA5. Furthermore, USP17\u2010deficiency inhibits the RNA\u2010virus\u2010induced expression of type I IFN and antiviral responses.176 US Mechanistically, USP15 interacts with TRIM25 during the late stage of a viral infection, removes the LUBAC\u2010induced K48\u2010linked polyubiquitination of TRIM25 at its SPRY domain, and ultimately leads to sustained type I IFN expression. Combined with the negative role of USP15 mentioned above, the opposing roles suggest that USP15 exerts disparate effects depending on substrate recognition or infection severity, thereby underscoring the tight regulation of cellular immune responses.In addition to preventing signaling proteins from degradation by removing K48\u2010linked polyubiquitin chains, USP15 was shown to upregulate RLR signal transduction by indirectly attaching K63\u2010linked polyubiquitin chains on RIG\u2010I. As mentioned previously, the E3 ligase TRIM25 is responsible for K63\u2010linked polyubiquitination of RIG\u2010I and it can be degraded by LUBAC\u2010mediated polyubiquitination. A study has demonstrated that USP15 deubiquitinates TRIM25, thus counteracting the LUBAC\u2010dependent degradation of TRIM25.181 Me3.3 The main pathway that mediates the immune response to DNA is governed by cGAS. cGAS recognizes DNA and is activated through direct binding, which catalyzes the formation of cGAMP. cGAMP then binds to and activates the stimulator of interferon genes , an endoplasmic reticulum (ER)\u2010localized adaptor. Upon activation, STING translocates to the Golgi and interacts with TBK1 and IKK, which phosphorylates IRF3 and NF\u2010\u03baB inhibitor I\u03baB\u03b1, respectively. The TBK1/IKK\u03b5 and IKK pathways are subsequently activated, thus leading to the induction of type I interferons and proinflammatory cytokines  catalyzes K27\u2010linked polyubiquitination of STING, which serves as an anchoring platform for recruiting TBK1 and promotes its translocation to the perinuclear microsomes. In addition to STING, cGAS can also be ubiquitinated to facilitate signaling. For example, the E3 ligase RNF185 attaches K27\u2010linked polyubiquitination to cGAS, thereby promoting its enzymatic activity. TRIM56 targets cGAS through monoubiquitination, which results in a remarkable increase in its dimerization, DNA\u2010binding activity, and cGAMP production. Another E3 ubiquitin ligase that mediates the monoubiquitination of cGAS is RING finger\u2010interacting protein with C kinase , which is critical for cGAS activation.Ubiquitination is equally important in STING signaling. TRIM56, an interferon\u2010induced E3 ubiquitin ligase, interacts with STING and targets it for K63\u2010linked ubiquitination, which induces STING dimerization and recruitment of TBK1.197 Li TRIM29, and TRIM30, all of which target STING for K48\u2010linked ubiquitination and subsequent proteasomal degradation. Interestingly, the E3 ligase RNF26 competitively promoted K11\u2010linked polyubiquitination of STING at the same residue targeted by RNF5, thus protecting it from RNF5\u2010mediated degradation. Besides, K48\u2010linked ubiquitination of cGAS has been reported as a recognition signal for p62\u2010dependent selective autophagic degradation, although the E3 ubiquitin ligase(s) involved has not been determined yet.The negative role of ubiquitination in the cGAS\u2010STING pathway is executed by the E3 ligases RNF5,203 TR Apart from targeting RIG\u2010I, USP27x was previously identified as another DUB that regulates the stability of cGAS. Unlike USP14, USP27x directly interacts with cGAS and releases the K48\u2010linked polyubiquitin chains during viral infection. Recently, a study reported that USP29 interacts with cGAS, hydrolyzes K48\u2010linked polyubiquitin chains on cGAS, and stabilizes cGAS in uninfected cells, or after HSV\u20101 stimulation. Following HSV\u20101 infection, USP29 knockout mice were shown to be hypersensitive to HSV\u20101 and produced less type I IFNs and proinflammatory cytokines than the wildtype control. Moreover, Trex1 deficiency resulted in the hyperproduction of type I IFNs and proinflammatory cytokines and have been associated with autoimmune disorders, such as Aicardi\u2010Goutieres syndrome (AGS) and systemic lupus erythematosus (SLE). Additionally, Trex1 knockout mice have been previously characterized to exhibit severe systemic inflammation and even lethal autoimmune responses. In the study, genetic ablation of USP29 in Trex1 knockout mice attenuated the related pathological symptoms, highlighting the crucial role of USP29 in the regulation of cGAS. Similar to cGAS, extensive studies have been performed on the discovery of DUBs involved in the cleavage of the K48\u2010linked ubiquitin chain on STING. For instance, USP20 was shown to interact with and remove K48\u2010linked ubiquitin chains from STING after HSV\u20101 infection, thereby stabilizing STING and promoting cellular antiviral responses. Although deubiquitinating STING alone, USP20 can be recruited by USP18, thus causing USP20 to remove K48\u2010linked polyubiquitin chains more efficiently, even at a low dose. Furthermore, a deficiency of either USP20 or USP18 has been reported to affect the stability of STING, and subsequent production of type I IFN, upon viral infection. Notably, this process is independent of the enzymatic activity of USP18. CYLD is another DUB that removes the K48\u2010linked polyubiquitin chains from STING at K150. CYLD knockout mice were shown to be more susceptible to HSV\u20101 infection than their wild\u2010type littermates. More recently, a study identified USP44 as a novel positive regulator of STING. Upon DNA virus infection, USP44 was recruited to the membrane\u2010localized STING and selectively removed its K48\u2010linked polyubiquitin moieties at K236. Additionally, USP44\u2010deficient mice were more susceptible following HSV\u20101 infection. All three DUBs seem to prevent STING from proteasome\u2010dependent degradation through uncoupling K48\u2010linked ubiquitination after DNA virus infection. However, compared to USP20 and USP44, CYLD\u2010mediated deubiquitination lacks target specificity in the control of innate immune signaling, as it also targets TRAF2 and RIG\u2010I. USP20 seems more complicated, as it has also been shown to deubiquitinate and stabilize Unc\u201051 like autophagy activating kinase 1 (ULK1), which is a negative regulator of STING. Besides, reconstitution experiments demonstrated that USP44\u2010mediated regulation of STING signaling is independent of USP20 and CYLD. In light of these, it is likely that USP20, CYLD, and USP44 are not functionally redundant. They may target STING spatially and temporally to fine\u2010tune the onset and termination of innate immune responses against DNA viruses.The K48 ubiquitin linkage type of cGAS is further supported by USP14, which can be recruited by the E3 ligase TRIM14 to cleave the K48\u2010linked ubiquitin chain of cGAS, thus promoting its stability Figure\u00a0. TRIM14 207 Ap DUBs can also cleave atypical polyubiquitin chains on STING, as demonstrated by USP13 deconjugating the K27/33\u2010linked polyubiquitin chains from STING, which impairs the recruitment of TBK1 to STING. In line with this, USP13\u2010deficient mice restricted HSV\u20101 replication in vivo and were more resistant to HSV\u20101 infection. USP21 is another DUB that hydrolyzed the K27/K63\u2010linked polyubiquitin chain on STING following p38\u2010mediated phosphorylation, thus negatively regulating the production of type I interferons induced by the DNA virus. STING signaling can also be negatively regulated by USP22, which interacts with USP13 and specifically cleaves K27\u2010linked ubiquitin chains on STING, while the catalytic activity of USP22 is dispensable for this effect.DUBs also play a role in downregulating the STING pathway Figure\u00a0. K63\u2010lin217 DUIn light of these studies, it is conceivable that STING is regulated by numerous DUBs in a spatial and temporal manner so as to initiate and terminate innate antiviral responses more accurately against non\u2010self. However, it remains to be investigated how these ubiquitination and deubiquitination processes are spatially and temporally coordinated.4\u03baB and IRF3. IKK phosphorylates I\u03baB, the inhibitory subunit of NF\u2010\u03baB that sequesters NF\u2010\u03baB in the cytoplasm, and the phosphorylated I\u03baB leads to its own ubiquitination and subsequent proteasomal degradation, thus enabling the release of NF\u2010\u03baB to the nucleus and the transcription of proinflammatory cytokines. Conversely, TBK1, along with its homolog IKK\u03b5, mediates phosphorylation and activation of IRF3. Upon activation, IRF3 dimerizes and translocates to the nucleus, thus leading to the production of type I IFN , and TRAF\u2010interacting protein (TRIP) conjugate K48\u2010linked polyubiquitin chains on TRAF3 and TBK1, resulting in their proteasomal degradation and inhibition of type I IFN production. DUBs targeting TBK1 cannot be ignored as several DUBs have been shown to be involved in the regulation of TBK1 activity , a protein of the Importin \u03b1 family that is involved in the import of proteins into the cell nucleus, thus efficiently boosting nuclear translocation of IRF3 and subsequent antiviral responses.As for the downstream molecule of TBK1, increasing evidence has shown the regulation of IRF3 activity via the ubiquitin system. Multiple E3 ubiquitin ligases, such as TRIM26, Pin1, RBCC protein interacting with PKC 1 (RBCK2), Ro52, and RTA\u2010associated ubiquitin ligase  target IRF3 for K48\u2010linked polyubiquitination, thus negatively regulating the antiviral innate immune response.243 To5Table\u00a0Table\u00a0Our discussion in the preceding sections of this review focused on how the host performs antiviral immune responses with DUBs. A list of host DUBs targeting key molecules in three major innate immune pathways is summarized in USP). Specifically, UL36USP has been reported to act as a viral protein\u2010mediated immune antagonist by deubiquitinating TRAF3 and dampening IFN\u2010\u03b2 signaling. The UL36USP knockout virus leads to increased production of IFN\u2010\u03b2 compared to wild\u2010type virus. UL36USP was also shown to restrict NF\u2010\u03baB activation in the DNA sensing signaling pathway to evade host antiviral immunity. Mechanistically, UL36USP cleaves polyubiquitin chains from I\u03baB\u03b1 and abrogates proteasomal degradation of I\u03baB\u03b1, thus inhibiting NF\u2010\u03baB activation. In addition, UL36USP dampens the IFN\u2010\u03b2\u2010induced activation of interferon\u2010sensitive response element (ISRE) promoter and the transcription of ISGs, by specifically binding to the interferon \u03b1 and \u03b2 receptor subunit 2 (IFNAR2) subunit, and blocks the association between Janus kinase 1 (JAK1) and IFNAR2. This study underscores the UL36USP\u2010IFNAR2 interaction as a novel evasion strategy exploited by HSV\u20101. Homologs of UL36USP have also been identified in other viruses in the Herpesviridae family, such as human cytomegalovirus (HCMV), murine \u03b3herpes virus 68 (MHV\u201068), Marek's disease virus (MDV), and Kaposi's sarcoma virus (KSHV). Although the DUB activity was identified, the role of these viral DUBs in hijacking host immune systems remains unknown. ORF64USP is the tegument protein of Kaposi's sarcoma\u2010associated herpesvirus (KSHV). ORF64USP was found to process K48\u2010 and K63\u2010linked polyubiquitin chains in vitro, and to target and deubiquitinate RIG\u2010I, thus leading to an inhibition of IFN\u2010I signaling. HBx, encoded by hepatitis B virus, another DNA virus, deubiquitinates both RIG\u2010I and TRAF3 for K63\u2010linked ubiquitin chains and inhibits the production of type I interferon. Epstein\u2010Barr virus (EBV) is a human oncogenic herpesvirus that presents a prolonged latent infection in the host. It has been found that EBV\u2010encoded BPLF1 interacts with and deubiquitinates TRAF6, inhibiting NF\u2010\u03baB signaling, and leads to viral lytic DNA replication upon lytic infection. BPLF1 was also shown to inhibit TLR signaling via the deubiquitination of downstream signaling components, such as NEMO and I\u03baB\u03b1.Virus\u2010encoded DUBs have been identified in DNA viruses. An example of a DNA virus\u2010encoded DUB that interferes with the host antiviral immune responses is the herpes simplex virus 1 (HSV\u20101) ubiquitin\u2010specific protease  encoded PLP2 was reported to strongly inhibit cellular type I interferon expression. Further investigation revealed that MHV PLP2 achieves this by targeting both IRF3 and TBK1, and affects both activities. Additional CoV PLPs include Porcine epidemic diarrhea virus (PEDV) replicase encoded PLP2, which was identified as an antagonist to both RIG\u2010I\u2010 and STING\u2010mediated IFN signaling by the reduction of RIG\u2010I and STING ubiquitination via its deubiquitinating activity that cleaves both K48\u2010 and K63\u2010linked polyubiquitin chains.In addition to DNA viruses, RNA viruses have also evolved DUBs that involve in virus\u2010host interplay. Coronaviruses (CoVs) are a large family of RNA viruses that cause respiratory tract infections, ranging from the mild common cold to serious diseases such as severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS), and the recent outbreak of the novel coronavirus, coronavirus disease 2019 (COVID\u201019). These three diseases result from SARS\u2010CoV, MERS\u2010CoV, and SARS\u2010CoV\u20102, respectively. Both SARS\u2010CoV and MERS\u2010CoV encode a papain\u2010like protease (PLP), featuring both deubiquitinating and deISGylating activities, which subsequently blocks the innate immune signaling of the proinflammatory cytokines.260 Se Previous studies have reported that the arterivirus\u2010 and nairovirus\u2010encoded DUBs resemble the OTU family of DUBs, and in the case of arteriviruses, the domain was characterized as PLP2. Notably, PRRSV\u2010encoded PLP2 also targets I\u03baB\u03b1. The foot\u2010and\u2010mouth disease virus (FMDV) is among the picornavirus family. Its product, Lpro, targets not only RIG\u2010I, but also TBK1, TRAF3, and TRAF6, thereby collectively suppressing the host innate immune signaling pathways. For the Seneca Valley virus (SVV), another picornavirus, a study has shown that SVV 3C protease (3Cpro) harbors deubiquitinating activity and acts on both K48\u2010 and K63\u2010linked polyubiquitin chains. The 3Cpro deubiquitinates RIG\u2010I, TBK1, and TRAF3, and consequently abolishes IFN\u2010\u03b2 induction and IFN stimulated gene 54 (ISG54) mRNA expression. We have listed these viral DUBs in Table\u00a0In addition to coronaviruses, several other RNA virus\u2010encoded DUBs identify RIG\u2010I as solid targets. These DUBs are encoded by the Equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), simian hemorrhagic fever virus (SHFV), and lactate dehydrogenase\u2010elevating virus (LDV), all belonging to the arterivirus family, as well as the Crimean\u2010Congo hemorrhagic fever virus (CCHFV), belonging to the nairovirus family.273 Pr M1\u2010linked ubiquitination is indispensable in antiviral responses elicited by hosts. Recently, a unique M1\u2010linked ubiquitin chain\u2010specific effector DUB was identified in pathogenic bacteria; however, whether viral pathogens have evolved DUBs to cleave M1\u2010linked ubiquitin chains remains a question to be answered. The interactions between hosts and invading viruses are an emerging research field that provides deeper insights into viral pathogenic mechanisms. Thus, further research to elucidate the viral manipulation of DUBs is of particular importance.Although it has been shown that various viral DUBs regulate the same target protein or pathway as their host cell counterpart, these virus\u2010encoded DUBs share no sequence or structural homology, which reflects the diversity and complexity of host\u2010virus interplay.279 M16Given the important roles of DUBs in innate antiviral immune response, and since DUBs harbor a catalytic domain or active center functioning as the target of inhibitors, it is not surprising that DUBs will be considered as potential therapeutic targets for a wide range of diagnoses and treatments. The compound IU1, a reversible small\u2010molecule inhibitor of USP14, was shown to target the USP14 catalytic site. Functional assays revealed that IU1 inhibits replication of several flaviviruses, especially the Dengue virus. Similarly, WP1130, directly inhibiting the DUB activity of USP5, UCH\u2010L1, USP9x, USP14, and UCH37, shows broad\u2010spectrum antiviral activity for several RNA viruses. Evidence showed that WP1130 inhibits USP9x activity through covalent modification of critical cysteine residues important for USP9x catalytic activity. PR\u2010619 is a general DUB inhibitor that nonetheless does not target other cysteine proteases like cathepsin B or calpain 1. In addition, P22077 was shown to covalently modify the catalytic cysteine of USP7 and induce a conformational switch in the enzyme associated with active site rearrangement, thus impairing HIV Gag processing and reducing the infectivity of released virions.In emerging basic studies, DUBs have shown promising prospects as drug targets for infectious diseases.33 The To illustrate, GRL\u20100617 was shown to be a potent, selective, and competitive noncovalent inhibitor of SARS\u2010CoV PLP. Several compounds, both clinical and preclinical, targeting SARS\u2010CoV PLP have been previously reviewed. The worldwide outbreak of SARS\u2010CoV\u20102 compelled researchers to struggle with countermeasures to fight infection and prevent the spread of the virus. Since PLP inhibitors may be effective in reducing coronavirus infections, we propose that PLP inhibitors should be considered as a latent therapeutic strategy for COVID\u201019. Recently, sequence analysis showed that the PLP present in SARS\u2010CoV\u20102 has a high degree of similarity to that in the extensively studied SARS\u2010CoV. Given that many active compounds against SARS\u2010CoV PLP have been clearly identified, evaluation of these inhibitors in targeting SARS\u2010CoV\u20102 PLP should be put on the agenda. Intriguingly, several recent studies demonstrated that GRL\u20100617 not only inhibits SARS\u2010CoV PLP but also inhibits the activity of SARS\u2010CoV\u20102 PLP, thus impairing the virus\u2010induced cytopathogenic effect, facilitating the antiviral signaling, and reducing viral replication in infected cells.As mentioned above, viral DUBs play pivotal roles in the regulation of host cellular processes, which provides us with novel insights into the possibility and validity of developing inhibitors that target the viral DUBs rather than the host DUBs. Indeed, several studies support the idea that PLP inhibitors may be effective in reducing coronaviruses infection.294 To Third, previous inhibitors against viruses were designed to inhibit viral replication and inherent survival. The targets of these inhibitors are essential proteins for virus replication, assembly, and survival, which are effective in killing viruses in a short time. However, due to strong selection pressure, the virus mutates or evolves to obtain long\u2010term resistance. Therefore, how to improve antiviral drugs to counter drug resistance is a key point.Despite the promising insights on targeting both host and viral DUBs as an antiviral strategy, challenges exist in the following aspects. First, as previously mentioned in this review, DUBs function in delicate mechanisms with complexity. For instance, different DUBs may target the same substrate, while some may target more than one substrate. DUBs may perform opposing roles depending on the context. As an example, USP7 would be a plausible target in the oncology context, but inhibiting USP7 may have a potential impact on inflammatory responses. Hence, given the complexity and pleiotropy of DUBs, comprehensive approaches and diverse strategies are needed to develop these inhibitors. Second, there are still many unknowns about how DUBs work and are modulated. To date, many studies merely link DUBs to their functions in vitro, which is unconvincing, as the bona fide DUB activity in vivo may not be reflected.300 ThNevertheless, based on the pivotal role of DUBs in pathogenesis, the druggable feature, and the existing positive evidence against viral infection, developing inhibitors targeting DUBs, especially viral DUBs, should not be underestimated and might deserve at least a further evaluation.7Figure\u00a0As a key regulator of the ubiquitin system, DUBs play a pivotal role in antiviral innate immunity at different layers. Generally, DUBs act alone or in a complex to strengthen their catalytic activities. Specifically, the mechanisms underlying the function of DUBs in the antiviral signaling pathway can be divided into six working models, according to the pattern of altering the ubiquitination status Figure\u00a07: i) moThe effects of DUBs are intricate. While some DUBs positively regulate antiviral pathways, others may function as negative regulators. Still, some DUBs play dual roles (either inhibitory or activating) depending on specific contexts, such as subcellular localization or the type of substrate proteins. Some even act on identical substrates with opposing outcomes (such as USP4 and USP15).Significant efforts have been dedicated to unveiling the virus\u2010encoded DUBs that are responsible for hijacking the cellular ubiquitin system to counteract host defense and evade host surveillance. While every effort was made to include in this Review all DUBs that have been identified, to date, in innate antiviral immunity, many more undoubtedly remain to be characterized. Many studies included in this Review relied on molecular and cellular approaches, which fail to accurately reflect the authentic circumstances that occur in vivo. Hence, further research is needed to ascertain the cellular targets and function of DUBs with in vivo animal experiments.Given the growing evidence showing the importance of DUB activity in innate antiviral immunity, it is reasonable to consider DUBs as promising drug targets for infectious disease treatment. Such strategies should focus on tightly regulating the antiviral immune responses and enhancing infected cell death by modulating the DUBs involved. Additionally, virus\u2010encoded DUBs may provide attractive therapeutic targets for antiviral therapy. Given the importance and urgency of finding treatments for COVID\u201019, the recommendation to evaluate potential drugs targeting SARS\u2010CoV\u20102 PLP should not be underestimated. Overall, given the success of DUB inhibitors for cancer therapies, it is promising that the development of small\u2010molecule inhibitors or agonists targeting DUBs is on the right path.There are still many gaps in our current knowledge and understanding of DUB mechanisms, which hinders progress in the therapeutic application of research in this area. First, unlike the designated roles of the canonical K63\u2010 and K48\u2010linked polyubiquitin chains, some atypical linkage types of polyubiquitin chains may exhibit quite distinctive roles. Yet, these atypical polyubiquitin chains are rarely reported in innate antiviral immune signaling. Thus, further research endeavors to elucidate the atypical polyubiquitin chains recognized by DUBs are required. Second, the activation and inhibition of key signaling molecules are determined by both E3 ligases and DUBs. How these E3 ligases and DUBs cooperate in a dynamic, temporal, and spatial manner to strictly regulate the immune response needs to be clarified further and deeper. Moreover, compared with the vast number of human DUBs, the number of virus\u2010encoded DUBs in control of host immunity appears limited. Therefore, identifying more viral DUBs that are responsible for modulating host immune system warrants increasing efforts to propel this field forward.The authors declare no conflict of interest.Z.Z., Z.K.Z., and L.W. contributed equally to this work. Z.Z. and Z.K.Z. conceived and drafted the manuscript. Z.Z. drew the figures. L.W. discussed the concepts of the manuscript. F.Z. and L.Z. provided valuable discussion and revised the manuscript. The authors apologize to those researchers whose related work were not able to cite in this review."}
{"text": "The aim of the present study was to identify factors associated with h delayed initiation of post-exposure prophylaxis (PEP) among animal bite victims.This cross-sectional study assessed biting patterns among 3032 cases that were referred to Tabriz Rabies Center. The delay was described as the initiation of PEP more than 48 hours (h) after possible exposure to the rabies virus. Determinants of delay in initiating PEP were recognized by a decision tree model.Totally, 8.5% of the victims who were bitten by an animal had a delay of more than 48 h in the initiation of PEP. The relative frequency of a delay more than 48 h in females was higher than in males (12.9% compared to 8.5%) . Relative frequency of a delay of more of 48 h from carnivorous  was significantly less than others . Of the decision tree, the overall classification accuracy was 89.5%, with 44.1% sensitivity and 92.3% specificity. The identified variables included gender, biting place , and type of animal.according to the results of the present study, among the various variables that affect the delayed initiation of PEP, rural residents and being female, in particular, were the major factors associated with a delay in the initiation of PEP for rabies prevention. We found relatively low rates of vaccine completion. Our findings indicat that providing training and patient education are required to ensure the completion of appropriate treatment. Animals attack is still a major health and social issue worldwide. An animal bite is the main source of transmission of rabies to humans, which has not yet been controlled in most parts of the world . ApproxiAfter\u00a0the\u00a0onset\u00a0of\u00a0clinical\u00a0symptoms,\u00a0there\u00a0is\u00a0no\u00a0effective\u00a0treatment for rabies. Therefore, the currently recommended intervention strategy is to remove and neutralize the infectious virus before it enters the nervous system . InapproAnnually, 180000 animal bite cases are reported in Iran, all of whom receive PEP because all biting animals such as cats, dogs, and wolf are considered to be reservoirs of rabies .In this regard, the community needs to be adequately educated to ensure them to take care of themselves in preventing rabies and the consequences caused by animal bites . BesidesTreating a rabies exposure, where the average cost of rabies PEP is US$ 40 in Africa, and US$ 49 in Asia, can be a catastrophic financial burden on affected families whose average daily income is around US$ 1\u20132 per person . PEP is Study designThis cross-sectional study investigated the patterns of animal bites among the cases referred to the rabies center of Tabriz between March 1 2013 and February 29 2019. A sequential sampling of 3032 patients with animal bite history visiting the rabies center was performed. The data were taken from rabies surveillance forms used in the district. These forms are used to identify and follow-up the suspected bites. When a bite case is admitted to a health center, a health worker for communicable disease control performs an examination, decides whether prophylaxis is necessary, fills out the form and follows-up the case until the end of PEP. Data mining was used via the decision tree model to identify factors affecting delay in vaccination 48 h subsequent to bites to prevent rabies. Study Site Post-prophylactic rabies centers have been established in provinces and even in small towns in Iran under the supervision of the Ministry of Health and Medical Education. The study was conducted at the rabies center of Tabriz which is affiliated to Tabriz University of Medical Sciences.Study populationAll cases of animal bite in all age and gender groups which had been referred to the Rabies Center of Tabriz were investigated.An animal bite was defined as any animal bites caused by mammals. Data were extracted for the patients who were registered at the health center and referred to the health center of Tabriz city to receive a rabies vaccination. Ethics approval and consent to participateThe institutional ethical review board reviewed and approved the study protocol in Tabriz, Iran (IR.TBZMED.REC.1397.1096). All data have been anonymized and treated confidentially.Data collectionWe collected data per span (year) on the number of animal bite injuries. To collect the data, we used the following a structured data abstraction tool to extract the required data form recoded forms: age ; sex ; occupation ; biting place ; being stray (yes/ no); place of injury in human body ; puncture wounds ; animal status ; time of event . The dependent variable was: PEP initiation delay which was defined as the initiation of PEP more than 48 h after an animal bite. We looked into the time of injury and time to visit with the respondents. In the present study, PEP delay was defined as the initiation of PEP more than 48 h after a possible exposure to the rabies virus. The variable was coded according to: 0= \u201cprompt PEP\u201d, 1= \u201cdelay of initiation of PEP\u201d.Input variablesAfter cleaning and preparation of the data, the final dataset consisted of 3032 records. The outcome of interest was the delay more than 48 h in initiation of PEP which was assessed by the model based on several input variables presented in Construction of decision treeAs the number of instances in this study was enough, the decision tree was built with the holdout method in which data are randomly partitioned into two independent sets, a training set and a test set.We divided the original dataset into two parts using stratified random sampling based on the target variable, with the training dataset containing about 70%, and 30% of the participants as the testing dataset. The Gini impurity index was chosen as the attribute selection measure. Where pi is the probability that an example in D belongs to class Ci, and is estimated by [Ci, D|/|D|. The sum is computed over m classes. The Gini index considers a binary split for each variable. In the decision tree, the first variable (root) is the most important factor and variables far away from the root are the next important factors in classifying the data. For easy understanding, the decision tree can be converted to a set of If-Then rules by tracing the path from the root node to each terminal (leaf) node. All the variables in one path are considered as predictors (If part) and the class label of the leaf node is expected outcome (Then part). These rules are extracted just by top down tracing of the path and there is no rank or weight for the rules. For classifying a new person, we should start with the root node of the decision tree and moving along the path that the person belong it until the leaf node is reached. The decision about the person is determined based on value of leaf node which is usually positive or negative with a certain probability .In addition, missing values for numerical features were handled by setting their values to average value, and replacing the most frequent value for nominal features.Classification is the process of finding a model (or function) describing and distinguishing data classes on concepts using the model to predict object classes . ClassifWhere J is the number of classes or the target variables, \u03c0(j) is the prior probability for class j, p (j|m) is the probability that node m includes observations of class j, and Gini (m) is the Gini index, which indicates impurity in node m. The Gini index is 0, if all the observations in a single node belong to a unique class that displays the least impurity, and is equal to 1-1/i, if results in different classes in one node are of the same proportion. In this situation, the maximum tree that overfits the training data has been created. Reducing the complexity of the end tree and generating simpler trees, based on a cost-complexity algorithm, the tree will be \u201cpruned\u201d. In the CART method, the decision tree is getting bigger and more so until each terminal node has the same observations. Gini index the degree or probability of a particular variable being wrongly classified when it is randomly chosen.Statistical analysisMean \u00b1 standard deviation (SD) for continuous variables and frequencies (%) for categorical variables were used to demonstrate baseline characteristics of the participants. Factors associated with PEP treatment with dichotomized and categorical variables were tested using chi-square test as a univariate study.In total, 3032 animal bites were recorded through 2013-2019; with no human rabies cases. The mean age of subjects was 33.71\u00b1 18.50 years. Cases ranged in age from 1 to 91 years, and 2438 (80.4%) were males. Demographic and injury Characteristics of the study population, and the number of victims bitten by the animals are shown in According to the results of the chi-square test, the relative frequency of a delay of more than 48 h in females was higher than the males (12.9% vs. 8.5%) . Relative frequency of a delay of more of 48 h from carnivorous  was significantly less than others . There was a significant difference between the animal status and delay in initiation of PEP according to the chi-square test  to be more specific the relative frequency of delay was higher in cases that the biter animal is dead after bite., animal status and biting place, respectively [The Gini index, as an impurity function of the CART algorithm, showed that the most important variables for predicting the delay of PEP include: type of animal, genderectively .We developed a decision tree with instruction set (3032 records). The criteria for building the tree included minimum record number per node, the pruning process and attribute selection measures; minimum number represents a stopping condition for further data partitioning at decision nodes. Briefly, various decision trees with different \u2018minimum records\u2019 were built and the value of 50 was chosen which resulted in best performance. The Gini index was selected as measure of attribute selection, and the tree were kept unpruned. The overall classification accuracy was 89.5%, with 44.1% sensitivity and 92.3% specificity.According to If the biting animal is other animals, 20.5% will have a delay of more than 48 h.If the biter animal is a dog or cat and gender of the victim is female, 10.9% had a delay of more than 48 h.If the biting animal is a cat or dog and the gender of the victim is female, as well as biting place is rural area, 15.3% will have a delay of more than 48 h.If the biting animal is a cat or dog and gender of the victim is male, as well as animal status is escaped or dead, 9.1% will have a delay of more than 48 h.Post-exposure vaccination against rabies is essential for prevention of this fatal disease. Obviously, several essential factors in the proper implementation of PEP, are available. Many factors influence timely access to PEP and its administration. This study investigated the factors causing delay in receiving anti-rabies PEP.In our study, 8.5% of the victims did not receive timely PEP treatment. The WHO advises that immediate washing and flushing of wounds with soap and water for at least 15 minutes, or water alone, and disinfection with substances with anti-viral activity is essential after exposure to rabies virus . Taking The present study found a higher rate of delay among women compared to men victims of animal bite (11.4% compared to 7.8%). A study conducted in shiraz province, Iran , which hAs demonstrated in a previous study, some people may think that domestic animals are less dangerous than roaming ones . On the On the presence of watchdogs in most rural households in the study area, appropriate education programs should be provided for the teaching of behavioral skills in high-risk groups. Similarly, those who live far from health centers, and are in lower socioeconomic classes undergo longer delays in receiving PEP, which increases the risk of developing rabies .In our study, delay more than 48 h belonged to the rustic region. Because of development of the primary health care (PHC) in Iran in past decades, and availability of health houses and health centers in almost all villages, it seems that there is no physical barrier to access the care animal bite victims . It is rBased on various variables that affect the initiation of PEPbeing bitten by a carnivorous, being bitten in the rural places, and especially being a female. Development of educational programs for dog owners, especially in rural areas, and increasing public awareness may help us prevent the delay in initiation of PEP for animal bite victims. First, given the limitations of the cross-sectional study design, it is not possible to claim causal effect. Due to the study\u2019s retrospective design, authors were unable to collect other variables such as education level, past history of rabies vaccination, socioeconomic status, and other variables related to delay in receiving PEP. Possible recall bias by the animal bite victims in remembering the time of bite may result in information bias in calculation of delay of treatments.This study was supported by the Tabriz University of Medical sciences, Tabriz, Iran. And the fund number was 60198."}
{"text": "We investigated the prevalence of retinal vascular occlusion and intraocular bleeding and compare their risks in patients undergoing anticoagulant therapy, either with non-vitamin K-antagonist oral anticoagulants (NOAC) or warfarin. We performed a cohort study (January 2015 to April 2018) in 281,970 patients with nonvalvular atrial fibrillation (AF) using health claims in the nationwide database of the Health Insurance Review and Assessment service of Korea. A Cox-proportional hazard regression was used to calculate the hazard ratio (HR) for retinal vascular occlusion or intraocular bleeding. The HR of retinal vascular occlusion was estimated to 1.59  for NOAC users compared to that with warfarin users. Among the various types of NOACs, all NOACs showed higher risk of retinal vascular occlusion than did warfarin. For intraocular bleeding, the HR was estimated to be 0.86  for NOAC users compared with that with warfarin users. The risk of retinal vascular occlusion was higher in NOAC users than in warfarin users, while the risk of intraocular bleeding was lower with NOAC therapy. NOACs were not found to be as effective as warfarin for retinal vascular occlusion, but safe in terms of intraocular bleeding. Warfarin can effectively prevent stroke and systemic embolism in patients with AF but increases the incidence of major bleeding such as intracranial hemorrhage2. Non-vitamin K-antagonist oral anticoagulants (NOACs) have been recently approved for prevention of stroke in patients with non-valvular AF, and do not require anticoagulation monitoring3. Since NOACs are at least as effective and safe compared to warfarin, they have become essential for patients with non-valvular AF4.Non-valvular atrial fibrillation (AF) is a global health burden and the prevention of AF-related thromboembolic events is a major concern5; retinal vein occlusion (RVO) is associated with thromboembolism due to degeneration of vascular walls and compression or vasospasm6. Retinal vascular occlusion is not only associated with predisposing factors for atheroembolic diseases7, but also with the risk of stroke, myocardial infarction, and total mortality8.Retinal vascular occlusion is a major vascular disease of the retina that can lead to severe visual impairment. Retinal artery occlusion (RAO) results from retinal thromboembolic events originating in the ipsilateral carotid artery, aortic arch, or cardiac origin9. Hence, several studies on ocular diseases have examined the impact of anticoagulant therapy with NOAC, with a focus on ocular bleeding as a major safety issue11. While NOACs benefit patients with stroke and systemic thromboembolisms12, their efficacy on microvascular conditions, such as retinal vascular occlusion, requires further evaluation.In terms of the safety in patients taking anticoagulants, intraocular bleeding is not a life-threatening \u2018major\u2019 complication, but is critical and can lead to severe visual loss and decrease vision-related quality of life. Previous studies have reported that there is an increased risk of intraocular bleeding with warfarin or other oral antithrombotics, raising concerns over the safety of these medicationsThis study is to verify the efficacy and safety of NOACs in retinal disorders in terms of the incidence of retinal vascular occlusion and intraocular bleeding. We compared these incidences in NOAC and warfarin users by using a Korean cohort study.The patients were initially categorized according to anticoagulant treatment. Among patients with non-valvular AF, 93,691 used NOAC and 27,496 used warfarin , and lowest with apixaban (2.66).The average follow-up period was 2.68\u2009\u00b1\u20091.33 years for warfarin users and 1.24\u2009\u00b1\u20090.84 years for NOAC users. The baseline characteristics are listed in Table\u00a0P for interaction\u2009=\u20090.0017). Among the different types of NOAC, when compared with warfarin, the HR for retinal vascular occlusion was 1.49, 1.47, 1.66, and 1.64 for dabigatran, rivaroxaban, apixaban, and edoxaban, respectively  for warfarin users and 0.82%  for NOAC users. Person-years were taken into account as the follow-up period differed by patient, and the NOAC users showed higher incidence rate (IR) compared to warfarin users (IR 6.03 vs. 3.33 per 1000 person-years). The NOAC group had a higher risk of retinal vascular occlusion than the warfarin group (hazard ratio (HR), 1.61; 95% confidence interval (CI) 1.37\u20131.91), which was consistent after adjusting multiple covariates  but not for RAO with NOAC . These were also consistent after adjusting for multiple covariates . There were no differences for other retinal diseases, including age-related macular degeneration, choroidal neovascularization, and retinal vascular occlusion.In patients with underlying retinal vascular disorders related to intraocular bleeding, more patients with diabetic retinopathy were administered warfarin than NOAC . Among the different types of NOAC, the HR of intraocular bleeding was 0.84, 0.85, 0.80, and 0.81 for dabigatran, rivaroxaban, apixaban, and edoxaban respectively when compared with warfarin 13. A cohort study reported that the incidence of intraocular bleeding was less in NOAC (dabigatran and rivaroxaban) users compared to that of warfarin users9. Other meta-analyses also revealed a lower risk of intraocular bleeding with NOAC use15, suggesting the safety of NOAC for intraocular bleeding.NOACs are known to be non-inferior or even superior to warfarin for prevention of stroke and systemic embolism in patients with non-valvular AF, and also associated with lower rates of bleeding, especially life-threatening hemorrhage , so that NOACs may preserve hemostatic mechanisms by selectively targeting thrombin and not interfering tissue factor-VIIa complexes16. Warfarin has limitations related with the diet and drug interaction and the need for anticoagulation monitoring, and the pharmacokinetics of warfarin is less predictable due to the polymorphisms in the genes 19. Accordingly, the lower risk of bleeding with NOACs may be associated with the selective actions on coagulation pathway compared to warfarin3.Our study also showed that NOAC users have a lower association with intraocular bleeding than did warfarin users. The mechanism for lower bleeding risk is not fully understood, but several points suggested in studies on major bleeding might be applied. Warfarin inhibits vitamin K-dependent proteins , thereby suppressing production of factor VIIa and formation of tissue factor-VIIa complexes21. There are also cohort studies which have reported that retinal vascular occlusion increases the risk of stroke24. Interestingly, our study found that the risk of retinal vascular occlusion was higher in NOAC users than in warfarin users in Korea. Although this study did not investigate mechanisms associated with the higher risk of retinal vascular occlusion in NOAC users, the selective inhibition of specific coagulation factors by NOACs might be responsible4. NOACs are safe in terms of bleeding complications by preserving hemostatic mechanism3, while this would work the other way around as more retinal vascular occlusion in NOAC users. The lower trough level of NOACs compared to that of warfarin might be another factor for the inefficacy of NOACs25. It should be noted that patients with newly diagnosed retinal vascular occlusions are often referred to internists in clinical practice, who then evaluate systemic risk factors for future cardiovascular events and often prescribe anticoagulants26. However, there is a lack of high-quality evidence to support the routine use of anticoagulants or antithrombotic drugs for retinal vascular occlusions, and the benefits and risks of this therapy need to be considered in clinical settings27. Based on our findings, the appropriate anticoagulant should be selected in each patient based on the risk of intraocular bleeding and retinal vascular occlusions.Retinal vascular occlusion is often associated with cardiovascular disease and have many of the same risk factors28. On the other hand, the precipitating factors for RVO are more variable: vasospasm, inflammation, compression, and localized thrombosis6. Typical atherosclerosis risk factors are commonly associated with all types of RVO, while hyperviscosity and hypercoagulability are also related risk factors26. Second, RAO is relatively uncommon as compared to RVO20, which may affect statistical significance. Finally, the exact doses for anticoagulants were not investigated in this study. There is a tendency in Asian countries to use the lower doses of NOACs in clinical practice30, which may have affected the results of this study.The risk of RAO was comparable between the NOAC and warfarin groups, while the risk of RVO was higher in the NOAC group compared to the warfarin group. There are possible reasons for this difference in efficacy of NOACs, besides the pharmacokinetics of NOACs acting on specific factors in coagulation pathway. First, RAO is related with atherosclerotic thromboembolism similar to stroke, and retinal artery emboli are commonly associated with AF, hypertension, and coronary artery disease31. Most studies showed comparable major bleeding rate in patients without significant interaction with diabetes31, while there was also a report that diabetic patients showed a reduced rate of intracranial hemorrhage when treated with dabigatran than with warfarin32. The mechanisms for this lower risk of bleeding in diabetic patients are not clarified, but less diet and drug interactions with NOACs might explain one of possible reasons for the safety of NOACs in diabetic patients3. In terms of retinal vascular occlusion, female patients showed a higher risk of retinal vascular occlusion with NOACs than male patients. Our study did not investigate the dosage by sex, while more female patients are known to be prescribed lower dose of NOACs in Korea30. This may result in a higher risk of retinal vascular occlusion with lower efficacy. Similarly, off-label low dose of NOAC was associated with increased risk of cerebral ischemic events33, and these tendencies of ischemic event need further investigation along with the gender difference.There were some interesting points in subgroup analyses. Diabetic patients had a remarkably lower risk of intraocular bleeding with NOACs than the opposite group. Diabetes is a well-known risk factor for ischemic stroke, while there were controversies on hemorrhagic risk associated with NOAC treatment in diabetic patients with AFi.e. intraocular bleeding, all the NOACs showed a lower risk of bleeding than did warfarin. This is similar to previous cohort studies which reported a reduced risk for intraocular bleeding15. Few guidelines have addressed the issue of anticoagulant use for treatment of retinal vascular occlusions and the possible risk for intraocular bleeding26. No specific medication has been established to directly improve perfusion, and current ophthalmic treatments focus on the complications of retinal vascular occlusions such as macular edema, ocular neovascularization, and intraocular bleeding7. All NOACs have a merit in terms of safety for intraocular bleeding; however, efficacy for the prevention of retinal vascular occlusion was not clarified in this study. As the incidence and prevalence of AF increases with advancing age, further studies are needed to investigate the efficacy of NOACs for preventing microvascular disease.There was no definite superiority among the different type of NOACs. All the NOACs showed a higher risk for retinal vascular occlusion and a comparable risk of intraocular bleeding than did warfarin. Regarding ocular safety in the present study, 29. We could not classify the patients according to dose as many patients changed the dose during the study period. Third, detailed information on patients\u2019 history of socioeconomic status, smoking, or alcohol consumption, and laboratory profiles including international normalized ratio (INR) of prothrombin time were not available from the HIRA database. Some of the patients on warfarin could be in supratherapeutic range of INR, but this is less likely as the proportion of warfarin users in supratherapeutic range is lower than those in subtherapeutic range in Asia34. Although we adjusted for various confounding factors using diagnostic and procedure codes from the HIRA database, more studies in real clinical practice are needed to confirm the effects of NOACs in retinal microvascular diseases. Lastly, the findings in this study need cautious interpretation due to the retrospective nature. Although the association of NOACs and higher incidence rate of retinal vascular occlusion was found in this study, further studies prospective in nature are needed to verify this association.This cohort study has some limitations. First, the presence of retinal vascular occlusion was defined by the presence of diagnostic codes from HIRA database. Although the diagnostic codes are mandatory for any patient, the accuracy of diagnoses was not confirmed by the medical records. However, this limitation might affect both NOAC and warfarin groups and therefore, a one-sided application might be prevented. Second, we could not investigate the difference between standard-dose NOAC users and low-dose NOAC users. As mentioned above, Asia shows a tendency for prescribing lower doses than do Western countriesIn conclusion, the present study showed that use of a NOAC was not superior to warfarin in terms of retinal vascular occlusions, unlike its effect on the treatment of stroke or systemic embolism. However, NOACs were safe in terms of intraocular bleeding. Further studies in clinical practice may help to identify the most appropriate anticoagulants for patients with microvascular diseases.This retrospective cohort study protocol was reviewed and approved by the Institutional Review Board of Ajou University Hospital (AJIRB-MED-EXP-18-380). The requirement for informed consent was waived by the Institutional Review Board of Ajou University Hospital as the data in this database were de-identified. All methods were performed in accordance with relevant guidelines and regulations.th revision (ICD-10). The inclusion criteria were: (i) patients aged \u226520 years, (ii) \u2265 1 criteria for non-valvular AF  in the primary or within 3rd order of secondary diagnosis, and (iii) \u22651 prescription of NOACs or warfarin between January 2015 and April 2018. The exclusion criteria were patients who had: (i) been diagnosed with AF in 2014 to select the patients who were newly diagnosed with AF from 2015, (ii) both warfarin and NOAC during the study period, (iii) mitral stenosis and/or prosthetic heart valve, (iv) prior diagnosis of retinal vascular occlusion, and (v) aged <20 years at the time of AF diagnosis. Those who were diagnosed with AF prior to 2014 but received a prescription between January 2015 and April 2018 were excluded. These criteria are summarized in Fig.\u00a0The Health Insurance Review and Assessment (HIRA) service reviews all of the health claims in Korea, including those submitted through the Korean National Health Insurance service which covers 97% of the population. Diagnoses are coded using the International Classification of Diseases, 10Each participant\u2019s index date was set to the date of earliest prescription. The follow-up for each event extended from the index date to the earliest incidence of retinal vascular occlusion, or intraocular bleeding, or the end of the observation date , if events did not occur.The presence of retinal vascular occlusion was determined on the basis of diagnostic codes of retinal vascular occlusions in the primary or within 3rd order of secondary diagnosis, including RAO , RVO , and other retinal vascular occlusions . Similarly, the presence of intraocular bleeding was determined using the diagnostic codes for non-traumatic hyphema , vitreous hemorrhage , retinal hemorrhage , and choroidal hemorrhage (H31.3). For retinal disorders leading to intraocular bleeding, the presence of the previously stated retinal vascular diseases was investigated. The NOACs included those available in Korea and covered by the national insurance service such as dabigatran, rivaroxaban, apixaban, and edoxaban.2DS2-VASc score, and calendar index year. The CHA2DS2-VASc score, which is used in guidelines for stroke prevention in AF implying comorbid diseases , was used in this study including AF patients35. A list of variables with corresponding ICD-10 codes, procedures, operation codes, and anatomical therapeutic chemical codes is provided in Supplementary Table\u00a0The following covariates were included for baseline adjustments to minimize confounding factors: age at diagnosis of AF, sex, hypertension, dyslipidemia, chronic kidney disease, diabetes mellitus, coronary heart disease, stroke, venous thromboembolism, CHAt-test for continuous variables.The baseline characteristics were summarized and compared for the patients with NOAC and warfarin using a chi-square test of homogeneity for categorical variables and a two sample 2DS2-VASc score, calendar index year, and comorbidities . The time from initiation of NOAC or warfarin to primary events was assessed using the Kaplan-Meier survival curve and compared using the log-rank test. Additionally, the unadjusted cumulative incidence was calculated according to the type of medication.The IRs were stratified by each medication and estimated with the number of retinal vascular occlusion or intraocular bleeding events per 1,000 person-years, and the 95% CI was calculated by the Mid-P exact test. The cox proportional-hazards regression model was used to calculate the HR and 95% CI. The HR with 95% CI was presented with separate statistical models; (i) unadjusted, (ii) adjusted with age and sex, and (iii) adjusted with age, sex, CHAP value of <0.05 was considered significant.In the secondary analysis, the same analysis was performed with different types of NOACs, such as dabigatran, rivaroxaban, apixaban, and edoxaban, and compared with warfarin. Those who switched medications (within the NOAC group) at any point in the time were excluded in the secondary analysis. Additionally, the unadjusted HR was presented by subgroups according to age \u226565, sex, diabetes, and stroke. All statistical analyses were performed using SAS software . A Supplementary information."}
{"text": "As neuronal activity can be achieved using non-invasive measures, it may be of interest to utilize the innate ability of neuronal activity to instruct myelination as a novel strategy for myelin repair in demyelinating disorders such as multiple sclerosis (MS). Preclinical studies indicate that stimulation of neuronal activity in demyelinated lesions indeed has the potential to improve remyelination and that the stimulation paradigm is an important determinant of success. However, future studies will need to reveal the most efficient stimulation protocols as well as the biological mechanisms implicated. Nonetheless, clinical studies have already explored non-invasive brain stimulation as an attractive therapeutic approach that ameliorates MS symptomatology. However, whether symptom improvement is due to improved myelin repair remains unclear. In this mini-review, we discuss the neurobiological basis and potential of enhancing neuronal activity as a novel therapeutic approach in MS.Enhanced neuronal activity in the healthy brain can induce  The most accepted mechanism of remyelination consists of both recruitment and proliferation of oligodendrocyte precursor cells (OPCs) as well as the differentiation of these new OPCs into mature myelinating oligodendrocytes (OLs) . Howeverin vivo, it became clear that activation of neurons can stimulate the proliferation of OPCs as well as their differentiation into myelinating OLs. For example, optogenetic stimulation of layer V projection neurons leads to increased OPC proliferation and differentiation and an increase in myelin thickness (in vivo has different effects on OPC proliferation and differentiation (Initial studies in the 90\u2019s demonstrated that neurons communicate with OL lineage cells at different stages of OL maturation, thereby modulating the myelination process . From rehickness . Moreovehickness . When pahickness . Notablyhickness . In thishickness . Taken tntiation . Indirecntiation . This isntiation . It shountiation . As suchvia mechanotransduction (de novo myelination resulting in behavioral changes. Consequently, this makes stimulation of neuronal activity an interesting approach for myelin repair in demyelinating disorders such as MS.How electrically active axons communicate with OPCs and OLs to promote myelination remains unclear. However, there are multiple synaptic and non-synaptic signaling mechanisms that may play a role . Electrisduction . Taken t+ channel blocker in demyelinated lesions induced by ethidium bromide showed a decreased differentiation of OPCs into myelinating OLs, an increased OL apoptosis and a reduced remyelination (in vivo (While activity-driven myelination is accepted as a key mechanism in the CNS, literature on the effect of neuronal activity on myelin repair is still scarce. Nevertheless, a limited number of studies have reported on the potency of manipulating neuronal activity as a remyelination strategy in animal models. The intracerebral infusion of AMPA/kainate receptor antagonists and a voltage-dependent Nalination . These rlination . Several(in vivo . Distinc(in vivo . This stRecent studies in animal models have addressed the effects of indirect methods of neuronal stimulation on remyelination. As indirect neuronal stimulation methods such as TMS, tDCS and behavioral training are readily applicable in the clinic, it is worthwhile to investigate different stimulation methods in rodents. Sixty Hz electromagnetic stimulation increases remyelination in toxin demyelinated lesions of the rat corpus callosum  and low Behavioral paradigms recruiting remyelinating brain regions might be able to accelerate the remyelination process, likely through increased neuronal activity. A recent study reported increased neuronal firing rates in the motor cortex following cuprizone demyelination that recovered to normal during the same period in which remyelination was completed, suggesting a correlation between the level of neuronal activity and remyelination . Behavio2+ and Ca2+ channel blocking agents have been shown to reduce damage to neurons in demyelinating lesions by inhibiting a neuronal cell membrane depolarizing mechanism (via neuronal activity (Although neuronal activity can be enhanced in a controlled manner to limit deleterious effects such as excitotoxicity, it cannot be excluded that apart from benefits for remyelination, enhanced neuronal activity may negatively affect the demyelinated neurons themselves. Indeed, Naechanism . Furtherechanism . Future activity .in vitro study has shown that magnetic field stimulation induces calcium influx in OPCs and enhances OPC differentiation and OL myelination (per se may also induce direct effects on oligodendroglia in the absence of neuronal activity. Taken together, these findings suggest that complex mechanisms implicating calcium signals in OL lineage cells may be involved in neuronal activity-induced remyelination and that further research is necessary to unravel the associated signaling pathways.Although emerging lines of evidence point to a role of neuronal activity in modulating oligodendroglia dynamics in lesions, few papers have investigated the signaling mechanisms that underlie the communication between electrically active axons and oligodendroglia during remyelination. Since the frequency of calcium transients is increased during remyelination , it is llination , suggestAs emerging evidence suggests that increased neuronal activity can aid remyelination, it is worthwhile to examine studies in which MS patients with demyelinating lesions undergo neuronal stimulation. Deep brain stimulation is the only direct stimulation method that has been tried in MS patients. Several studies have shown that thalamic deep brain stimulation significantly reduces tremor and improves quality of life in MS patients . HoweverOther than non-invasive brain stimulation, exercise and behavioral paradigms have also been shown to positively affect MS symptoms and their effects might be mediated by enhanced neuronal activity. For example, 8 weeks of high intensity exercise improve clinical outcome  and bothde novo myelination in the healthy brain. The facts that neuronal activity can initiate myelination and that neuronal stimulation can be achieved relatively easily make activity-dependent myelination an attractive strategy for myelin repair in demyelinating disorders such as MS. Research into neuronal activity-dependent myelin repair is still scarce, however, the few in vivo preclinical studies that have been conducted suggest that enhanced neuronal activity in a demyelinated lesion indeed has the potential to improve remyelination (see Neuronal activity-dependent myelination can take place regardless of neuronal identity, but the effects of neuronal activity on oligodendroglial cells likely depend on the cumulative pattern of activity of a neuronal network. This means that both direct stimulation of neurons by for example optogenetics and indirect stimulation via non-invasive methods such as TMS and behavioral training can achieve tion see . In rodetion see .It is known that electrically active axons can communicate with oligodendroglia cells via synaptic and non-synaptic sites involving a number of receptors and messenger molecules that often lead to a depolarization of oligodendroglia and subsequent intracellular calcium signals. The nature of the communication from neurons to oligodendroglia and the frequency and amplitude of intracellular calcium increases in oligodendroglia are thought to determine the OL response to neuronal activity. Future studies need to reveal the molecular pathways involved in, and the calcium response characteristics of oligodendroglia to neuronal electrical activity.In conclusion, the stimulation of neuronal activity is a promising strategy for myelin repair in demyelinating disorders such as MS and future research into the exact neurobiological mechanisms as well as clinical evaluation of changes in the brain after neuronal stimulation in patients will aid the development of this novel therapeutic approach to myelin repair.DM and MCA wrote and corrected the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
{"text": "Ichthyosis is a heterogeneous group of Mendelian cornification disorders that includes syndromic and non-syndromic forms. Autosomal Recessive Congenital Ichthyosis (ARCI) and Ichthyosis Linearis Circumflexa (ILC) belong to non-syndromic forms. Syndromic ichthyosis is rather a large group of heterogeneous diseases. Overlapping phenotypes and genotypes between these disorders is a major characteristic. Therefore, determining the specific genetic background for each form would be necessary.A total of 11 Tunisian patients with non-syndromic (8 with ARCI and 2 with ILC) and autosomal syndromic ichthyosis (1 patient) were screened by a custom Agilent HaloPlex multi-gene panel and the segregation of causative mutations were analyzed in available family members.NIPAL4) and 4 already reported mutations . Yellowish severe keratoderma was found to be associated with NIPAL4 variations and brachydactyly to TGM1 mutations. Two novel variations (c.5898G\u2009>\u2009C and c.2855A\u2009>\u2009G in ABCA12) seemed to be features of ILC. Delexon13 in CERS3 was reported in a patient with syndromic ichthyosis.Clinical and molecular characterization, leading to genotype\u2013phenotype correlation in 11 Tunisian patients was carried out. Overall, we identified 8 mutations in 5 genes. Thus, in patients with ARCI, we identified a novel (c.118T\u2009>\u2009C in Our study further extends the spectrum of mutations involved in ichthyosis as well as clinical features that could help directing genetic investigation. Ichthyosis is a heterogeneous group of Mendelian cornification disorders that includes syndromic and non-syndromic forms. Autosomal Recessive Congenital Ichthyosis (ARCI) and Ichthyosis Linearis Circumflexa (ILC) belong to non-syndromic forms. ARCI is characterized by abnormal desquamation over the whole body due to a dysfunctional skin permeability barrier and altered lipid composition. It is a rare skin disease affecting around 1 in 200,000 individuals , AL 607800) , SDR9C7  609769) , PNPLA1  604125) , CYP4F222 MIM: 6195 [14], S3 MIM: 6276 [15], 603741) , LIPN (M 613924) , SLC27A4 604194) , and ST1A major characteristic of ARCI is partially overlapping phenotypes and genotypes between LI and CIE  Table 11. TherefThe current study focused on 11 newly recruited Tunisian patients affected with either non-syndromic ichthyosis forms  or autosomal syndromic ichthyosis (1 patient). We tried to highlight some clinical features related to specific genes that could facilitate genetic diagnosis. We reported three novel missense mutations in two different consanguineous families, as well as 5 known disease-causing variants in 4 different genes in the remaining subjects. In silico analysis was performed in order to predict the effect of novel mutations on each corresponding protein level.PatientsA total of 11 patients with different ichthyosis phenotypes were enrolled. Among them, five belonged to two consanguineous families: A  and F (F1 and F2). The other six cases  were sporadic. Ten out of 11 patients were affected with non-syndromic ichthyosis . Only one patient suffered from autosomal syndromic ichthyosis.2.DNA extractionThe study protocol conforms to the approval of the local ethical committee of CHU Hedi Chaker, Sfax, in compliance with the Declaration of Helsinki. Written informed consent was obtained from all study participants or the parents for minor children.3.Next generation sequencingTotal genomic DNA was extracted from patients and available members\u2019 family using the phenol\u2013chloroform standard procedure . The DNA4.Sanger sequencingNext generation sequencing (NGS) was performed using a custom Agilent HaloPlex multi-gene panel with 32 ichthyosis linked genes comprising a target size of\u2009~\u200964 kbp. The average sequencing depth was 420.3x. A rate of 97.6% of the target position had at least 20\u2009\u00d7\u2009in depth. Sequencing was performed on an Illumina MiSeq sequencer using MiSeq reagent kit v2 (2\u2009\u00d7\u2009150\u00a0bp). Reads were aligned using bwa (v. 0.7.17)  against PCR amplification was performed using a thermal cycler GeneAmp PCR System 2720 , in a final volume of 15\u00a0\u03bcl using 100\u00a0ng DNA, 10\u00a0\u03bcM of each primer, 2\u00a0mM dNTP, 10\u2009\u00d7\u2009PCR buffer, 1U of Taq DNA polymerase (Qiagen) and Q-solution. Primers were generated with primer3 and corresponding sequences are mentioned in Table NIPAL4 (ENST00000311946.7) and ABCA12 (ENSG00000141527.18) by BLAST online software.5.Bioinformatic predictionsPCR products were sequenced using Applied biosystem . Sequences were analyzed, using Bio Edit Program and compared with wild type sequences: Protein sequence alignment across species was performed using CLUSTALW and PolyPhen2 software. The potential functional impact of p.(M63T) mutation on NIPA4 and ABCA12 proteins was predicted using PolyPhen2, SIFT, Mutation Taster, Panther and CADD software.The current study included 11 ichthyosis patients with 3 phenotypes. Among them, five belonged to two consanguineous families: A  and F (F1 and F2). The other six cases  were sporadic  displayed at the age of 3\u00a0years larger scales on the forehead and limbs, while the scales were fine and brown\u00a0on the trunk on an erythematous skin. However, their first cousin (A7), who was affected with LI demonstrated darker, thicker and plate-like scales all over the body, without erythema Fig.\u00a0a. PatienThe F family included 2 patients F1 and F2). The proband F1, aged 35\u00a0years, had a similar phenotype to her 4\u00a0year-old daughter (F2). Both patients presented multiple polycyclic erythematous and squamous plaques, with brownish fine scales  presented mild erythematous skin. Scales were white and fine on the face, the trunk and the upper members, while they were thicker and larger on the knees and the legs. White scales were also located on the dorsal aspect of the hands and feet and in the folds. Mild palmoplantar keratoderma and brachydactyly were also observed. There was no ectropion and ears were normal. Interestingly, ophthalmological examination revealed bilateral myopia and microspherophakia.NIPAL4, TGM1, CYP4F22, ABCA12 and CERS3)  in exon 1, which substitutes a methionine to a threonine . It was reported in family A affected members , with two ARCI phenotypes (CIE and LI). To confirm the segregation of this mutation and its association with ARCI, Sanger sequencing was performed in all available family members and control population. This mutation was in heterozygous state for parents and absent in unaffected members and control individuals . It was identified in a CIE patient  who presented a mild erythema of the skin, with small white scales sparing the folds.The second disease-causing variant  occurred in exon4 of ABCA12 gene in family F (F1 and F2 patients) with ILC  were identified (data not shown).Two novel missense mutations were detected at the homozygous state: c.5898G\u2009>\u2009C (ENST00000389661.4)  and c.2855A\u2009>\u2009G (ENST00000389661.4)  in TGM1 with two different homozygous nonsense germline mutations (Table TGM1 gene was c.1042C\u2009>\u2009T (ENST00000206765.6) located in exon 7  and detected in patient Y1 aged 19\u00a0years, who showed LI phenotype.In three patients with non-syndromic ichthyosis , the involved gene was ns Table . InteresCYP4F22 gene. This variation was also reported only in heterozygous state in gnomAD database.K1 patient with LI had rather a single homozygous missense mutation  in exon 8 which substitutes arginine to tryptophan  in CERS3 gene in a 15-year old patient (E1) affected with syndromic ichthyosis (CIE with ocular defect).We also identified a homozygous deletion in exon 13 of NIPAL4 gene and p.Glu1966Asp, p.Tyr952Cys in ABCA12 gene. We have to mention that all new variants were not reported before in the gnomAD database.As mentioned in Table Moreover, multiple alignments of corresponding proteins showed that these residues were located within a highly conserved region Fig.\u00a0b, d.NIPAL4, TGM1, CYP4F22, ABCA12 and CERS3). Among these, three were novel . Using bioinformatic tools, these variations were predicted as pathogenic and causing a structural protein modification suggesting their involvement in ichthyosis. Moreover, already described mutations were pathogenic according to ClinVar database, except for c.844 C\u2009>\u2009T in CYP4F22 gene which was classified as an uncertain significant variant. However, this variant was associated to both LI and CIE phenotypes [We collected detailed phenotypic data from 11 Tunisian patients with different ichthyosis forms. In order to identify causative gene(s) mutation(s), we used a custom multi-gene panel. One of the most important aspects of this panel is its ability to be easily upgradable in view of novel discoveries. Moreover, compared with whole exome sequencing, analysis of the multigene panel is easier and faster. Overall, we identified 8 disease-causing variants in 5 different genes  with two missense mutations (c.534A\u2009>\u2009C and c.118T\u2009>\u2009C). This discordance could be explained by the small number of patients investigated in our study.Unlike ARCI series in literature, where ble gene \u201333, in oABCA12 gene. These variations and clinical related aspects have never been reported before. In fact, this gene is involved in either HI phenotype [SPINK5 gene [SPINK5 gene, neither in genes involved in erythrokeratoderma variabilis (EKV) were found in ILC patients (data not shown), confirming then different etiologies. Recently, Leersum et al. [ABCA12 mosaicism. This particular form of ichthyosis was explained by the combination of a germline mutation and an acquired postzygotic mutation in this gene. ABCA12 gene was reported also in a keratosis pilaris with missense mutation and in Nevus comedonicus with a high protein expression level in the sebaceous gland without any variation in ABCA12 coding region [Phenotype-genotype correlation was one of the major objectives of the current investigation. Besides finding new variations in genes causing ichthyosis, we tried to highlight novel clinical characteristics associated with particular genotypes. In this context, a particular form of non-syndromic ichthyosis (ILC) with multiple localized polycyclic erythematous and squamous plaques and brownish fine scales was found in patients carrying two novel missense mutations in henotype , with hohenotype \u201337. On tNK5 gene . As cleam et al.  also repg region .NIPAL4 mutations presented with a phenotype of non-syndromic CIE. Concerning patients with TGM1 mutations, 66.67% of them showed a phenotype of LI. These observations confirmed other studies showing that NIPAL4 mutations were usually associated with a moderate phenotype with fine grey/white scales, while TGM1 mutations were associated with a more severe phenotype with darker, adherent and plate-like scales [Otherwise, our investigation showed that 75% of our patients with e scales .TGM1 gene. This phenotype with \u2018long palm/short fingers\u2019 appearance was also reported in two Tunisian patients with LI carrying TGM1 mutations [TGM1 mutations. To the best of our knowledge, this phenotype, noted in E1 patient also, has never been reported before in ARCI patients with CERS3 mutations. We suggest that this symptom may be caused by the deletion of ADAMTS17 gene [CERS3, complete sequence of non-coding RNA FLJ42289 and the first three exons of ADAMTS17) was reported in three consanguineous Tunisian families affected with ichthyosis associated with ocular, cardiac and skeletal anomalies [Our findings showed that some clinical features were associated with particular genotypes Table . Thus; wutations . Even thS17 gene . Indeed,nomalies . Since Enomalies . In ordeTGM1 (3/7), NIPAL4 (3/7), or CYP4F22 (1/7) genes. In the literature, this clinical feature was significantly associated with TGM1 [NIPAL4 mutations with varying frequencies (28\u201373%) [ABCA12, CERS3, ALOX12B, ALOXE3, CYP4F22, lipase N, PNPLA1 and SDR9C7 genes [In our cohort, 7 patients were born as collodion babies and were mutated either in ith TGM1 , and NIP(28\u201373%) , 45. ButC7 genes , 47.TGM1 mutations, and 50% of those with NIPAL4 mutations. This finding was consistent with literature data and confirmed the previous observation that ARCI patients bearing TGM1 mutations were more likely to develop bilateral ectropion [Ectropion was noted in all studied patients with ctropion .NIPAL4 variation and in the patient with CYP4F22 variation. Although reported in the literature [NIPAL4 and CYP4F22 genes.In our study, skin folds were spared in 2 patients with terature  as a preNIPAL4, TGM1, CYP4F22 mutations and CERS3 deletion, but with varying severity. Thus, in patients with p.Met63Thr NIPAL4 variation PPK was either moderate or absent. However, PPK was rather yellowish and severe in patients with p.Glu178 Asp NIPAL4 mutation as reported in the literature. Indeed, yellowish PPK has been reported in NIPAL4 series and, to the best of our knowledge, never associated with other ARCI genes [NIPAL4 mutation in ARCI patients. In addition, patient C1 showed a constricting band around his right fifth finger, suggestive of pseudoainhum  and alopecia in one patient (M1) carrying CI forms . AlopeciTGM1 gene was found in both CIE (M1 patient) and LI (B1 patient), suggesting the effect of a modifier gene as it was already reported [FLG gene coding the filaggrin protein. We identified 44 missense polymorphisms in M1 not present in B1 (data not shown). Prediction effect of these polymorphisms showed that the majority were benign.Nevertheless, we also revealed that diverse phenotypes were caused by the same gene with an identical mutation. Notably, c.788G\u2009>\u2009A in reported . In an aABCA12 gene responsible for ILC, a rare clinical form of ichthyosis, as well as novel clinical characteristics associated with particular genotypes.To summarize, we report three novel mutations, two of which were located in TGM1 mutations and associated to deletion of ADAMTS17 gene associated to deletion of CERS3 exon 13.Phenotypic-genotypic correlation suggests that brachydactyly could be related to NIPAL4 and ABCA12 genes, we added new insights to the already reported particular phenotypes linked to specific genes. The involvement of such genes in a particular ARCI form remains discussed. In order to better explain its phenotype heterogeneity, investigation of whole genome is necessary to search for responsible modifier genes.Besides the new reported variations in http://bioinfo.ut.ee/primer3-0.4.0/http://www.ncbi.nlm.nih.gov/blast/https://www.genome.jp/tools-bin/clustalwhttp://genetics.bwh.harvard.edu/pph2/https://sift.bii.a-star.edu.sg/http://www.mutationtaster.org/http://www.pantherdb.org/tools/index.jsphttps://cadd.gs.washington.edu/snv"}
{"text": "Iodine status, including Iodine Deficiency (ID) of the children aged 12\u201359 months of Jaffna District, Sri Lanka, have never been studied. This study thus aimed to assess ID among children aged 12\u201359 months by monitoring the Urinary Iodine Concentrations (UIC), the prevalence of goitre, and the factors causing ID.A cross-sectional study was conducted among 846 children aged 12\u201359 months in Jaffna District, Sri Lanka. Sociodemographic characteristics and other factors were collected using an interviewer-administered questionnaire. Dietary pattern of children was obtained using semi-quantitative food frequency questionnaire. We performed urinary iodine estimation and physical examinations to detect the goitre, according to the World Health Organization criteria. A multivariate logistic linear regression model was used to identify the factors that causing ID.The median UIC was 146.4 \u03bcg/L (interquartile range = 112.6\u2013185.3 \u03bcg/L). Based on the UIC (<100 \u03bcg/L), 17.8% had ID, of which 15.7% and 2.1% had mild and moderate ID. The mean consumption of iodine from food was 128.7 (\u00b120.2) \u03bcg/day. Gender variation had no influence on ID (p>0.05). Median UIC was significantly associated with living area, wealth status, type of drinking water, and method of iodized salt usage. A higher percentage of ID was significantly associated with younger age [AOR 2.32 (95% CI: 1.31\u20134.10)], urban area [AOR 1.94 (95% CI 1.27\u20132.96)], inland regions [AOR 3.20 (95% CI 1.85\u20135.55)], improper method of iodized salt usage [AOR 3.63 (95% CI: 1.38\u20139.56)], and low consumption of iodine-containing foods. The neck palpation revealed that only three children had goitre (0.4%).This study revealed that high ID among the children in Jaffna children was due to improper usage of iodized salt, even though the iodized salt is freely available in the region, living area, and age, while the prevalence of goitre was not significantly identified as a public health problem. Deficiency of essential micronutrients, including iodine, has substantial impacts on the health and development of growing children \u20133. IodinID in a geographic area is caused by low iodine content of soil, water or crops . ID, incIodine status, including prevalence of goitre among the children less than five years and the causative factors of ID have still not been reported from Jaffna District, Sri Lanka. Hence, the children from Jaffna District were selected to find the iodine status, including ID and prevalence of goitre, and the factors contributing to ID were studied.All the chemicals used in this research were of analytical grade.2, and a coastline of 160 km  were selected as Secondary Sampling Units (SSU). A total of 10 households were selected randomly from an SSU. If more than a child resides in a house, a child was selected based on his or her recent birthday at the date of data collection.Age, sex, and birth weight of each child were obtained from the growth chart book, Child Health Development Record (CHDR). The weight of the child was measured with lightweight cloth by using an electronic weighing scale (SECA 811) with an accuracy of \u00b1100g. The height of the child was measured with a portable stadiometer (SECA 213) and Infantometer (SECA 417) with an accuracy of \u00b10.5 cm according to standard World Health Organization (WHO)\u2019s procedures .Information on the socio-demographic and economic characteristics, drinking water source, and information regarding usage of iodized salt of the selected participants were obtained by a structured interviewer-administered questionnaire. The details of households that fall into the category of urban or rural areas based on the administrative divisions of Jaffna District Secretariat and coastal or inland regions based on the households within the coastal divisions of Grama Niladhari divisions were obtained. A spot morning urine sample of 10mL was collected after first voiding between 08:00 and 12:00 using a sterile wide-mouth plastic urine container with a screw cap covered with black paper. The containers were immediately transferred to a cool box and transported to Biochemistry Research Laboratory, Faculty of Medicine, University of Jaffna for iodine estimation. Collected urine samples were kept at 4\u00b0C and analysed within 24 hours.Training to data collectors (pre-intern doctors) was given by principal investigator and a community physician. The training mainly focused on the study\u2019s objective, the technique of interview, administration of questionnaires, collection, storage, and transport of urine samples, and maintaining ethical issues. The questionnaire prepared in English was translated into the native language, Tamil and then back-translated to English to ensure consistency of translation. A pilot study was carried out to pre-test the questions. During the pre-test, the questionnaire was evaluated for suitability and applicability during the interview between parents and data collectors. All questionnaires were regularly monitored for completeness and consistency by the research supervisors. The overall research activities were coordinated by principal investigator.The principle of Sandell-Kolthoff reaction was used to determine the iodine concentration in urine samples ,29. BrieClinical examination of neck for goitre was performed for all children described by Smith et al. (1990) . Goitre TM) , food products of rice flour [67.75 (\u00b128.70) g], formula milk [8.58 (\u00b15.21) g], and a variety of legumes daily. They consumed other main foods, including fish , egg , meat , and cow\u2019s milk  per day. The mean consumption of iodine from food was 128.72 (\u00b120.18) \u03bcg/day, which was higher than the World Health Organization\u2019s recommendation [0.82 mL] Among the children who consumed iodine-containing nutrient supplements (n = 796), the mean consumption of iodine from nutrient supplements was 10.77 (\u00b17.6) \u03bcg/day. The mean consumption of total iodine by the study population was 139.5 (\u00b122.12) \u03bcg/day (128.72 \u03bcg/day from food and 10.77 \u03bcg/day from nutrient supplements). The mean iodine consumption from nutrient supplementation was not significantly different between coastal [9.98 (\u00b15.66) \u03bcg/day] and inland areas [11.05 (\u00b18.17) \u03bcg/day]. The mean total iodine consumption (from foods and supplements) of coastal children [146.43 (\u00b118.33) \u03bcg/day] was significantly higher than the children who live in inland areas of the District [136.93 (\u00b122.71) \u03bcg/day] (p\u22640.0001). Children who had ID consumed mean of 10.46 (\u00b17.6) \u03bcg/day iodine from supplements, whereas mean of 10.83 (\u00b17.6) \u03bcg/day iodine by non-iodine deficient children (p>0.05). Mean iodine consumption from both food and nutrient supplements of iodine deficit children [123.56 (\u00b120.47) \u03bcg/day] was lower than the non-iodine deficit children [142.95 (\u00b120.79) \u03bcg/day] (p\u22640.001).Out of 846 children screened for goitre, only 3 (0.4%) had goitre. On the basis of WHO criteria, among those three children  who had goitre, two children had grade 1, and a child had grade 2 goitre . HoweverIn contrast, 12%, 5.6%, and 0.6% of children consumed goitrogenic substances containing foods, namely manioc (cassava), cabbage, and sweet potato, respectively. The ID was significantly high (28.3%) in children who consumed goitrogenic substances containing foods than the children who consumed non-goitrogenic substances containing foods (15.6%). Children who consumed goitrogenic substances containing foods had the median UIC of 137.9 \u03bcg/L than that of children who consumed non-goitrogenic substances containing foods (151.2 \u03bcg/L) (p\u22640.05). A child with goitre was from coastal areas and consumed seafood predominantly. Only a child affected with goitre consumed cassava, while the other two children did not consume the goitrogenic foods.In the present study, the ID, prevalence of goitre and factors influencing ID, including social determinants and dietary patterns in Jaffna children aged 12 to 59 months, were evaluated. This study demonstrated that the median UIC of children of 12 to 59 months was within the normal range (146.4 \u03bcg/L), between 100 \u03bcg/L and 200 \u03bcg/L ,21. In aWe noticed that 22.5%, 18.8%, 16.7%, and 12.4% of children aged 12\u201323 months, 24\u201335 months, 36\u201347 months, and 48\u201359 months, respectively, had ID. The increased risk of ID among children of 12\u201323 months was more when compared with those of 47\u201359 months. This was supported by a previous study . It was On the other hand, the studies among children aged 6\u201312 years revealed that the older children had a higher risk of getting ID than those of younger ages ,48. The Females aged 6\u201312 years had higher median UIC than that of male counterparts . We founThe weight and height of the children were not significantly affected by iodine-deficient children compared to non-iodine deficient children. Morales-Su\u00e1rez-Varela et al. (2018)  observedThe children from rural areas had a higher median UIC and a lower ID than those of children in urban areas. The present study was consistent with previous studies ,53. FurtThe income and family type of the households were not associated with iodine status. V\u00f6lzke et al. (2013)  reportedThis study also revealed that utilization of iodized salt to the food preparation by mothers was 100%, which was an ideal value and was higher than the WHO recommendation (>90%) . HoweverEven though the mean consumption of iodine from the food and supplementation was higher than the WHO recommendation of \u226590 \u03bcg/day , 14 chilID in a population is a public health problem if the total goitre rate is more than 5% . Further-) and is entirely absorbed via the small intestine. However, two-third of the ingested iodine is excreted by the kidney, and the remaining one-third is taken by the thyroid gland [Three children with goitre had excreted the normal level of iodine in their urine [185.74 \u03bcg/L]. The children with goitre might have less iodine uptake or excess iodine intake by the thyroid gland or any defect in the thyroid gland in the synthesis of thyroxin hormone; thus, the absorbed iodine is excreted through urine . In the id gland , where iid gland . The preid gland  that goiid gland  and goitIDDs prevalence is high in the Jaffna District under the present study settings was related to poor dietary intake of iodine-containing food and importer usage of iodized salt. The present study showed that children who consumed the seafood had less ID. Seafood contain a higher amount of iodine than other foods, including crops . Fish coThe present study concluded that the prevalence of ID was high (17.8%). Among the children, 15.7% and 2.1% of children were affected with mild and moderate ID, respectively, while none were affected with severe ID. The mean consumption of iodine from food was (128.7 \u03bcg/day) was higher than the World Health Organization\u2019s recommendation. Median UIC was significantly associated with living area, wealth status, type of drinking water, and method of iodized salt usage. Children having ID decreased with age, namely, children aged 12\u201323 months had a two-fold chance of getting ID than those of 48\u201359 months. Less ID was observed among the children from rural areas (15.9%) than those from urban areas (23.9%), while children from the inland areas had higher ID (20.2%) than the children from the coastal areas (11.4%). The risk of ID among children in urban areas and inland regions was approximately two- and three-fold higher than those of counterparts, respectively. The increasing educational level of the mothers marginally increased the iodine status of the children. Significantly higher median UIC (159.0 \u03bcg/L) was observed in children of mothers who added iodized salt after cooking the foods than those who added iodized salt by other methods. This study also showed that the risk of ID was approximately three times higher in children when the mothers added the iodized salt after washing with water or solubilized with water than that in the children of mothers who added the iodized salt after cooking the foods. The present study also revealed that the consumption of less iodine-rich foods contributed to the ID in Jaffna children. Conversely, ID was not associated with height, weight, and birth weight of the children. Moreover, family income, wealth index, type of drink water, and nutrient supplements were not associated with ID. Consumption of iodine-containing nutrient supplements was insufficient, and it did not improve the iodine status of children. As our study limited to estimate the amount of iodized salt consumed, we recommend incorporating the iodine availability from the table salt. The present study found a low (<5%) prevalence of goitre, and did not have a significant impact on public health."}
{"text": "The recently proposed Cognitive Experiential Leadership Model (CELM) states that leaders\u2019 preference for rational thinking and behavioral coping will be related to their level of transformational leadership. The CELM was based on research that principally used cross-sectional self-report methods. Study 1 compared both self-ratings and follower-ratings of leadership styles with leaders\u2019 self-rated thinking styles in 160 leader-follower dyads. Study 2 compared both self-ratings and coworker-ratings of leadership styles with leaders\u2019 self-rated thinking styles for 74 leaders rated by 607 coworkers. In both Studies, leaders\u2019 rational thinking, imaginative thinking, and behavioral coping correlated positively with their self-rated transformational leadership. However, only behavioral coping, but not rational thinking, was correlated with follower-rated (FR) transformational leadership in Study 1, and thinking styles were unrelated to other-rated transformational leadership in Study 2. These results partly support and partly challenge the CELM. Practically, this study suggests that leadership may be improved by leaders developing their capacity for behavioral coping. Would anyone argue with the proposition that leaders who think better are likely to lead better? In order to be testable, however, this proposition requires a definition of good thinking and good leadership. Recently, transformational leadership , and underlies most behavior. Like other dual-process theories, according to CET rational thinking is slow, conscious, relatively affect-free, and more useful in novel situations. In contrast, the experiential system is defined as a broad intuition-based system that encapsulates emotion, concrete reasoning, generalization, spontaneity, and imagination  and Defensiveness (>1.5 SD above the mean) scales, as specified in the CTI Manual (N = 320).Each participant was issued with a unique code number that they entered when completing measures online. Leader and follower code numbers were retained by the researcher so that paired data could be matched. A total of 245 leaders and 239 followers, among whom there were 191 matched pairs completed online questionnaires. Thirty-one pairs were omitted from the sample because of incomplete responses (>5%) or for falling outside the acceptable bounds of the Constructive Thinking Inventory\u2019s (CTI) Validity  was higher than the average age of their followers . Gender was similar in both the leader  and follower samples . Leaders had an average tenure of 5.75 years (SD = 6.37) in their current position. Followers had an average tenure of 3.19 years (SD = 3.73) in their current position. The participants were from a range of industries with the largest sub-groups being from retail, sales, and marketing (20.0%); healthcare (15.6%); engineering, mining, and construction (14.4%); and education (9.4%).Among the 160 dyads, the average age of leaders  and CTI, and they also completed the Multifactor Leadership Questionnaire ; followers completed the MLQ-5X (other report) and other measures that are not the focus of the present study . The experiential-thinking scale contains 30 items that represent three facets of experiential thinking: imagination, emotionality, and intuition . The imagination subscale measures respondents\u2019 preference for visual stimuli and tendency to think visually . The emotionality subscale represents respondents\u2019 emotional reactivity . The intuition subscale represents respondents\u2019 tendency to base their decisions on how they feel . An overall experiential thinking scale is calculated by combining these subscales. All items were presented in five-point Likert format with response options ranging from 1  to 5 (Definitely True). The REIm\u2019s scales had good Cronbach\u2019s alpha internal consistency reliability coefficients except for emotionality  and behavioral coping . The emotional coping factor consists of four subscales: self-acceptance, absence of negative overgeneralization, non-sensitivity, and absence of dwelling. Behavioral coping consists of three subscales: positive thinking, action orientation, and conscientiousness. Facets of destructive thinking are represented on four scales: personal superstitious thinking , categorical thinking , esoteric thinking , and na\u00efve optimism . The CTI also includes two lie scales: defensiveness  and Validity . As noted, participants scoring outside the acceptable bounds for Validity or Defensiveness were excluded from the sample. Items were in a five-point Likert format with scores ranging from 1  to 5 (Definitely True). The CTI scales had good Cronbach\u2019s alpha internal consistentcy reliabilities , transactional , and passive-avoidant . Transactional leadership has two subscales (contingent-reward and management-by-exception-active). Passive-avoidant leadership has two subscales , we calculated Pearson\u2019s correlations between thinking styles and both self-rated and other-rated leadership styles. In addition, where multiple thinking styles correlated with transformational leadership, we regressed transformational leadership on thinking styles in order to examine the unique contribution of thinking styles to transformational leadership.via a logarithm transformation. No other statistical assumption breaches were found, and, for ease of interpretation, descriptive statistics are reported for the untransformed data. Principal components analysis with direct oblimin rotation was used to statistically investigate potential common methods bias for the self-report measures. There was little evidence for common methods bias, with the largest single component only accounting for 21.57% of the variance  versions of the MLQ. For self-rated passive-avoidant leadership, the skew was corrected by removing three outliers, for follower-ratings, the skew was corrected variance . Descripr = 0.26, p = 0.001). However, self-rated and follower-rated transactional  and passive-avoidant leadership were not significantly correlated . Thus, there was a weak alignment between how followers evaluated their leaders\u2019 styles and how leaders evaluated themselves.Pearson\u2019s product movement correlations were calculated between leaders\u2019 self-rated and follower-rated leadership styles in order to assess the level of agreement in their ratings of leadership styles. Self-rated and follower-rated transformational leadership correlated significantly but weakly . These analyses were supplemented with relative importance analysis, with relative weights calculated in order to determine the unique contributions of each thinking style variable as a predictor of leadership style . The resF = 15.48, p < 0.001] and 11.8% of the variance in follower-rated transformational leadership . Looking at the significance of the \u03b2s and relative weights in The regression analyses found that the thinking styles accounted for 48.2% of the variance in self-rated transformational leadership  but did not account for significant variance in other-rated transformational leadership . For self-rated transformational leadership, only behavioral coping was a significant predictor in the regression , and no thinking style variables significantly predicted other-rated transformational leadership.The regression analyses found that the thinking styles accounted for 30.2% of the variance in self-rated transformational leadership  but was approaching significance in Study 2 .Theoretically, the CELM expects that rational thinking will be connected with transformational leadership because rational thinking contributes to adaptation by leaders, and the selection of a transformational leadership style is an effective adaptation . The absIt is possible that a relationship exists between leader\u2019s preference for rational thinking and the extent of their transformational leadership that other raters \u2013 especially followers \u2013 do not detect. Rational thinking, which engages a slower thinking system, may be associated with more deliberative and less action-focused decision-making that followers may perceive as evincing less transformational leadership. This possibility is in contrast to the relationship between leaders\u2019 preference for behavioral coping and transformational leadership. Behavioral coping is a component of how constructively leaders\u2019 experiential thinking is used . ExperieA limitation of the current studies was the low internal consistency reliabilities obtained for some measures that were of focal theoretical interest \u2013 emotionality (Study 1) and behavioral coping (Study 2). via coaching (e.g., The studies presented in this paper aimed to provide a more methodologically robust test of the CELM  than hasng e.g., . TherefoThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Murdoch University Human Research Ethics Committee. Written informed consent for participation was obtained for this study in accordance with the national legislation and the institutional requirements.GC conceptualized the studies, collected and analyzed the data, and wrote the manuscript. SW analyzed the multi-level data in Study 2, and reviewed and edited the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
{"text": "Hepatocellular carcinoma (HCC) is the most common malignant disease worldwide. Although the diagnosis and treatment of HCC have greatly improved in the recent years, there is still a lack of accurate methods to predict the prognosis of patients. Evidence has shown that Hippo signaling in tissues adjacent to HCC plays a significant role in HCC development. In the present study, we aimed to construct a model based on the expression of Hippo\u2010related genes (HRGs) in tissues adjacent to HCC to predict the prognosis of HCC patients.Gene expression data of paired normal tissues adjacent to HCC (PNTAH) and clinical information were obtained from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. The HRG signature was constructed using four canonical Hippo\u2010related pathways. Univariate Cox regression analysis was used to screen survival\u2010related HRGs. LASSO and multivariate Cox regression analyses were used to construct the prognostic model. The true and false positive rates of the model were confirmed using receiver operating characteristic (ROC) analysis.The prognostic model was constructed based on the expression levels of five HRGs  in PNTAH. The mortality rate of HCC patients increased as the risk score determined by the model increased. Furthermore, the risk score was found to be an independent risk factor for the survival of patients. ROC analysis showed that the prognostic model had a better predictive value than the other conventional clinical parameters. Moreover, the reliability of the prognostic model was confirmed in TCGA\u2010LIHC cohort. A nomogram was generated to predict patient survival. An exploration of the predictive value of the model in HCC tissues indicated that the model is PNTAH\u2010specific.We developed and validated a prognostic model based on the expression levels of five HRGs in PNTAH, and this model should be helpful in predicting the prognosis of patients with HCC. We built a prognostic model based on the Hippo signaling for the first time, and found that the expression of the Hippo signaling in adjacent tissues can better predict the survival of liver cancer patients. Paired normal tissues adjacent to tumor (PNTAT) are often used as a normal control for cancer research because of the shortage of healthy samples. However, whether PNTAT is truly \u201cnormal\u201d is controversial. Recent studies have shown that the transcriptomic profiles of PNTAT are distinct from those of healthy and tumor tissues.Hippo signaling was primarily discovered for its control of organ size in Drosophila and is highly conserved in mammals.GSE14520 using univariate Cox regression analysis. In addition, we screened out five key HRGs  to construct a prognostic model using least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analyses. We further confirmed that the model is good at predicting prognosis through survival and ROC curve analysis and found that the risk score calculated by the model formula was an independent risk factor through univariate and multivariate Cox regression analyses. Finally, we generated a nomogram to provide clinicians with a quantitative method for predicting survival. Moreover, the reliability of this model was validated by analyzing the PNTAH expression profiles from TCGA\u2010LIHC. Exploration of the predictive value of these five HRGs and the constructed model in HCC tissues demonstrated that the model is PNTAH\u2010specific. In summary, the present study may help reveal the underlying role of Hippo signaling in PNTAH and provide a useful prediction tool for HCC survival.In this study, we first constructed a Hippo\u2010related gene (HRG) signature using four canonical Hippo\u2010related pathways. We then identified 14 prognostic HRGs based on PNTAH expression profiles from 22.1GSE14520 dataset , GSE102079 dataset , and GSE112790 dataset (15 NL samples) were downloaded from the GEO database (www.pubmed.com/geo). The ComBat function from the sva package in R was used to remove the batch effects of the three datasets. TCGA\u2010GTEx cohort was downloaded from the UCSC Xena browser (http://xena.ucsc.edu/), among which 110 normal liver samples of GTEx, 50 PNTAH samples, 371 HCC samples, and the corresponding clinical data of TCGA liver cancer hepatocellular carcinoma (TCGA\u2010LIHC) were selected for the next analysis. To make data from different sources more compatible, the UCSC Xena project recomputed all expression raw data based on a standard pipeline to minimize differences from distinct sources. Principal component analysis (PCA) was applied using the PCA function implemented in the FactoMineR package. The 232 PNTAH samples from GSE14520 were used to construct the prognostic model, and 50 PNTAH samples from TCGA\u2010LIHC were used for validation.Expression profiles from the 2.2http://software.broadinstitute.org/gsea/msigdb/). After merging the four canonical pathways, a Hippo\u2010related signature was constructed. The heatmap was plotted using the pheatmap package in R to show the expression levels of the HRG signature in different samples. The protein\u2013protein interaction (PPI) of the HRGs was predicted using STRING (https://string\u2010db.org/) and visualized using Cytoscape (v3.7.2). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were carried out using the R clusterProfiler package. Enrichment results were visualized using the enrichment plot package. A p\u2010value\u00a0<0.05 was set as the cutoff criterion for both GO and KEGG functional analysis.Four Hippo\u2010related canonical pathways, GO_HIPPO_SIGNALING, KEGG_HIPPO_SIGNALING_PATHWAY, REACTOME_SIGNALING_BY_HIPPO and WP_HIPPOYAP_SIGNALING_PATHWAY, were obtained from the Molecular Signature Database \u00a0+\u00a0(the expression of gene2 in PNTAH\u00a0\u00d7\u00a0regression coefficient of gene2)\u00a0+\u00a0\u2026\u00a0+\u00a0(the expression of genen in PNTAH\u00a0\u00d7\u00a0regression coefficient of genen).To screen out the prognostic HRGs, the association between PNTAH HRG expression and overall survival (OS) was evaluated using univariate Cox regression. Genes with a Based on the median risk score calculated using the PNTAH expression profiles, the patients were divided into low\u2010 or high\u2010risk groups. The difference in OS between the two groups was analyzed using the Kaplan\u2013Meier method and log\u2010rank test. The risk score distribution, number of patients examined, and the heatmap of the prognostic HRGs in different risk groups were displayed. Univariate and multivariate Cox regression analyses were performed to explore whether the risk score could be an independent indicator of OS. The true and false positive rates of the prognostic model were analyzed using the receiver operating characteristic (ROC) and the area under the curve (AUC). A nomogram was constructed to estimate the 1\u2010, 3\u2010, and 5\u2010year survival rates of HCC patients using the rms package in R, and calibration of the nomogram was measured using calibration curves. Moreover, the predictive value of the constructed model was further confirmed using independent data from TCGA\u2010LIHC.2.4GSE14520. HCC patients were divided into high\u2010 or low\u2010expression groups based on the median expression of the key HRGs in HCC tissues. Moreover, the risk score was calculated with the established formula for PNTAH, using the HCC tissue expression profiles, and the patients were divided into high\u2010 or low\u2010risk groups based on the median risk score. The survival curve was drawn using the Kaplan\u2013Meier method, and the difference in the survival rate between different groups was verified by the log\u2010rank test. Similarly, the prognostic value of the key HRGs and the constructed model for the HCC tissues of TCGA\u2010LIHC cohort were also analyzed.To investigate whether the prognostic model built using PNTAH also had prognostic value in HCC tissues, the expression profiles of HCC tissues were extracted from 2.5https://www.r\u2010project.org). Univariate Cox regression analysis was conducted to estimate prognosis\u2010related HRGs. The Kruskal\u2013Wallis rank sum test was used to determine whether the HRGs were differentially expressed among NL, PNTAH, and HCC. LASSO regression analysis was used to prevent overfitting. Multivariate Cox regression analysis was performed to construct a prognostic model. An independent t\u2010test was performed to analyze the association between the risk score and conventional clinical characteristics. A nomogram was created using the rms package in R. ROC analysis was performed to test the true and false positive rates of the model. The survival curve was plotted using the survival and survminer package of R. Forest maps were plotted using the forsetplot package of R. The survivalROC package was used to generate the ROC curves, and the AUC values were calculated according to the ROC curves. All tests were two\u2010tailed and considered significant when p\u00a0<\u00a00.05.Statistical analysis was carried out using R 4.0.1 , suggesting that the expression of NF2 in PNTAH was of great significance in HCC patients. However, since the HR of NF2 was much higher than that of other genes, it was temporarily excluded from the forest map of the univariate regression analysis. In addition, these 14 genes were found to be differentially expressed among NL, PNTAH, and HCC  had a lower survival rate than the low\u2010risk group (n\u00a0=\u00a0105)  Figure\u00a0. The risn Figure\u00a0. The res3.5p\u00a0=\u00a02.949e\u201002)  Figure\u00a0. The ris) Figure\u00a0.3.6p\u00a0<\u00a00.001) and tumor stage  remained as independent prognostic factors for patients , advanced stage (p\u00a0=\u00a00.005), and more recurrence (p\u00a0=\u00a04.009e\u201006). To show the relationship between individual predictors and survival rate, a nomogram model was developed based on the data of the GEO cohort and converted to scale within a certain range  to construct the prognostic model. High expression levels of BIRC3, CSNK1E, MYC, and NF2 in PNTAH were associated with poor prognosis in HCC patients, while MINK1 expression was associated with a good prognosis. We divided patients into high\u2010 or low\u2010risk groups based on the median risk score calculated by the model formula and found that the high\u2010risk group had a lower survival rate. The AUC calculated by the ROC curve was 0.750, indicating that the model could predict the survival of HCC patients. We then confirmed that the risk score was an independent prognostic indicator after adjusting for other clinical parameters. ROC curve analysis demonstrated that the risk score had a better predictive value than other clinical parameters. Finally, we established a nomogram that predicted the survival of HCC patients well. The use of this tool could help clinicians dynamically assess a patient's prognosis based on different levels of clinical parameters and implement more targeted interventions accordingly. Furthermore, we also observed a similar trend in the survival analysis and ROC curve analysis of an independent dataset from TCGA, which further confirmed the reliability of this prognostic model. Given that PNTAH might be a precancer state, the five HRGs and the constructed model could have better prognostic performance in HCC tissues. We investigated the prognostic ability of the five HRGs and constructed a model using the expression profiles of HCC tissues from GEO and TCGA\u2010LIHC. Interestingly, the results showed that neither the five HRGs alone nor the constructed model had prognostic value in HCC tissues. Thus, we concluded that the prognostic model based on HRGs had a prognostic value specific for PNTAH.We further explored five selected HRGs. NF2 is a well\u2010established tumor suppressor and an essential upstream regulator of Hippo signaling.Some limitations of this study should also be considered. First, while the expression profiles were downloaded from the GEO and TCGA databases, the sample size was not large enough. Second, we only focused on the mRNA levels of these genes, and the five genes at the protein level should be further investigated. Third, the results of our study are descriptive, and the potential molecular mechanisms of these five genes warrant additional functional experiments. In addition, it has not been determined whether the median risk score we chose in the GEO and TCGA databases can be used as the threshold in real\u2010world clinical practice to identify high\u2010risk and low\u2010risk patients.5In the present study, we constructed and validated a prognostic model using PNTAH expression profiles, and this model could predict the survival of patients with HCC. The differentially expressed HRGs may provide a new perspective for the elucidation of the molecular mechanisms of HCC.The authors have no conflicts of interest to declare.(I) Conception and design: Y Zeng; (II) Administrative support: H Ren; (III) Provision of study materials or patients: QB Pan; (IV) Collection and assembly of data: BN He, N Yang, YT Zhang, HY Yuan; (V) Data analysis and interpretation: QB Pan, FB Qin; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All data were publicly available and downloaded from online databases; therefore, this study did not require additional ethical approval.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file."}
{"text": "GRM8, SPC25, FSD1L, SLC386A, FAM72A and SLC39A10) were screened via LASSO Cox regression, which provided the basis for developing a novel prognostic risk model. Based on the risk model, HCC patients were subdivided into high-risk and low-risk groups. Kaplan-Meier curve indicated that patients in the high-risk group have a lower survival rate compared with those in the low-risk group. The prognostic model showed good predictive efficacy, with AUCs reaching 0.802 at 1\u00a0year, 0.694 at 2\u00a0years, and 0.672\u00a0at 3 years. Univariate and multivariate cox regression analysis demonstrated that the risk score had significant prognostic value and was an independent prognostic factor for HCC. Moreover, this model also showed a good diagnostic positive rate in the ICGC-LIRI-JP and GSE144269. Finally, we demonstrated the efficacy of the AF-risk model in HCC patients following sorafenib adjuvant chemotherapy. And revealed the underlying molecular features involving tumor stemness, immune regulation, and genomic alterations associated with the risk score. Based on a large population, we established a novel prognostic model based on 6 AFs to help identify HCC patients with a greater risk of death. The model may provide a reference for better clinical management of HCC patients in the era of cancer precision medicine.Hepatocellular carcinoma (HCC) is the most common primary liver cancer with poor prognosis. An optimized stratification of HCC patients to discriminate clinical benefit regarding different degrees of malignancy is urgently needed because of no effective and reliable prognostic biomarkers currently. HCC is typically characterized by rich vascular. The dysregulated vascular endothelial growth factor was proved a pivotal regulator of the development of HCC. Therefore, we investigated the capability of angiogenic factors (AFs) in stratifying patients and constructed a prognostic risk model. A total of 6 prognostic correlated AFs ( Hepatocellular carcinoma (HCC), as the most common primary liver cancer, is amalignant tumor with poor prognosis . HCC is Tumor\u2019s access to the blood system is mainly accomplished by sprouting angiogenesis . AngiogeFor example, WNT2 has been confirmed to correlate with prognosis and considered to be an angiogenic growth factor that promotes liver regeneration . ExpressIn this study, we constructed and verified an effective prognostic risk model based on the expression of informative AFs. The investigation of the risk score deepened further understanding of the divergence of molecular features underlying different risk groups. This model was also proved to be suitable for patients following sorafenib adjuvant chemotherapy and we created the predictive nomogram. As a whole, this prognostic model might help guide the prognostic status of patients with HCC.http://xena.ucsc.edu/). We also obtained two independent validation cohorts of HCC patients from the ICGC portal  and the GEO , with transcriptomic and clinical data available.The raw counts of RNA-Seq data and corresponding clinical information of TCGA-LIHC patients were collected as the training cohorts. Data were downloaded from UCSC Xena (http://www.gsea-msigdb.org/gsea/msigdb/collections.jsp). The final AFs gene set studied here was the combination of genes from the two resources  and FDR of each gene was analyzed by DESeq2 (version 1.34.0) package . The twop-value < 0.01 and HR > 1 or HR < 0.5. Kaplan-Meier analysis was also performed to screen the prognostic candidate AF genes using R package survival (version 3.2-13) (Univariate Cox regression was performed for each AF genes to obtain the prognostic genes with  3.2-13) . MultivaThe prognostic candidate AF genes were screened by univariate cox regression and log-rank test. Tumor samples of the TCGA-LIHC were used as the training cohort to establish the LASSO model. A lasso penalty was used to find the best gene model utilizing an R package glmnet. The risk score for each sample can be calculated with the final LASSO model coefficient as follows:The median risk score was used as cutoff for high-risk group (with higher risk score) and low-risk group (with lower risk score).CIBERSORT algorithm was used to evaluate the infiltration of 22 immune cell types . The immMutation comment file (MAF) of TCGA-LIHC cohort was downloaded from the GDC client. Differential analysis and visualization of somatic mutations were performed using maftools package. The Fisher\u2019s exact test was used on all genes between two groups to detect differentially mutated genes. Segment file of the TCGA-LIHC cohort was downloaded from FIREHOSE and analyzed using the GISTIC 2.0 pipeline .AFs-derived risk scores, TNM stages, clinical stage, gender, age and grade were used as independent prognostic factors through univariate cox regression and AFs-derived prognosis risk model. Nomogram was finished based on the results of multivariate cox regression analysis. The calibration curves of the nomogram were constructed to test consistency between 1-, 3- and 5-years survival probability prediction and actual observation. The performance of the nomogram was evaluated using the concordance index (C-index) and time-dependent receiver operating characteristic (ROC) curves. Nomograms analysis and visualization were performed using R packages rms (version 6.2-0)  and survFirst, the large population of liver hepatocellular carcinoma (LIHC) patients from the TCGA database was used as the training cohort. We downloaded the transcriptomic and clinical data for 371 tumor samples and 50 adjacent normal samples. A total of 8,250 differentially expressed genes were found between tumor and normal samples . We then systematically collected AF genes from NCBI and MSigDB (see Methods) and noticed 1,038 AFs were differentially expressed  > 1 or HR < 0.5, p-value < 0.05, EGF, GRM8, TRPM6, SLC38A6, BLM, BARD1, CLSPN, PRIM2, MSH2, FAM72A, SPC25, IGF2BP3, CENPP, GTF2IRD1, TMC7, FSD1L, and SLC39A10. The distribution of expression levels and Kaplan-Meier curves of the four genes with top significance were displayed  and low-risk group (n = 159) according to the median cut-off value of risk score. The overall survival (OS) time of patients in the high-risk group was remarkably decreased  and GSE144269 (N = 68) were collected. Patients were all separated into high- or low-risk groups according to the risk score. The high-risk group of the ICGC-LIRI-JP validation cohort also showed significantly lower survival rate than the low-risk group . The predictive capacity was proved as AUC reaching 0.602\u00a0at 1-year, 0.632\u00a0at 2-years, and 0.709\u00a0at 3-years in ICGC-LIRI-JP cohort , tumor stage, virus infection status, etc. . Chronicetc. . We obsep-value < 2.2e-16, Next, we investigated the associations between the AFs risk score and the immune response in tumors. The mutation burden (TMB) was not differed between high- and low-risk groups , while tp-value <0.05). Among them, the mutation frequency of HCC driver gene TP53 was enriched in the high-risk group , this observation suggested the classic role of TP53 in cell-cycle regulation and guarding genome stability might also contribute to the malignant progression of HCC . Previously, multi-omics integration analysis revealed three HCC subtypes, one of which exhibited few CTNNB1 mutations companied by poor prognosis  was analyzed through maftools and GISTIC 2.0. As shown in n of HCC . While trognosis . This wap-value < 0.0001, Accumulating evidence supported that sorafenib was effective in extending the time of progression in HCC . In ordep-value < 0.05, TIDE is the tumor immune dysfunction and rejection score, depicting the primary mechanisms of tumor immune evasion. It was proved to predict the clinical response and outcomes of patients following immunotherapy . We usedFinally, a graphic prognostic nomogram based on the 6-AFs genes was developed for 1-, 3-, 5- and 10-years prediction of OS for HCC patients from TCGA. The tumor stage, grade, age, and gender were also included . MeanwhiIn the present study, we established an important prognostic model based on 6 DE-AFs genes significantly related to the prognosis of HCC, and further verified it in two independent validation datasets. The patients in the high-risk group showed poor prognosis, which was consistent in the three cohorts. Through univariate and multivariate Cox regression analysis, the risk score had significant prognostic value and was an independent prognostic factor of HCC. The model suggested that high risk may cause the regulation of immune mechanism, and these 6 gene signatures in the model could be used as potential prognostic molecular markers of AFs in HCC.Several prognostic staging systems have been built for liver cancers, such as the Japan Integrated Staging score , the CanGRM8, SPC25, and FAM72A was negatively correlated with favorable outcomes and also observed in other cancer types such as lung cancer (GRM8) was elucidated to promote the survival of squamous cell lung carcinoma (LUSC) tumor cell through inhibiting cAMP pathway and activating MAPK pathway and the transcription level of GRM8 was reversely correlated with the prognosis of LUSC cases  is a complex ecosystem consisting of various types of cells and the extracellular matrix with obvious heterogeneity . Tumor cSorafenib is an oral multikinase inhibitor, its action mechanism includes inhibition of both MAPK/ERK-mediated cell proliferation and angiogenesis driven by VEGF signalling . SorafenNomograms have been widely used as prognostic devices in oncology and medicine . ConstruIn summary, we constructed and validated a novel risk model consisting of 6 prognostic-associated AFs genes. This risk model showed effective and independent prognostic power, thereby providing important insight into the survival prediction of HCC. To our knowledge, this is the first study to predict prognosis of HCC patients based on the expression levels of AFs. Therefore, our study provided novel insights into the relationship between the regulation of AFs and development of HCC. In addition, we also revealed the underlying molecular features involving tumor stemness, immune regulation and genomic alterations between high/low-risk groups in this model. We expected further verification and mechanism exploration by the accumulated datasets in the future."}
{"text": "Galinsoga quadriradiata, across populations at different elevations in the Qinling and Bashan Mountains in central China. Seed mass\u2013area ratio (MAR), an important seed dispersal-related trait, of 45 populations from along an elevational gradient was measured, and genetic variation of 23 populations was quantified using inter-simple sequence repeat (ISSR) markers. Individuals from four populations were then planted in a greenhouse to compare their performance under shared conditions. Changing patterns of seed dispersal-related traits and populations genetic diversity along elevation were tested using linear regression. Mass\u2013area ratio of G. quadriradiata increased, while genetic diversity decreased with elevation in the field survey. In the greenhouse, populations of G. quadriradiata sourced from different elevations showed a difference response of MAR. These results suggest that although rapid evolution may contribute to the range expansion of G. quadriradiata in mountain ranges, dispersal-related traits will also likely be affected by phenotypic plasticity. This challenges the common argument that dispersal ability of invasive plants increases along dispersal routes. Furthermore, our results suggest that high-altitude populations would be more effective at seed dispersal once they continue to expand their range downslope on the other side. Our experiment provides novel evidence that the spread of these high-altitude populations may be more likely than previously theorized and that they should thus be cautiously monitored.Detecting shifts in trait values among populations of an invasive plant is important for assessing invasion risks and predicting future spread. Although a growing number of studies suggest that the dispersal propensity of invasive plants increases during range expansion, there has been relatively little attention paid to dispersal patterns along elevational gradients. In this study, we tested the differentiation of dispersal-related traits in an invasive plant,  Galinsoga quadriradiata across populations at different elevations in Qinling and Bashan Mountains, China. Our results suggest that the dispersal ability of G.\u00a0quadriradiata decreases along elevational dispersal routes as a result of genetically based rapid evolution and phenotypic plasticity, challenging the common argument that dispersal ability of invasive plants increases along dispersal routes. Our findings provide new insight into how the dispersal ability of invasive plants may change along the elevational gradient. This is important for revealing the upward expansion dynamic of invasive plants in high mountains.In this study, we tested the differentiation of dispersal-related traits in an invasive plant  Invasive plants, which tend to spread uncontrollably and cause environmental or economic damage , land-usPlants have been shown to do this via two main strategies: phenotypic plasticity and genetic adaptation . Other eSolidago canadensis, for which genetic diversity decreased significantly with elevation  How do population-level traits of G. quadriradiata related to dispersal vary across an elevational gradient? (ii) Are these changes attributable to phenotypic plasticity, genetic adaptation or a combination of both? (iii) What do these changes suggest about the future expansion of G. quadriradiata in the mountains of central China?In this study, we investigated how dispersal-related traits of invasive Galinsoga quadriradiata  is an annual herbaceous plant originating in Central and South America. It is a harmful agricultural invasive weed, mainly established in moist, warm temperate and subtropical zones around the world on abandoned land or farmland  are characterized by complex topography and distinct environmental conditions between the northern and southern regions . To calculate HSW, we randomly selected 100 ripe seeds from the collected seeds of each population and weighted them. At least five replicates were made for each population. We used WinSEEDLE Pro  to measure seed length, pappus length and pappus width. Each time, we randomly selected 30 ripe seeds from an elevational population and scanned and analysed them. We repeated this 10 times for each population.G. quadriradiata, is typically approximated by morphological characteristics . Seven primers  were used to amplify the 233 samples from 23 of the 45 populations. The polymerase chain reaction (PCR) system for ISSR analysis was as follows: total volume 25 \u00b5L; 12.5 \u03bcL 2 \u00d7 Es Taq MasterMix (Dye), 1 \u03bcL 10 \u03bcmol L\u22121 primer, 1 \u03bcL 50 ng \u03bcL\u22121 DNA, 10.5 \u03bcL ddH2O. All reactions were performed on a gradient PCR instrument (Agilent SureCycler 8800). The PCR procedure was as follows: pre-degeneration at 94 \u00b0C for 2 min, degeneration at 94 \u00b0C for 30 s, annealing at 50.5\u201355 \u00b0C for 30 s , extension at 72 \u00b0C for 1 min, 6 cycles; degeneration at 94 \u00b0C for 30 s, annealing at 50.5\u201355 \u00b0C for 30 s , extension at 72 \u00b0C for 1 min, 32 cycles; final extension at 72 \u00b0C for 3 min and preservation at 4 \u00b0C. The amplification products were separated in 2 % agarose gel (containing 0.5 mg mL\u22121 ethidium bromide) in 1 TBE (Tris-Borate-EDTA), and the separated bands were visualized under UV light by using an Electrophoresis Documentation and Analysis System 120 (Eastman Kodak Company). DL2000 ladder (Dongsheng Biotech Ltd) was used as DNA molecular weight markers.We used the inter-simple sequence repeat (ISSR) markers to analyse the genetic diversity of B method , and dil\u22121, respectively. Eight replicate plants from each population were grown, yielding a total of 32 pots. Pots were randomly arranged in an 80-m2 greenhouse, and the positions of the pots were changed at random every 2 weeks. Seedlings were watered daily and were replaced if mortality occurred within 1 week of the start of the experiment. The plants began to bloom in early June and the seeds began to disperse in late June. We collected mature seeds every 2 days until all plants no longer yielded seeds. All seeds were stored in envelopes and air-dried under laboratory conditions until measurement. We then analysed seed dispersal-related traits using the same method as described above for the field survey.To explore the influence of genetic differentiation on dispersal ability of different populations in a common environment on the basis of field survey, we randomly selected populations from four elevations. In late April 2016, seeds from the four populations (BS1 (223 m asl), QL5 (680 m asl), QL22 (1307 m asl) and BS15 (1756 m asl); We constructed linear mixed-effects models to evaluate the effects of elevation on diaspore-related traits , using the packages \u2018lme4\u2019 and \u2018lmerTest\u2019 in R-3.5.3 . ElevatiI) is usually used as an evaluation index, with higher values reflecting higher genetic diversity. Expected heterozygosity (He) is often used to measure the genetic diversity of a population, with higher values indicating richer the genetic diversity.Only distinct, reproducible and well-resolved PCR fragments were included in the statistical analysis for genetic differentiation. Inter-simple sequence repeat bands were scored as presence (1) or absence (0) characters, to construct the binary matrix. To compare the amount of total genetic variation partitioned within and among populations, three methods were employed, namely the hierarchical analysis of molecular variances (AMOVA), the analysis of Shannon\u2019s diversity  and Nei\u2019Linear mixed-effect models were then constructed to test for associations between genetic diversity and elevation (fixed factor) for the field seedlings, using mountain (Qinling and Bashan) as a random factor. We then constructed the phylogenetic tree of UPGMA based on Nei\u2019s genetic distance using MEGA-X . We alsoseeThe results of the linear mixed-effect models revealed that elevation has significant effects on HSW, NSC, seed length, pappus length, pappus width and MAR see, NSC Fisee, seed leI and He were significantly negatively correlated with population elevation . Furthermore, these trends were associated with reductions in genetic diversity at high elevations, suggesting that adaptation is largely responsible for the observed patterns. However, some of contradicted results from our greenhouse study showed greater dispersal-related traits of higher-elevation population in unstressed environment, suggesting that trait plasticity could still play an important role in the range expansion of invasive species in mountain ranges.Elevational variation associated with seed dispersal-related traits of invasive species has not yet been fully explored, creating a knowledge gap of how phenotypic plasticity and genetic diversity affect the invasive success of plants at high altitudes. In this study, we examined the phenotypic plasticity and genetic diversity of dispersal-related traits in invasive Spatial selection theory hypothesizes that dispersal phenotypes are spatially separated and that only the best dispersers will accumulate towards the range front . This thFor wind-dispersed Asteraceae seeds, dispersal ability is often approximated by measuring seed MAR, which is correlated with terminal seed velocity . Higher G. quadriradiata seeds exhibiting positive correlations between MAR and elevation  and the rate of gene flow (m), are strongly influenced by the demography and spatial distribution of populations, with optimal parameter values in central populations and less optimal values in marginal populations  and other dispersal-related traits has also been detected by others (G. quadriradiata in central China. This differentiation was also associated with dispersal-related traits of this species, suggesting that dispersal ability may be genetically based. Moreover, differentiation in MAR between elevational populations in the greenhouse experiment (seeG. quadriradiata in mountain ranges. Although our results support the idea that mountain ranges can act as natural barriers to plant invasions, the plasticity demonstrated in the greenhouse experiment implies that once acclimated to high elevations, acclimating back to low elevations on the other side will occur quickly and therefore should be cautiously monitored. Further research is needed to link the traits from this study directly to dispersal ability as well as to investigate these patterns across broader geographic gradients.Trait variation among populations has been associated with changes in natural selection pressure along elevation . Long-teiment see. Our resThe following additional information is available in the online version of this article\u2014I and He.plab008_suppl_Supplementary_MaterialsClick here for additional data file.plab008_suppl_Supplementary_DataClick here for additional data file."}
{"text": "In the past decade the treatment of patients with heart failure with reduced ejection fraction has quickly gained momentum. Numerous randomised controlled trials (RCTs) have proved the benefit of angiotensin receptor-neprilysin inhibitors (ARNIs) and sodium-glucose transport protein\u00a02\u00a0inhibitors (SGLT2i) in addition to the current \u2018guideline-directed medical therapy\u2019 (GDMT) \u20133.The novel European Society of Cardiology (ESC) guideline on heart failure (HF) that will be published this year will undoubtedly recommend a\u00a0substantial change in GDMT, presumably in accordance with the previously published ESC position papers and the American College of Cardiology consensus document , 5. UntiNetherlands Heart Journal.Despite the significant improvement in pharmacotherapy for HF, previous studies have demonstrated that real-life patients do not reach the target doses that lead to the beneficial results observed in RCTs, on which the guidelines base their recommendation , 7. It iThe authors retrospectively analysed the effectiveness of GDMT in terms of improvement of left ventricular function and prognosis after a\u00a0mean follow-up of 3.3\u00a0years in a\u00a0cohort of 378 HF patients with reduced left ventricular ejection fraction (LVEF) between 2012 and 2018. The maximal tolerated dose of each drug type was also recorded, including the reason for intolerance. All patients were newly diagnosed with HF and GDMT was initiated and/or up-titrated according to the ESC guideline , in an oThe mean age of the patients was 65.5\u00a0years and just under 35% were women. After up-titration 85% of patients used a\u00a0beta-blocker, 81% a\u00a0renin-angiotensin-system (RAS) inhibitor and only 53% a\u00a0mineralocorticoid receptor antagonist (MRA). Of the patients on medication 73% reached >\u202f50% of the target dose of beta-blockers, for RAS inhibitors 73% and for MRA 77%. The most important reasons reported for not reaching the target dose were bradycardia, hypotension and renal dysfunction.p\u202f=\u20090.002) for mortality and 0.85  for the combined endpoint of mortality and/or HF hospitalisation.After an arbitrarily chosen 9\u2011month follow-up period, LVEF on average improved from 28% to 39%. In patients with a\u00a0non-ischaemic cardiomyopathy, higher plasma levels of N\u2011terminal pro-B-type natriuretic peptide or older age, the LVEF was more likely to improve. The 1\u2011year mortality rate was 10% and a\u00a033% mortality rate was observed after a\u00a0median follow-up of 3.3\u00a0years. Each 5% increase in LVEF was associated with a\u00a0hazard ratio of 0.84  or revascularisation remains unclear in this cohort. Potential survivorship bias exists, since 35\u00a0patients died before the actual follow-up at 9\u00a0months and these patients were not included in the overall analysis. Lastly, the change in functional  class was not described. It was mentioned that the most important reason for not reaching the recommended dose of MRA was that there was no indication at the time of the follow-up visit because patients\u2019 functional status had improved to NYHA functional class\u00a0I. However, no percentages are provided.Nederlandse Hart Registratie (NHR) will provide more insight into this matter in the future.Despite these limitations, the article by Nauta et\u00a0al. gives the reader a\u00a0clear overview of the up-titration of GDMT, LVEF improvement and prognosis of real-life HF patients, albeit the relatively young age and the fact that the treatment is performed in a\u00a0tertiary care centre suggest that the results could differ from an outpatient population in secondary care. The heart failure registry of the Ultimately this study underlines that it is essential to verify the feasibility of implementing findings from large RCTs in daily clinical care. It shows that the majority of real-life HF patients are able to tolerate GDMT and, more importantly, seem to benefit from this regimen."}
{"text": "Amyloidosis is a disease of deposits classified into systemic and localizedBecause of the severity of the systemic form of the disease and its association with plasmocytoma and multiple myeloma, it is important to distinguish these manifestationsWe present here a case of a 42-year-old patient with ear amyloidosis.\u00ae and codeine. Upon the exam, there were hyperemia and edema of the outer ear and pinna , once a week, for 4 weeks, evolving with symptom improvements.Today, the patient is being observed.Amyloidosis is a rare disease, with deposits of protein fibrils. Such protein build up in the tissues may compromise the function of organs, such as the heart,In head and neck amyloidosis, systemic involvement must be ruled out by laboratory tests, such as protein electrophoresis, renal function test, electrocardiogram and abdominal ultrasoundIn this paper, we report partial and temporary stenosis of the external auditory canal, affecting the pinna - a rare manifestation of amyloidosis. In the few reports present in the literature, fullness of the ear and hearing loss are constant findings, which did not happen with pain - which was not reported by other papers.In most cases, amyloidosis is systemic and follows multiple myelomaThere are but a few studies on amyloidosis. Among the localized forms, the ear form is rare. The definition of clinical and laboratory characteristics is important for its diagnosis. It is important to investigate multiple myeloma in the systemic form of the disease."}
{"text": "In the present scenario, the challenge of emerging antimicrobial resistance is affecting human health globally. The increasing incidences of multidrug-resistant infections have become harder to treat, causing high morbidity, and mortality, and are posing extensive financial loss. Limited discovery of new antibiotic molecules has further complicated the situation and has forced researchers to think and explore alternatives to antibiotics. This has led to the resurgence of the bacteriophages as an effective alternative as they have a proven history in the Eastern world where lytic bacteriophages have been used since their first implementation over a century ago. To help researchers and clinicians towards strengthening bacteriophages as a more effective, safe, and economical therapeutic alternative, the present review provides an elaborate narrative about the important aspects of bacteriophages. It abridges the prerequisite essential requirements of phage therapy, the role of phage biobank, and the details of immune responses reported while using bacteriophages in the clinical trials/compassionate grounds by examining the up-to-date case reports and their effects on the human gut microbiome. This review also discusses the potential of bacteriophages as a biocontrol agent against food-borne diseases in the food industry and aquaculture, in addition to clinical therapy. It finishes with a discussion of the major challenges, as well as phage therapy and phage-mediated biocontrols future prospects. Globally, throughout the ages, the primary factor that leads to mortality is infectious diseases. Although infectious disease incidences have been reduced with the advancement in sanitary and hygienic conditions, diseases continued to be a significant threat until the discovery of antibiotics in 1928. The commercialization of antibiotics is regarded as a medical miracle leading to a rise in average life expectancy up to 78.8\u2009years in the United States , and in Bacteriophages are believably one of the most ancient and ubiquitously existing biological entities (viruses) that are capable of infecting and replicating within bacteria and therefore play an important role in sustaining the equilibrium of an ecosystem where bacteria reside. After injecting their DNA, phages use the host machinery to replicate and translate the necessary information required to produce viral progeny (in the case of lytic phages) and enzymes such as spannin, holin, and endolysin. In the case of gram-negative hosts, caudovirales use holin-endolysin or pinholin SAR (Single arrest release) mechanisms for breaking the peptidoglycan bond of the cell wall, resulting in host lysis and the release of viral progeny during lytic cycle . While iE.coli and K. pneumoniae, cystic fibrosis with chronic Pseudomonas aeruginosa infections, and prosthetic joint and osteomyelitis infections due to Staphylococcus aureus . The Shanghai Institute of Phages in China has also started providing clinical treatment with phages to patients suffering from multidrug-resistant infections /lysins are used in the therapy and biocontrol. GMO phages are considered Advanced Therapy Medicinal Product (ATMP) by the European Medicines Agency. For their access to the European market, they must comply with Directive 2001/83/EC, Directive 2001/18/EC (Article 12.2), and Regulation (EC) 726/2004 (Articles 6.2 and 6.3) under current good manufacturing practices (cGMP) . HoweverSeveral studies have indicated the successful treatment of bacterial infections with phage therapy . HoweverThe main rule for finding a bacteriophage for a specific host is to use an environmental sample where the host is located. For example, phages for fish pathogens are generally isolated from coastal or fish farm waters . SimilarPhage characterization generally includes the determination of the plaque morphology, host range and the multiplicity of infection. Structural properties of phages are determined by transmission electron microscopy, thermal and pH stability for therapeutic purpose, and genome/proteome analysis are carried out for checking their suitability for therapy. A plaque assay is performed to assess the lytic efficiency of a bacteriophage where phages producing clear plaques are generally considered lytic/virulent while those forming turbid plaques are considered lysogenic/temperate. Phages can be additionally screened for the presence of integration/recombination/excision/toxin genes that are associated with the temperate life style and have the potential for transduction, which can be tested either by nucleic acid hybridization/PCR or by whole phage genome sequencing . Whole-g\u22121 h\u22121  for their use in human, veterinary, aquaculture, and processed foods has increased, but only a limited number of phage products are developed according to guidelines under Good Manufacturing Practices (GMP). To prepare a phage for its use in therapy, it needs to be free of bacterial endotoxins and other gross impurities while concurrently maintaining the phage efficacy. Endotoxins and protein toxins are the main impurities present in phage lysate that need to be removed from therapeutic preparations. Chromogenic Limulus Amebocyte Lysate (LAL) assay  is used \u22121 h\u22121 . The tra\u22121 h\u22121 . Therefo\u22121 h\u22121 , EndoTra\u22121 h\u22121 , and LPS\u22121 h\u22121 . Dependi\u22121 h\u22121 . For int\u22121 h\u22121 . For the\u22121 h\u22121 .A phage susceptibility test is carried out to simultaneously check hundreds of phage candidates selected from different phage banks and phage laboratories against the bacteria isolated from a patient. A phage susceptibility test can be performed by conventional agar overlay assay, or by direct spot of liquid phage suspension on the targeted bacterial lawn (Direct spot assay). Further, the productivity of phage infection can be evaluated by the Efficiency of Plating (EOP) assay, by studying time kill growth kinetics in for planktonic cultures and the biofilm inhibition/eradication assay . A phageCaudovirales, S. aureus, including MRSA strains and it was found that trehalose and sucrose (0.5\u2009M) were the best additives for protecting the phage particles   and accoterature . Later tterature . Bangladterature . Similarterature . A similterature . Throughterature  it was dterature . Similarfections . Also, tfections . DetaileS. aureus, P. aeruginosa, and E. coli, and to a lesser extent Enterococcus spp. induced infections of different body organs. However, there is a need to develop a relatively standardized protocol to drive the phage therapy beyond its compassionate use to a more accessible frontline therapy.Compassionate therapy refers to using non-standard medicines to treat a patient for which authorized medicines have run out. Article 37 of the \u201cHelsinki Declaration\u201d summarizes the doctrine of the compassionate use of phage therapy and emphasizes the physicians consent to use best practice to treat the patient along with the patients or guardians consent . The regvia pattern recognition receptors  restored healthy gut function and suggested fecal filtrate transfer (FFT) as an alternative and effective approach in comparison to fecal microbiota transplantation (FMT). Similarly, C. difficile infection (RCDI) eliminated the symptoms of infection and restored a healthy gut microbiome.In a recent report by in vivo. So far, most of the phage therapy studies are based on single phage-host pairs or a cocktail of a limited number of phages against the host in vitro. Therefore, understanding the phage interactions with their host and immune response in vivo lays the first stepping stone for the oral therapeutic application of phages in humans.Similarly, E. coli, Campylobacter spp., non-typhoidal Salmonella enterica, Shigella spp., Vibrio cholera, and Listeria monocytogenes were responsible for 96% of the food-borne illnesses. The food industry routinely utilizes several antimicrobial interventions such as chemicals, physical disruption techniques, and irradiation to eradicate the pathogens of the contaminated foods . Similarly, in vitro experiment of phages against A. salmonicida, which showed significant lysis at low MOI in comparison with high MOI , a very limited number of phages  have been characterized completely , and Enterococcus. Bacteriophage or lysins induced cell lysis in the case of gram-negative bacteria may lead to the release of endotoxins which are responsible for inflammatory response and, in severe cases, may cause septic shock. While the use of another phage-derived enzyme, polysaccharide depolymerase helps to overcome this problem because, as such, they do not lyse the bacterial cell but only remove the surrounding polysaccharide layer, thus exposing the bacteria to immune cells  and immune reaction studies should be carried out extensively. The regulations involved in phage preparation and the legal framework should be set up decisively. Also, there is a need to formulate universal and favorable regulations to promote phage or phage-based products for human benefits. In addition, popularizing phage therapy and its benefits over other therapeutic agents will help in the acceptance of phage therapy by medical practitioners. Awareness should be generated among clinicians to offer phage therapy as a treatment option for patients where antibiotics have failed altogether. The areas that need to be strengthened, of course, include expanding phage biobanks/repositories for the timely offering of specific and usable phages, improvements in phage production protocols to provide stable and safe phage preparations, and to relax the regulatory protocols for phage therapy. Looking at the extensive damage to the environment, animal, and human health that antibiotics have posed since their discovery, and simultaneously the benefits that the naturally occurring phages (the living drugs) have offered, it does not seem relevant to completely reject phages.The inclusion of bacteriophages in the treatment of human diseases and food biocontrol has witnessed a significant surge in the last few decades. A large number of studies employing single phages, cocktails, phages in combination with antibiotics, and simultaneous improvements in phage production protocols and the ease of genetic manipulation technologies, have broadened the versatility of phage application. However, there is a requirement for the adoption of favorable regulations to promote phage or phage-based products for human/livestock benefits. The phage therapy provides hope against ever growing menace of antimicrobial resistance however, a major boost is required for widening its application through the involvement of researchers, clinicians, industry, and policy makers. Use of phage therapy also aligns with the goal of one health approach to sustainably benefit the environment with simultaneous improvement in the treatment strategies. Incorporation of phage therapy in medical education and willingness among physicians to consider and apply phages will help accelerate its acceptance. Further, making phage therapy cost-effective by supporting medical tourism and relaxing the stringent regulatory guidelines associated with its compassionate use will help in improving the accessibility of phage therapy as a frontline medical intervention to treat resistant bacterial infections.AJ and TA: conceptualization. AJ: methodology and software. AJ, MV, TA, and RV: validation. NV, BB, RV, and BT: formal analysis. TA: resources and funding acquisition. AJ and MV: data curation. AJ and RV: writing - original draft. MV, TA, NV, and BB: writing - review and editing. AJ, MV, and RV: visualization. TA and BT: supervision. TA and RV: project administration. All authors have agreed to the published version of the manuscript.This work was supported by the National Agricultural Science Fund, Indian Council of Agricultural Research, New Delhi, India and Council of Scientific and Industrial Research, New Delhi, India.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
{"text": "Bacteriophage therapy holds promise in addressing the antibiotic-resistance crisis, globally and in Germany. Here, we provide an overview of the current situation (2023) of applied phage therapy and supporting research in Germany. The authors, an interdisciplinary group working on patient-focused bacteriophage research, addressed phage production, phage banks, susceptibility testing, clinical application, ongoing translational research, the regulatory situation, and the network structure in Germany. They identified critical shortcomings including the lack of clinical trials, a paucity of appropriate regulation and a shortage of phages for clinical use. Phage therapy is currently being applied to a limited number of patients as individual treatment trials. There is presently only one site in Germany for large-scale good-manufacturing-practice (GMP) phage production, and one clinic carrying out permission-free production of medicinal products. Several phage banks exist, but due to varying institutional policies, exchange among them is limited. The number of phage research projects has remarkably increased in recent years, some of which are part of structured networks. There is a demand for the expansion of production capacities with defined quality standards, a structured registry of all treated patients and clear therapeutic guidelines. Furthermore, the medical field is still poorly informed about phage therapy. The current status of non-approval, however, may also be regarded as advantageous, as insufficiently restricted use of phage therapy without adequate scientific evidence for effectiveness and safety must be prevented. In close coordination with the regulatory authorities, it seems sensible to first allow some centers to treat patients following the Belgian model. There is an urgent need for targeted networking and funding, particularly of translational research, to help advance the clinical application of phages. The decreasing sensitivity of clinically relevant bacteria to antibiotics, as stated in the WHO surveillance reports of recent years, also affects Germany ,2,3. TheStaphylococcus aureus (MRSA), ~4000 with vancomycin-resistant enterococci , ~8000 with MDR Escherichia coli, ~2000 with MDR Klebsiella pneumoniae and ~4000 with MDR Pseudomonas aeruginosa). Of these, ~1500 cases (0.3%) are caused by isolates that are pandrug-resistant to almost all antibiotic classes [The proportion of patients acquiring infections in hospitals is ~3.6%, corresponding to an estimated 400,000\u2013600,000 nosocomial infections in Germany per year [ classes .Nosocomial and community-acquired infections by MDR pathogens, according to projections, amount to ~54,500 people in Germany annually. A 2018 study on the disease burden due to MDR pathogens published by the European Centre for Disease Prevention and Control (ECDC) found the number of infection-associated deaths for Germany to be ~2400 people per year   and 2018The regulatory situation or legal framework for phage products in Germany;The extent of the existing clinical application of phages;Existing activities to produce phages for this purpose;The status of phage banks;The technical status of sensitivity testing (\u201cphagogram\u201d).The current situation of phage therapy in Germany is presented primarily from the perspective of clinicians, cooperating research institutions, and regulatory authorities , with whom effective exchange on the topic has been in place since 2017. Thus, an interdisciplinary network of physicians, pharmacists, microbiologists, bioengineers, bioinformaticians and regulators\u2014all with a patient-oriented focus\u2014were involved in describing the current situation in Germany. The goal of taking translational aspects into account in the present analysis seemed achievable within this group of authors, following scientific exchange at national and international meetings over the last five years, as well as joint research activities. An attempt was made to include only one author from each working group. The following aspects are addressed in particular:For an insight into further developments, ongoing research projects, the activities for a one-health approach, as well as the existing Germany-wide network structure and activity will be presented.To avoid a limited overview of the phage landscape in Germany, literature covering at least the years 2020\u20132022 was reviewed for the academic and industrial working groups that, according to the authors, have contributed to translational phage research. In addition, the authors of this article provided their opinion on the most urgent changes needed in the German-phage landscape, the most important research projects, and the biggest hurdles for the implementation of phage therapy. Multiple answers were possible.Within the European Union, phages for therapeutic use in humans are defined as biological medicinal products, in accordance with Directive 2001/83/EC. This implies the need for a marketing authorization (MA) for market access of phage-therapy medicinal products (PTMP). Article 3 of Directive 2001/83/EC (and paragraph (\u00a7) 21(2b) of the German Medicinal Products Act, \u2018AMG\u2019) define exemptions from the need for an MA, including prescription medicinal products prepared in a pharmacy for a specific patient . According to \u00a755(8) AMG, these products must be manufactured in compliance with the recognized pharmaceutical rules . A Ph. Eur. general chapter on PTMPs is currently being drafted that will provide harmonized quality standards. According to \u00a713 AMG, a manufacturing authorization is required for PTMPs produced on a commercial/professional basis and intended to be marketed. An exemption exists (\u00a713(2) AMG) which includes manufacturing in pharmacies  or by physicians . PTMP manufacturing must also follow the recognized pharmaceutical rules, and is subject to notification of competent supervisory authorities (\u00a767(2) AMG). PTMPs therefore can be made available to patients using either way, MA  or formula magistralis.While MA would primarily pertain to standardized ready-to-use PTMPs, magistral phage products could provide a more personalized approach, adapted individually. The existing regulations are often seen as an obstacle for phage therapy. Nevertheless, as indicated above, varying routes for patient access to PTMPs are currently available. In any case, the development and modification of regulatory requirements will be based on the expansion of knowledge. Once the efficacy of PTMPs is demonstrated, the regulatory framework can be amended, if required, to address the particularities of these products. However, they can be used in cases of hardship. Thus, within the framework of an official compassionate-use program, the use of PTMPs that are currently included in clinical trials or in the approval process of an MA application can be authorized by the national authorities, and thus would be available for a defined group of patients .This officially authorized compassionate-use program is distinguished from an \u201cindividual treatment trial\u201d, which does not require notification. The latter is the application of PTMPs in an individual case, decided on by the treating physician, under his or her responsibility, within the framework of therapeutic freedom and with the patient\u2019s consent. The individual treatment is usually applied after all standard treatment options have been exhausted or are not available, i.e., in the case of an \u201cunmet medical need\u201d, according to Article 37  of the Declaration of Helsinki of the World Medical Association (WMA), and if the treating physician suspects a benefit of phage use for the patient, based on scientific findings. The focus of the individual treatment trial is not on obtaining research results, but on healing the individual patient. However, the regulatory authorities explicitly emphasize that precise documentation, observation of the patient and transparent communication of the individual case, as well as scientific justification, must be in place, which also corresponds to the requirement of Article 37, Declaration of Helsinki. No therapeutic phage products are currently clinically approved in Germany; an overview of ongoing and planned clinical trials is included in \u00ae against Shigella infections was the first phage preparation marketed by Behringwerke, near Marburg/Lahn, in July 1939 [\u00ae against Salmonella. Many German soldiers were subsequently treated with phages. Positive therapy results were reported, but also\u2014after the initial euphoria\u2014relevant failures. It has been speculated that many of these failures occurred because clinicians were not aware of the narrow host specificity of the phages and important application parameters, and, probably, temperate phages were also used. In addition, process-related impurities, and insufficient stability of the phage solutions impaired product quality. However, scientific proof for effectiveness as well as for the manifold speculative reasons for failure is lacking. Ultimately, these failures greatly reduced interest in phage therapy after the market launch of antibiotics (penicillin and sulfonamides) in the clinical field [At the beginning of the Second World War, phage therapy experienced a significant upswing in Germany. Dysentery-Polyfaginuly 1939 , followeal field .\u00ae, was available from 1959 to 1965 . This preparation was, however, ineffective against Shigella flexneri type 4A, which was responsible for an epidemic in 1962. Within a very short time, production capacities of 11,000 L per week were implemented for the prophylaxis of the population /mL). At that time, phages in the GDR were not subject to the \u201cOrdinance on the Regulation and Supervision of the Traffic in Medicinal Products\u201d, but to the \u201cOrdinance on the Traffic in Vaccines, Sera and Bacteriophages\u201d. Phage prophylaxis began in Berlin on 26 April 1962. Until June 14, 1962, a total of 174,906 people received the phage preparation [In the German Democratic Republic (GDR), a ready-to-use phage preparation, Intestolysin7\u2013108 PFU/mL; 30 min before eating; after taking an acid-binding drug). In addition, the phage lysate was introduced intraoperatively into the infected area (exposure time 30 min). From August 1979 to May 1981, 54 patients with implant-associated infections were treated , over a period of at least 5 years (unpublished data). For the treatment of periprosthetic infections, the phage lysate was administered to the patient twice daily for 14 days before surgery and for 14 days afterwards  began to be applied topically to chronic limb wounds, post-traumatic infections and burn wounds at the Hanover Medical School (B. Wippermann), as part of individual treatment trials (unpublished data). Most recently, this treatment series was continued in Hildesheim. Since 2015, 33 patients with chronic periprosthetic infections (on average 11 previous operations) have been treated topically with phages by another working group (R. Ascherl) in the Chemnitz and Tirschenreuth hospitals and, with a follow-up period of 14\u201342 months at that time, a complete cure of infection without severe side effects (mostly fever and chills on the 2nd\u20134th day) was achieved in 25 cases. .PhagoFlow research project (see below), it will be possible to treat patients with P. aeruginosa and S. aureus infections on a larger scale with magistral phage preparations, from spring 2023 onwards (www.phagoflow.de). Additionally, at the German Heart Institute Berlin, Germany, six patients were treated as a last-resort measure between December 2018 and October 2021, in collaboration with Charit\u00e9\u2014Universit\u00e4tsmedizin Berlin, Germany, Center for Musculoskeletal Surgery, with phages acquired from the Belgian LabMCT and Eliava Institute of Bacteriophage, Microbiology and Virology, IBMV . Following skin closure, a 1:1 mixture of PYO-bacteriophage with 106 PFU/mL and staphylococcal phage Sb-1 with 107 PFU/mL  was applied locally\u2014and in one case also orally and intravenously [S. aureus) remained infection-free until the end of the observation period (\u226530 months) [P. aeruginosa infection, followed by Staphylococcus haemolyticus, received a magistral phage preparation applied both locally and intravenously, and remained infection-free for four months before succumbing to LVAD pump thrombosis. Another patient, with an S. aureus infection, was administered PYO-bacteriophage plus Sb-1 locally and, at a nine-month follow-up, showed no signs of a local infection [Berlin: At the Military Academic Hospital Berlin, three patients have been treated with phages from Georgia since 2016, partly in collaboration with the laboratory for molecular and cellular technology (LabMCT) at Queen Astrid Military Hospital in Brussels (Belgium) and the Charit\u00e9\u2014Universit\u00e4tsmedizin Berlin, Germany, Center for Musculoskeletal Surgery. As part of the venously ,14. Out nfection .S. aureus (n = 13), P. aeruginosa (n = 12), E. coli (n = 4), E. faecium (n = 3), K. pneumoniae (n = 3), Burkholderia multivorans (n = 2), E. faecalis (n = 1), Stenotrophomonas maltophilia (n = 1), Serratia marcescens (n = 1), and Proteus spp. (n = 1).Hannover: Nowadays, phage therapy is again based at the Department of Cardiac, Thoracic, Transplantation and Vascular Surgery of the Hannover Medical School. The department\u2019s portfolio includes 33 cases of personalized phage therapy since 2015 in critically ill patients, of which 31 were successful. The high clinical success rate (>90%) is due to a combination of modern principles of permission-free preparation using mostly self-isolated phages (see below), an interdisciplinary approach to administration of phages and an optimal, concomitant conventional treatment, including antibiotic therapy [\u00ae bacteriophage cocktail produced by the Eliava Institute was recommended, which is active against various strains of Shigella, Salmonella, E. coli, Proteus, S. aureus, P. aeruginosa, and Enterococcus. To improve local release kinetics, the phage cocktail was mixed with a hydrogel, as previously described [PhagoDAIR trial, a randomized, non-comparative, double-blinded phase I/II clinical study in patients with S. aureus knee or hip periprosthetic-joint infections with indication for debridement, antibiotic and implant retention (DAIR), combined with antibiotics .Regensburg: At the Department of Trauma Surgery University Hospital Regensburg, phage therapy has been performed since October 2022. The first case was an infected non-union of the proximal femur with different MDR gram-negative bacteria. Phage testing was performed by LabMCT at Queen Astrid Military Hospital and treatment with Intestiescribed . No locaRostock: Phage therapy is also performed at the Clinic for General, Visceral, Thoracic, Vascular and Transplant Surgery, Rostock University Medical Centre. In the field of vascular surgery, it is mainly patch- or bypass-associated infections in the groin area, followed by acute life-threatening aortic-prosthesis infections. In cardiac surgery, the most frequent indication is drive-line infection after LVAD insertion. In orthopedic-surgery patients, periprosthetic-joint infections in the hip area dominate, increasingly also after implantations of endo-exo systems in transfemoral amputations. Unfortunately, despite a high demand for treatment, there are fundamental problems with individualized phage therapy. Therefore, it has only been possible to treat patients with composite phage cocktails , which have so far been accompanied by therapeutic success of only ~30%. The focus on individualized phage therapy with on-site production planned for 2023 is expected to significantly improve success rates. Current and planned research projects are focusing on testing the thermostability of phages in bone cement, topical phage application to infected endo-exo systems at the stoma, and the planning of the study \u201cBacteriophage-related treatment of chronically infected Drive-Line/LVAD Systems: a randomised controlled multicentre trial\u201d (via a German Research Foundation (DFG) application), in collaboration with the Heart Center, Leipzig, Germany.The Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Braunschweig, is developing a platform-like manufacturing process for natural phages as APIs using host bacteria under GMP-conforming conditions. For this purpose, the starting materials, master cell banks (MCB) and master phage stocks (MPS), are first produced according to GMP. Phage drug production begins with cultivation (up to 10 L) of the production strain from the MCB, infection with phages (MPS) and subsequent cell lysis. The phage-containing lysate is first separated by depth filtration (to reduce cell fragments), diluted in a buffer suitable for chromatography (diafiltration) and then purified chromatographically to remove endotoxins and host-cell proteins. This is followed by a second diafiltration step with a physiological buffer and 0.2 \u00b5M filtration, to reduce bioburden. Finally, the phage agent is filled into the primary packaging. The challenge in developing the manufacturing process is to find suitable conditions that allow for high titers and yields and the reduction of endotoxins and host-cell proteins. After filling, the phages must meet international quality requirements and are tested for identity (sequencing), potency (plaque assay) and purity , for release. The justification of the batch-release specifications is carried out in close exchange with the competent authorities and process-immanent aspects. The challenge here is that phages represent a completely new class of active substances, for which official quality standards remain to be defined. The first manufacturing authorization for phage production in Germany was granted in August 2022. Three phages produced this way are set to be supplied to the Military Academic Hospital Berlin in Q1 2023.https://invitris.com, accessed on 12 January 2023), a spin-off company of the Technical University of Munich. For in vitro or cell-free protein synthesis (CFPS) the cellular-expression machinery is isolated from the cell, most commonly E. coli, and combined with metabolites, precursor molecules and a DNA template specific for the intended phage. CFPS has been used to produce E. coli phages such as T7, phiX174, and MS2 with titers of up to 1012\u20131013 PFU/mL [E.-coli phages, including those against clinically relevant pathogens such as Yersinia pestis and even gram-positive bacteria such as Bacillus subtilis [K. pneumoniae patient isolate, from isolation to in vitro production. Utilizing the advantages of in vitro bacteriophage production, a personalized phage-therapy pipeline can be implemented, and could be applied for on-site production [Klebsiella phages, most of which initiate infection through the highly diverse capsular polysaccharide of K. pneumoniae.An alternative way to produce phages, the expression in an in vitro system, is currently being developed by INVITRIS  AMG  that uses host bacteria is currently only in use at the Hanover Medical School  according to the quality and safety requirements for sustainable-phage-therapy products. The production process uses only strictly lytic phages, which do not contain known genes encoding integrases, bacterial virulence and antibiotic-resistance factors. Despite the absence of a conventional GMP process in the production consumables, nutrient media and reagents designed for either GMP production or for clinical use are employed. For example, nutrient media free of animal components are used for cultivation. The host bacterial strains are tested for absence of inducible prophages. Phage amplification is performed on a small-scale, using dense nutrient media, which has shown stable high-phage titers and does not require optimization of the production process. After amplification, the phage lysate undergoes a multistep purification process to remove unlysed cells, nutrient-medium components, and pyrogens, followed by control of sterility, phage titer, identity, and endotoxin content. Other clinics in Germany  are currently also aiming for this type of production.E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter spp.) are dominate, and are used in externally funded projects such as P. aeruginosa in Phage4Cure, some prioritized species in PhagoFlow, E. coli in IDEAL-EC and E. faecium in EVREA-Phage (there are 30\u2013150 phages per ESKAPE species). Other large phage panels exist for Achromobacter xylosoxidans and Stenotrophomonas maltophilia; phages in lower numbers were isolated on rarer patient isolates of, e.g., Bordetella bronchiseptica, Burkholderia cepacia or Mycobacterium abscessus. The focus of the DSMZ phage bank is defined by medical need. DSMZ can search for new suitable phages at any time, at the request of clinicians, and they can then be supplied according to material-transfer agreements. However, direct application in humans is excluded, for warranty reasons. For genomic-identity confirmation, the DSMZ was granted GMP certification according to \u00a764 (3f), German Medicinal Products Act, in 2022.In the early 2000s, a working group specializing in clinically relevant bacteria was founded at the Leibniz Institute DSMZ and, furthermore at the DZIF (German Center for Infection Research) strain repository. Both provide an essential basis for isolating, characterizing, and investigating phages at the DSMZ and for using them in the institute\u2019s so-far exclusive applicative-phage-research projects. The bank contains >1000 phages for more than 150 bacterial species; however, phages for the ESKAPE bacteria  [K. pneumoniae (>380 isolates with a large variety of capsule types (>80)) and their specific phages (>505 plaque-purified stocks) in Europe. The group is using its phage collection for studies to extend the host range [K. pneumoniae infections [Another large phage bank is located at the Bundeswehr Institute of Microbiology (IMB), Munich, with a focus on the isolation and characterization of  groups) . The IMBst range , and engfections .Salmonella (>120 phages), K. pneumoniae (>80), Yersinia (>50), Brucella (>40), P. aeruginosa (>30), Morganella (>15), Campylobacter (>15), Burkholderia (>15) and Vibrio (>10). However, due to the different clonality of the bacteria in the human and livestock/food sector, most of the phages are much more effective on isolates of the respective compartments than on human isolates (esp. Klebsiella).Phage research at the German Federal Institute for Risk Assessment (BfR) is based on fundamental investigation of the occurrence of phages (temperate/lytic), their genetic diversity and their potential for application, especially in the veterinary and food sectors. Therefore, the culture collections of foodborne pathogens as well as bacteria recovered from livestock during annual monitoring of zoonoses are used for phage testing. Phages against foodborne pathogens at the BfR mainly originated from samples of the food-production chain , but have also been recovered from environmental sources  and/or municipal/clinical wastewater. The current collection includes phages against Conventionally, phage\u2013host-susceptibility testing is based on the double-agar-overlay plaque assay , which ihttps://phage4cure.de, accessed on 12 January 2023, funded by the German Federal Ministry of Education and Research (BMBF)) is a first-in-human (FIH) study to investigate safety, tolerability, and preliminary efficacy of a phage cocktail in healthy volunteers and patients with chronic P. aeruginosa lung infection. The phages investigated in the clinical trial were selected with the help of the phage biobank of the Leibniz Institute DSMZ GmbH. Three phages from the order of Caudovirales were selected for the study: two myoviruses (JG005 and JG024), and one podovirus (Bhz17). JG005 and JG024 had been known previously, whereas Bhz17 was newly isolated. The phage selection aimed at providing a broad antibacterial spectrum and limiting the emergence of phage-resistant bacterial variants. Three individual IMP formulations of the respective phages were initially prepared, according to European GMP standards and will subsequently be combined in a cocktail prior to use. The cocktail will be administered as an aerosol through a CE-certified nebulizer. Efficacy and safety were previously tested, in animal models. The Phage4Cure study consists of two parts: part 1 is a classical single-ascending-dose (SAD) design in healthy volunteers, while part 2 is a multiple-dose design conducted in patients with chronic P. aeruginosa colonization of the lung, with demonstrated susceptibility to this particular phage combination. All documentation for phases 1a  and 1b  has been completed, allowing for phase 1a to start in Q2 2023. In the longer term, a scalable procedure for phage therapy will be developed, which, after initial establishment, can also be applied to other phage entities in its modified and adapted form.\u201cPhage4Cure\u201d . The project investigates whether, with today\u2019s biopharmaceutical possibilities, it is possible to adapt phage preparations in the hospital pharmacy to the individual patient and prepare them in time for therapeutic use\u2014thus, the practicability of magistral production of phage products is investigated . The project focuses on wounds on arms and legs infected by multidrug-resistant pathogens. In the first project phase, phages were isolated (DSMZ GmbH), characterized and preserved with all methodology following the OECD Best Practice Guidelines for BRCs. Subsequently, they will be produced in a biotechnological process (Fraunhofer ITEM) in such a way that they can be made available in a purified form to the hospital pharmacy, as API components. The second phase of the project is aimed at treating patients. First, pathogens from a patient\u2019s wound material are typified for phage sensitivity and then a tailor-made phage preparation is produced. Treatment, initially with phages against multidrug-resistant P. aeruginosa, will begin in February 2023, and against S. aureus from August 2023.\u201cPhagoFlow\u201d  characterize phage-resistance development during in vivo preclinical treatment, (2) decipher the impact of these cocktails on the respiratory and intestinal microbiota, as well as on microbiota-dependent immune responses, (3) evaluate the efficacy of the cocktails in penetrating biofilms produced in vitro and ex vivo on explanted human-lung tissue, (4) investigate the direct interaction of phages with the immune system, and the mechanistic basis for the synergy between innate immune cells and phages during therapy, and (5) characterize by mathematical modeling the efficacy of these cocktails in vivo, and propose optimized treatment regimens [\u201cMAPVAP\u201d . The MAPVAP project is a collaborative research program financed by France and Germany through the 2019 dedicated call on antimicrobial resistance ) . Ventilaregimens .IDEAL-EC project focused on evaluating phages against extended-spectrum beta-lactamase-producing clinical E. coli samples. The WHO has recently classified E. coli as a pathogen of international concern. Antibiotic-resistant E. coli strains are on the rise, and can cause severe infections, especially in immunocompromised patients. During the project period, an E. coli phage cocktail was developed, which is currently being investigated in preclinical models . The results of this project will serve as the basis for subsequent preclinical and clinical studies investigating the effects of E. coli phages on the human microbiota and their applicability for therapeutic purposes.\u201cIDEAL-EC\u201d . In collaboration with the Leibniz Institute DSMZ and DZIF, the EVREA-Phage envisages the characterization of new phages and the composing of a phage cocktail against E. faecium for oral application, to specifically decolonize intestinal VRE E. faecium in immunocompromised patients. Considering potential concomitant colonization by different E. faecium strains in individual patients involving not only VRE but also VSE (vancomycin-sensitive) or VRE+ strains being additionally resistant to linezolid, the phages selected for the cocktail will be tested against panels of all mentioned E. faecium variants, to ensure the broadest-possible host coverage. Large phage and strain panels were compiled to perform the full biological-phage characterization. Bacterial strains are clinical isolates from German university hospitals of the DZIF network and from the Robert Koch Institute, and all phages were newly isolated by the Leibniz Institute DSMZ. EVREA-Phage envisages the demonstration of phage efficiency by in vitro and in vivo gut models at the University Hospital Bonn and the Helmholtz Centre for Infection Research, Braunschweig, to confirm phage effects, according to regulatory requirements and the BfArM scientific advice. All derived data of EVREA-Phage will flow into the preparation of an IMPD  required for later authorization of a clinical trial, applying highly purified approved phage preparations.\u201cEVREA-Phage\u201d . The DZIF-funded, translational preclinical project www.sprind.org). The project is currently identifying top groups of applied research in Europe to form clusters of excellence and to focus efforts, for example, on innovative and rapid approaches to match patient isolates to phages, on improving the yield of phage-production processes and on exploiting synergies between phages and antibiotics. In addition, with a focus on ESKAPE pathogens, requirements for large-scale production of phage cocktails for bacteria with broad-spectrum phages  or small-scale on-site production for individualized phage therapy for pathogens with narrow-spectrum phages  are being evaluated.\u201cPhage2030\u201d  aims to identify feasible research efforts and manufacturing pathways, the coordination and targeted support of which would represent a \u201cleap forward\u201d in making phage therapy available to the German public by 2030. The validation study required for this is funded by the Federal Agency for Disruptive Innovation, SPRIN-D . Another project with close cooperation between the Leibniz Institute DSMZ and the DZIF will focus on the pre-clinical evaluation of phage\u2013antibiotic synergy in multidrug-resistant https://github.com/deng-lab/viroprofiler/, accessed on 12 January 2023\u201d) [PHARMS\u201d  aims to discover novel antibacterials against A. baumannii, Helicobacter pylori, and Haemophilus influenza, by studying phage\u2013host interactions using culture-independent and multi-omics approaches. \u201cCOVPHA\u201d  aims to identify the co-infecting MDR bacteria in COVID-19 patients using metagenomics and to isolate effective phages against them. In addition, multiple phage endolysins have been identified. The efficacy of the phages and endolysins will be tested in vivo and ultimately applied to patients, under compassionate use and in an early clinical trial. In addition, the Munich Phage Center led by L.D., which is part of the \u201cCenter for Integrated Infection Prevention (ZIP)\u201d at the Technical University of Munich , aims to develop innovative phage-based therapeutics against MDR pathogens in humans and animals. To this end, a high-throughput phage-culturomics pipeline to automatically isolate and characterize phages as well as a well-characterized phage bank against highly-critical bacteria, including ESKAPE pathogens, are currently being built.Helmholtz Centre Munich and Technical University of Munich (head L.D.) Conducting multiple projects funded by the European Research Council Innovative Training Networks , DFG priority program \u2018Novel Concepts in Phage Biology\u2019 (duration 2021\u20132024) and sequencing program \u2018Asthma-Phage\u2019(2023\u20132025) that aim to explore the cross-talk between virome, microbiome and the human host in dysbiosis-associated digestive and respiratory diseases  and to eradicate causative bacteria using synthesized-phage communities (phageome therapy). To this end, multiple tools have been developed, including Replidec to predict phage replication cycles  and Viroy 2023\u201d) ,30. The Klebsiella phages and their bacterial hosts are characterized, optimized and prepared for therapeutic applications with a one-health perspective . The aim is to expand the host range of phages, optimize cell-free production, support phage therapy in clinical trials and individual healing-trial protocols with personalized phage preparations, and improve therapeutic approaches of clinical partners [The \u2018Therapeutic Phage Group\u2019 of the Bundeswehr Institute of Microbiology, Munich (head J.B.): In collaboration with the European Commission, Salmonella enterica, Campylobacter spp.), but so far, no general permission for the use of phage products exists in Germany. As the clonality and the target bacteria in the veterinary/food sector are broadly overlapping, products for animal use  may also be suitable along the food chain. So far, various research projects have confirmed the beneficial effect of different applications, such as bioremediation, biocontrol and bio preservation between animal housing (pre-harvest) to the food product (post-harvest) [Salmonella, Campylobacter, Yersinia, Klebsiella, Vibrio) have been developed to gain access to a broad spectrum of phages suitable for potential treatment application. In addition, questions addressing the safety of phages are increasingly being studied to close the knowledge gaps for the final risk assessment.\u2018One-health\u2019 phage applications are increasingly discussed worldwide, but have so far hardly found their way into a holistic approach with respect to the German food chain and veterinary/environmental use. For over a decade, German experts of the different \u2018one-health\u2019 compartments and the legislative authorities have been discussing the routine implementation of phages. Major concerns hampering a final decision include esp. (A) a lack of risk assessment for phages released into habitats, (B) the bacterial diversity associated with low reduction and phage-efficiency rates, (C) a spread of phage-resistant bacterial subpopulations associated with a loss of treatment options, and (D) potential disadvantageous effects or changes in microbial communities affecting plant, animal, and human health . Neverthharvest) ,33,34, bOctober 2017: Two symposia, both organized by the University of Hohenheim in Stuttgart, brought the potential of the German phage-researchers\u2019 community to light. The first symposium, in 2017, with 170 participants from 20 countries, addressed the full spectrum of phage research and application. The plenary included experts from the two regulatory bodies BfArM and Paul-Ehrlich-Institut (PEI), as well as from the Federal Ministry of Education and Research (BMBF), who discussed \u201cquo vadis, German phage research\u201d. The regulators stated that there was no clearly defined product to prompt an immediate licensing activity, and urged interested companies to approach the authorities proactively for an early detailed exchange, to avoid costly procedures. They declared their openness to dialogue to support companies planning to develop a phage product. It was also stated that close international cooperation should compensate for the limited number of phage applications worldwide.www.sto.nato.int, accessed on 12 January 2023\u201d) was held at the Military Academic Hospital, Berlin. A total of 67 invited participants from 17 nations exchanged views on the topic of the re-introduction of phages into today\u2019s medical space. From individual working groups as well as from the plenary, it was emphasized that the creation of an international register and an easier exchange of already isolated phages would be particularly important to advance phage therapy.December 2019: A workshop within the framework of the NATO HFM-313 Research Task Group (RTG) \u201cRe-introduction of phage therapy in military medicine\u201d , developments in phage diagnostics and therapy were presented and discussed. The topics of talks included reporter-phage-based detection of bacterial pathogens, phage-receptor-binding proteins for pathogen identification, and an enzyme-linked receptor-binding-protein assay. Further topics included the genetic modification with bactericidal transgenes, non-cellular phage production, issues with GMP production of therapeutic phages in Germany, the efficacy of commercial phage preparations against MRSA, and the possibilities of overcoming the antibiotic resistance in dormant bacteria, as well as the genetic modification of phage TUN1, specific for 4 MRGN K. pneumoniae.October 2021: Phage therapy was also a topic at the 17th Medical Biodefense Conference in Munich, Germany 2021 (MBDC21). In several sessions , several externally funded projects emerged and DZIF launched campaign-like activity in the area. In addition, cooperation with the regulators has intensified considerably, as a result.DZIF TransPhage-Net) was founded and a roadmap for translational phage research was drafted.July 2022: To promote translational phage research, DZIF organized a first strategic meeting on \u201cBacteriophages in Science and Clinical Use\u201d. The symposium took place in Frankfurt am Main, with 75 national and international participants, including physicians, scientists and industrial partners. Following expert talks on important phage-related research aspects, regulatory requirements and first experiences with phage treatments, all participants actively engaged in further identification of specific needs to promote phage research and clinical application in Germany. As major results of the symposium, a DZIF Translational Phage-Network , originated in Germany in 2020. The combined efforts and expertise of phage scientists, physicians, pharmacists, microbiologists, bioengineers, bioinformaticians, and regulatory affairs officers have joined together to support physicians who intend to treat patients with phages. Topics of the monthly meetings are the presentation of activities of the individual network partners, preparation of funding applications and the identification of suitable partners for clinical and scientific studies. Invited presentations from members and guests on new approaches to phage therapy and the required regulatory processes aim to inform and integrate successful approaches in Europe, Israel, and the US.DZIF has a strong interest in promoting translational phage research, i.e., the successful transfer of research results from bench to bedside. Within the DZIF translational thematic unit \u201cHealthcare-Associated and Antibiotic-Resistant Bacterial Infections\u201d (TTU HAARBI) several initiatives have started up to support phage research in Germany. To provide physicians with an official directive for safe phage therapy, the German Society for Infectious Diseases initiated an official A more complete overview of the current phage landscape in Germany is provided through a literature review of recent years and the personal communication of various research groups. The working groups of institutions and companies contributing to translational/clinical phage research are compiled in Each author provided one or more answers to the three questions presented below in the legends to As seen in Research topics that the participants would like to see prioritized are mainly related to optimizing phage therapy and investigating safety aspects . SpecifiThe biggest hurdles towards implementation of phage therapy in Germany, as seen by the participants, include the current lack of approved phage therapeutics, missing uniform guidelines and quality standards, the limited access to phages for clinical use and the lacking infrastructure for phage production, sensitivity testing and application in the clinics . MoreoveThe aim of this article was to present the current German-phage-therapy landscape from the perspective of clinicians, researchers working closely with them, and those responsible for regulatory aspects, i.e., a group representing the present efforts of physicians, pharmacists, microbiologists, bioengineers, bioinformaticians and regulators.Phage4Cure and PhagoFlow, mainly with phages against P. aeruginosa and S. aureus. Due to the current and previous limitations, there are probably hardly 100 patients who have been treated in Germany in the immediate past, in marked contrast to the many thousands of phage applications between the 1930s and the 1960s. Nevertheless, the treatment volume will increase from 2023 onwards, due to the imminent start of clinical phases of the ongoing research projects. It is also becoming apparent that numerous clinics will take advantage of permission-free production of medicinal products (\u00a713 (2b) German Medicinal Products Act) in the foreseeable future, to produce phages for their patients.As phages are currently not clinically approved, phage therapy is only being applied to a limited number of patients in university hospitals and a German armed-forces hospital, which operate without exception under the umbrella of Article 37 of the Declaration of Helsinki. Either ready-to-use composite phage products are used  or phages are produced within the framework of permission-free production in the respective hospital for their own patients, under supervision of the treating physician. To date, there is only one place in Germany that can produce phages on a larger scale, according to GMP (Fraunhofer ITEM in Braunschweig). These GMP phages will initially benefit the publicly funded research projects It is striking that different phage banks exist in Germany, but, due to their different institutional backgrounds and the policies to which they are bound , they do not operate in comparable modes, and do not freely exchange all their phage holdings. There is also no full genome sequencing of the respective complete phage collections. Thus, a complete and common data collection of all bioinformatic data and the respective host areas is not yet available. However, the DSMZ envisages sequencing the genomes of all clinically relevant phages quickly.SPP2330\u2014aims to elucidate the regulation of the phage life cycle, identify new bacterial anti-phage defense systems, and study the impact of phages on viral communities and biofilms . These research efforts will also help to further improve the efficacy of phage therapy.A major limitation of the present analysis is certainly the arbitrary selection of authors, which cannot cover all the important groups contributing to phage therapy and research in Germany. However, the group encompasses stakeholders from most\u2014if not all\u2014relevant German phage-research consortia, so that overall a valid portrait of the situation in Germany could be generated. In addition, there are numerous research groups engaged in translational research, for example, to facilitate the bacteria\u2013phage matching process  or exploTherefore, although the funding situation and awareness require further improvement, important steps have been taken en route to addressing the antibiotic-resistance challenge with phage therapy. It must also be emphasized that large sums of public funding are already being used for research projects, and that the expert report on phage therapy commissioned by the German Bundestag in 2021\u2014expected to be published in 2023\u2014also underlines the serious interest of political decision makers. In the opinion of the authors, following the demonstration of the clinical efficacy of phage therapeutics, the current legal framework for the use and production of phages  should be adapted to the special requirements of phages, to ensure patient access in a timely manner. However, the present regulatory situation can also be viewed positively. The current relatively low level of information in the population and the legal situation mostly prevents uncritical use of ill-defined phage preparations. The broad application of phage therapy without appropriate rules and scientific exchange of treatment results would probably result in unnecessary failures, and may contribute to discrediting the therapy. The worst case would be that phage therapy would be abandoned again, before proper implementation\u2014a situation resembling the first half of the 1940s in the US and Germany. Thus, establishing controlled, high-quality phage production and therapy is paramount.Against the background of these considerations, the German regulatory authorities support phage therapy and the respective approaches for patient access, i.e., a pragmatic approach of magistral production for selected research, and clinical facilities in Germany. Options such as the Belgian model, i.e., a legal framework that allows for individualized phage therapy using magistral formulations (see reference  for detaA nation- or EU-wide common approach to modern phage therapy, developed together with international actors, is the ideal. Indeed, institutional dependencies and personally motivated efforts to commercialize phage production and therapy may lead to demarcations and limited exchange of information. Nevertheless, the current situation shows activity supported by great enthusiasm in investigating the potential and implementing phage therapy in Germany as quickly as possible. So far, the stakeholders have been substantially supported by the regulatory authorities. At present, there is an urgent need to have more phages available for clinical testing and use, to expand production capacities, develop quality standards for production and clinical use, establish a registry with structured documentation of all treated patients, and gain more scientific evidence for the effectiveness of phage therapy. Current important scientific questions should urgently be answered through targeted networking and funding of translationally oriented research institutions, so that the clinical use of phage products in Germany can take a crucial step forward in the fight against multi-resistant bacteria."}
{"text": "Streptococcus suis as a model, we describe the prevalence of prophages and the anti-viral defence arsenal in the genome of the pathogen as a means to define the genetic building blocks that are available for the (synthetic) development of phage-based treatments. The data and approach described herein may provide a framework for the development of therapeutics against an array of bacterial pathogens.Bacterial infections of livestock threaten the sustainability of agriculture and public health through production losses and contamination of food products. While prophylactic and therapeutic application of antibiotics has been successful in managing such infections, the evolution and spread of antibiotic-resistant strains along the food chain and in the environment necessitates the development of alternative or adjunct preventive and/or therapeutic strategies. Additionally, the growing consumer preference for \u201cgreener\u201d antibiotic-free food products has reinforced the need for novel and safer approaches to controlling bacterial infections. The use of bacteriophages (phages), which can target and kill bacteria, are increasingly considered as a suitable measure to reduce bacterial infections and contamination in the food industry. This review primarily elaborates on the recent veterinary applications of phages and discusses their merits and limitations. Furthermore, using  Campylobacter spp., Salmonella spp., E. coli, Clostridium spp., Listeria monocytogenes and Streptococcus suis take a significant toll on animal welfare and exert enormous financial losses on the industry [The global livestock industry is a major contributor to food security and economic development, with a value of about 1.4 trillion dollars . Howeverindustry . It has industry . In Austindustry .In the 20th century, the concept of \u201cOne Medicine\u201d became a popular topic in public health . This coStreptococci (GAS-SLOdm) and pilus 2a backbone protein of Group B Streptococci (GBS-BP-2a) were the selected prototypes of bacterial proteins (antigens) [Vaccination with autogenous bacterins, a suspension of attenuated or killed bacteria isolated from a specific herd and administered to nonimmune animals within the herd/flock, is a standard practice in infectious disease prevention . Althougntigens) . This vaThe administration of antibiotics is the most widely implemented measure to prevent (prophylaxis), control (metaphylaxis) and treat (therapeutic) bacterial infections. While the other strategies have proven effective, the use of antimicrobials on farms\u2014particularly as a therapeutic measure\u2014remains a widely adopted practice . TetracyS. suis.With recent increased stringency in regulatory frameworks pertaining to antibiotic usage, new prophylactic and therapeutic strategies are required and actively being explored. These include medium chain fatty acids (MCFA) ,39, lyso30 to 1032 particles on Earth [Bacteriophages or \u201cbacteria eaters\u201d are viruses that infect and kill a cognate bacterial host. These bacterial viruses are ubiquitous and have an estimated abundance of 10on Earth . Their pon Earth . The genon Earth ,46.After their discovery, many studies investigated the potential of phages as an antibacterial in clinical cases, often with contradictory treatment efficacies reported. However, some of the challenges encountered in early phage therapy trials have been attributed to knowledge gaps in phage biology, procedural errors in experiments, and inadequate technology to study host\u2013phage interactions . These cPhages target bacteria in a highly host-specific manner but do not infect eukaryotic cells and have no reported serious adverse effect on treatment subjects. These desirable properties render some theoretical advantages to phages relative to antibiotics as prohylactic or treatment candidates. Conventionally, phage application in therapy, biocontrol or decontamination began with phage isolation followed by screening against a panel of host strains. In vitro characterisation such as determining phage titres, pH, thermal stability and growth kinetics was carried out, and phage stocks were prepared and stored for future investigations or application A. Novel While the use of phages has not been approved for commercial use in most countries, they are used on a case-by-case basis in treating patients for whom currently approved therapeutics have failed (compassionate phage therapy). The world is currently moving toward \u201cgreener\u201d crops and animal food products, with an attitudinal shift in consumer preference for antibiotic-free and sustainably farmed and or processed products . On farmSalmonella spp. remains a leading aetiological agent of human food poisoning and gastrointestinal infections in swine, poultry and other livestock. As an enteric pathogen, intestinal contents are usually affected, which predisposes carcasses to contamination during slaughter and packing. To prevent this, controlling colonisation of the pathogen prior to slaughter is practised. Thanki et al. determined the efficacy of a two-phage cocktail in reducing Salmonella enterica colonisation in piglets. Weaner meals infused with spray-dried phages were fed to experimentally infected piglets over five days. Phage treatment significantly reduced Salmonella carriage in the stomach tissue by 1 \u00d7 101 CFU/g, duodenum tissue (1.05 \u00d7 102 CFU/g), colon content (1 \u00d7 101 CFU/g) and caecum (1 \u00d7 101 CFU/g). A 16 S rRNA gene sequencing analysis demonstrated no deleterious effect on the overall microbiome following phage treatment. However, a group that received a phage diet yielded higher abundance of Prevotellaceae\u2014a Bacteroidete that has been associated with disease resilience and growth performance in pigs [Salmonella in the caecal samples (95%) and ileal samples (90%) when pigs were orally administered a microencapsulated phage cocktail post-bacterial challenge [Salmonella Typhimurium phage in diets significantly reduced bacterial shedding in pig faeces while increasing growth performance [Salmonella and other infections associated with poultry has been extensively reviewed by Mosimann et al. [ in pigs . This st in pigs . A similhallenge . Gebru eformance . Assessmn et al. . Contrasn et al. ,66,67. TEscherichia coli (APEC) is an extra-intestinal E. coli that causes local and systemic infections in avian species such as chicken, turkey and ducks. Some common colibacillosis caused by APEC include egg peritonitis, pericarditis, salpingitis, airsacculitis, cellulitis and osteomyelitis [E. coli O78. Birds that were treated with phages following challenge presented less severe clinical manifestations compared to the challenged untreated group. In addition, administration of phages offered complete protection, whereas in the untreated group, a mortality rate of 26.7% was recorded [Avian pathogenic myelitis . Collectmyelitis . APEC mamyelitis ,71. Tawamyelitis . A similrecorded . The suprecorded .Campylobacter is a zoonotic pathogen responsible for ~25% of all human diarrheal illnesses globally [C. jejuni populations in the gut of broiler chickens by 2.4 log CFU/g without disturbing the resident gut microbiome [C. jejuni infection is the reported in vitro and in vivo emergence of phage-resistant isolates (up to 13% of isolates) [Campylobacter phages were grouped into I, II and III [C. jejuni counts in broiler chickens by 3.0 log units compared to a two-phage cocktail made of only group III phages (1.0 log unit). In addition to the higher reduction, combining group II and III resulted in lower levels of phage-resistant isolates compared to using a single phage or a homogenous phage cocktail [Campylobacter phage cocktail design has been validated in vitro using several group II and III phages [C. jejuni infection in chickens has been reported by other studies with varying degrees of efficacy [globally . The baccrobiome . One majsolates) . However and III . In a 31cocktail . The effI phages . The appefficacy ,81,82.Clostridium perfringens Type A strains form part of the resident microbiota of animals; however, toxin-producing strains are implicated in a number of livestock diseases, including necrotic enteritis in poultry, necrohaemorrhagic enteritis in cattle, haemorrhagic diarrhoea in swine, and pigbel in humans. The multi-host nature of this pathogen makes it an ideal target for phage therapy. The phage cocktail \u201cINT-401\u201d was examined for its effectiveness in chicken necrotic enteritis (NE). As part of the study, three methods of oral administration were tested. Throughout the 42 days of observation, all phage treatments significantly (p < 0.05) reduced mortalities due to NE to 0\u201314.0% compared to the mortality rate among the challenged untreated birds (64%) or the antibiotic/medicated group (50%). In-water delivery of phages was the most effective delivery method, with 0% mortality due to NE [C. perfringens via oral gavage. Phage treatment via similar routes significantly improved clinical parameters and reduced gross lesions compared to the infected untreated birds. The mortality rate was 30% in the phage-treated group compared to the infected untreated group (55%) [ue to NE . In a reup (55%) .Aeromonas hydrophila infection in Nile tilapia. A. hydrophila is a zoonotic pathogen reported as the leading cause of septicaemia in freshwater fish [A. hydrophila in fish as well as stimulated the development of specific IgM against the MDR A. hydrophila [Vibrio harveyi and administered in-feed phage [Aquaculture is one of the fastest growing industries in agriculture. The FAO reported that fish accounted for 17% of all animal protein consumed globally in 2017 . Aquaculter fish . In theidrophila . The immdrophila ,91,92. Hed phage . The difed phage . It is led phage . Other fed phage . Encapsued phage ,98. Phaged phage ,100,101.p < 0.05) and reduced the incidence of diarrhoea in weaned piglets (p < 0.001) [Phages have been demonstrated to play a significant role in growth promotion and improving gut health. Upadhaya et al.  assessed< 0.001) . Studies< 0.001) ,83,104.Pseudomonas syringae, Pectobacterium spp., Ralstonia solanacearum, Dickeya solani and Xanthomonas spp. are some of the leading causes of bacterial diseases in crops. Phages that target these plant pathogens have been isolated and characterised as safer alternatives to conventional broad spectrum chemotherapeutics. Bacterial canker caused by Pseudomonas syringae is an economically important disease in farming. The efficacy of single phages in controlling canker was evaluated by spraying the leaves of two-year-old cherry trees with P. syringae. Phage treatment almost completely cleared the bacterial population (down to < 10 CFU) by the fifth week [Dickeya solani and Pectobacterium spp. account for half of all Enterobacteriaceae soft rot (SRE) in angiosperms, including food and ornamental crops. A six phage cocktail was used to control soft rot in potato tubers following a 5-day incubation. Washing tubers in a phage preparation significantly reduced (p < 0.0001) maceration in D. solani infection [Pectobacterium atrosepticum [Phages have also been evaluated as biocontrol agents in crop production. fth week . Dickeyanfection . Phage tsepticum .After harvest, both animal and plant products are susceptible to bacterial contamination and spoilage while in storage or undergoing postharvest processing. The FAO estimates show that the global economy loses $220 billion annually due to plant diseases . MoreoveEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) bacteria have been characterised and assessed in vivo for their potential in veterinary medicine and the food industry, with several commercial phage preparations  based on the antigenicity of their capsular polysaccharides (CPS) [Streptococcus parasuis, 32 and 34 as Streptococcus orisratti, and 33 as Streptococcus ruminantium [S. suis. The prevalence of serotypes varies among different locations; however, most clinical cases worldwide have been associated with serotype 2 [ry tract  and, to ry tract . However piglets ,134. S. es (CPS) . DNA hominantium . For diarotype 2 .S. suis, proteins such as suilysin (Sly), DNase, muramidase-release protein (Mrp), surface antigen protein, VirA, and extracellular protein factor (Epf) contribute to the overall pathogenicity of the bacterium [In addition to the CPS, which is a critical virulence factor of acterium ,140,141.acterium . Colonisacterium .S. suis is the most prevalent bacterium implicated in systemic infections in swine [S. suis infections is not obligatory in many countries and hence data on global prevalence is limited [S. suis on Spanish, Dutch and German farms determined that the cost per pig was \u20ac 0.60\u20131.30. Using a stochastic model, the study reported higher morbidities and mortalities in nurseries than in any other production stage. This reflected the higher cost of metaphylactic  measures in piglets compared to that of autogenous vaccines for sows [S. suis type II infections alone were \u00a3100,000\u20131.3 million annually in Great Britain [in swine . Regarde limited . This ha limited . An evalfor sows . In 2005 Britain . The los Britain .S. suis is not limited to the porcine industry [S. suis infection among piglets, a report of a human infection was identified among meningitis patients in Denmark [S. suis meningitis and septicaemia among patients were recorded in the Netherlands and other European countries, suggesting that the pathogen may have crossed the species barrier long before it was first reported [S. suis genomes suggest the emergence of human-associated clade from swine isolates originating from western Europe. Using representative samples from six continents, Dong et al. traced the most common recent ancestry of human-associated clade to 1802\u20131855 [suis-carrying pigs in enclosed and often poorly ventilated spaces\u2014and the export of selectively bred pigs from Europe and United States to different continents [S. suis strains, new epidemic lineages appear to have emerged in Asia, where reports of human and porcine infections remain prevalent [S. suis infections among humans with no direct contact with pigs are becoming increasingly common [S. suis infections and S. suis isolates have been recently identified in cattle, lamb, dogs and other animals [As is the case with many viral and bacterial pathogens, the threat posed by industry . Within  Denmark ,150. In reported . Recent 802\u20131855 . A populntinents . Followirevalent . Cases oy common ,157,158. animals ,161,162.S. suis are limited. To date, only five lytic phages  have becreening . Other sisolates ,166,167.equences ,169. HowS. suis phage landscape, we analysed 133 publicly available whole genome sequences of S. suis for the presence of prophages and anti-phage defence systems. The sequences/strains (https://padloc.otago.ac.nz/padloc/) (accessed on 22 June 2022) [Understanding host\u2013phage interactions is key in the evaluation of phages as therapeutic agents against specific bacteria. These interactions include the preservation of phage populations through integration (prophage) in host genomes and the defence mounted by host bacteria via the expression of anti-phage systems. To shed light on the /strains  were ranne 2022) . Detectine 2022) . The prene 2022) . A phagene 2022)  and visuS. suis genomes encoded a total of 1890 anti-viral proteins representing 20 distinct anti-viral systems  of RM systems in most prokaryotic genomes [S. suis genomes could be grouped into three categories: DNA/RNA degradation, abortive infection, and systems of unknown mechanisms  per sequence (ranged 13\u2013141 CDS), among which, an average of 49.7% could be assigned a putative function. Bacterial proteins associated with virulence, such as toxin/antitoxin, efflux pumps, zeta toxin, and virulence-associated protein E, were detected in the prophages. In addition, anti-phage defence genes such as RMs were detected in prophage sequences, indicating that some bacterial defence systems are prophage-encoded . The funp < 0.0001). The influence of host genome size on the association between prevalence of prophages and number of defence systems was controlled using a correlation matrix (p < 0.0001). Similarly, a moderately positive correlation was observed between the host genome length and the number of prophages . Up until 2.5 Mb, the number of prophages increased with increasing genome length  [S. thermophilus, in which all strains possibly encode the system, or in S. mutans, where 95% encode at least one type of CRISPR-Cas [S. suis. We categorised the defence systems on the basis of their molecular mechanism. Nucleic acid degradation systems were the most common mechanism in the S. suis defence arsenal. This is in line with the estimation for prokaryotes [Persistent lysogeny and anti-viral mechanisms are critical phage\u2013host interactions that affect the success of phage infection, particularly in the application of phages as therapeutic agents. We investigated the prevalence of prophages in e system . Interesa (~50%) . MoreoveISPR-Cas . Recent karyotes .S. suis is higher than has been reported for other Streptococcal species such as S. pneumoniae (1.4 per genome) [S. mutans (1.5 per genome) [S. suis chromosomes. However, this observation could also be a function of the sample size. An all-against-all genomic similarity revealed little proteomic relatedness among full-length prophages suggesting a high genetic diversity. Two patterns can be described for phage groups with low gene relatedness. Firstly, along the genomes of phages of common ancestry, several gene homologs of little similarity exist. Alternatively, phage mosaicism may arise from genetic transfer between dissimilar phages such that only a small fraction of the genome would contain highly similar homologs. The latter trend is consistent with the low inter-cluster relatedness as we qualitatively show in a schematic representation of functional genome alignment   and S. m genome) . Of the lignment . Furtherosaicism . Future S. suis host genome size, the prevalence of prophages, and the abundance of defence systems exists. In Salmonella and E. coli, larger genomes were associated with more integration hotspots. In particular, there seemed to be a selection for integration sites within intergenic and regions of low gene expression rather than highly transcribed regions. In highly transcribed regions, spillovers from transcribed genes could trigger phage gene expression, which can reduce host abundance and consequently the temperate phage population following a switch to lytic cycle [S. suis and how this influences prophage prevalence among different strains. Following integration, temperate phages can confer fitness to the host. For instance, in the context of the correlation between defence systems and the number of prophages, annotations revealed the presence of phage-encoded RM systems , CRISPR-Cas systems, and in vitro synthetic genome assembly have been leveraged to custom-design phages with desired characteristics [Klebsiella sp. and Yersinia sp. in a mixed population [S. thermophilus has been used to introduce specific point mutations, large deletions, and ORF replacement, with relatively high efficiencies. This model was adapted for the construction of escape phages through the introduction of a methyltransferase gene such that the resulting phage mutants evaded the R/M system of the host. This modification significantly improved the bacteriolytic efficiency of the S. thermophilus phage 2972 [Mycobacterium abscessus in cystic fibrosis patients [S. suis using their prophages as building blocks.A potential approach to explore untapped (pro)phages against eristics . Modificpulation . Alternaage 2972 . A synthage 2972 , allowinpatients ,58. SimiK. pneumoniae infection [S. suis have demonstrated flexibility in engineering, low toxicity profiles, and broad efficacy against the pathogen and other streptococcal species, which support their potential for translational development [Moreover, co-evolution \u201cphage training\u201d experiments have been adopted to adapt phages to oppose host defences and widen the host range . The connfection . Howeverelopment ,193,194.Although naturally occurring phages possess a green appeal, the incorporation of genetically modified organisms (GMOs) in food may interfere with their approval by regulatory bodies. To date, the application of genetically modified phages in the treatment of infections in humans have been limited to rare cases with specific therapeutic circumstances meriting exemptions for their usage. Any additional barriers to approval will lessen commercialisation. Similarly, consumer reticence towards GMOs will decrease the likelihood of their application. Compelling research will be needed to prove the efficacy, safety and scalability of genetically engineered phages required by authorising bodies. Researchers also have a duty to engage with the public and policy groups to impress upon them the importance of phage solutions to tackle the catastrophe of antimicrobial resistance. As the prevalence of the problem continues to grow, this consumer reticence towards GMOs may become less of a challenge.S. suis, for which phage applications remain underexplored. We provide insights on the S. suis phage landscape and also paint a quantitative picture of the anti-phage arsenal of S. suis. Our findings reveal the diversity and abundance of S. suis prophages, which can be used as building blocks for synthesising safe lytic phages for application in food and medicine.The emergence of antibiotic-resistant zoonotic bacteria poses a threat that transcends the food industry, affecting the overall health of the environment and other animal populations. The efficacy and safety of phages have been demonstrated in several bacterial infections, with some commercial products already approved in several countries for use in food production. However, there are other zoonotic bacteria, including"}
{"text": "In the field of phage applications and clinical treatment, virulent phages have been in the spotlight whereas temperate phages received, relatively speaking, less attention. The fact that temperate phages often carry virulent or drug-resistant genes is a constant concern and drawback in temperate phage applications. However, temperate phages also play a role in bacterial regulation. This review elucidates the biological properties of temperate phages based on their life cycle and introduces the latest work on temperate phage applications, such as on host virulence reduction, biofilm degradation, genetic engineering and phage display. The versatile use of temperate phages coupled with their inherent properties, such as economy, ready accessibility, wide variety and host specificity, make temperate phages a solid candidate in tackling bacterial infections.  As the most prevalent and widely distributed group of viruses on earth, phages are estimated to be around 10iosphere . Moreovehia coli . In recehia coli , researchia coli ,7,8,9,10hia coli . Due to hia coli ,12,13,14hia coli . For inshia coli . Cell lyhia coli . Throughhia coli . Meanwhihia coli ,20. ThusTaxonomically, phages are categorized primarily according to their morphology and genome, and more refined and comprehensive classification is under way . Based oVibrionaceae family [Flavobacterium psychrophilum, which currently causes considerable economic losses in salmonid aquaculture [Temperate phages are detected in a large proportion of bacteria . When ine family , membersaculture . Around aculture , whereasaculture . E. coli, is perhaps the most thoroughly studied and widely applied temperate phage [To date, phage lambda, which infects te phage . The lamte phage . Geneticte phage . Althougte phage .Phage lambda could reproduce many generations in a lysogenic cycle. The lytic-driving genes persist in prophage, yet repressed, which is the key lysogenic maintaining force . Under cBordetella bronchiseptica and decreased the virulence of parental strain B. bronchiseptica Bb01 in mice [Siphoviridae temperate phage infecting S. aureus and shows stronger host inhibition activity than antibiotic ceftazidime [Of note, the interactions between temperate phages and the host genome are complicated. Prophage-induced lysis can impose beneficial effects on the bacterial population and, thus, is preferred in the interest of the overall situation . Most of in mice . vB_SauStazidime .Using lambda phage as a role model, we can obtain basic insights of the life cycle of temperate phages and the host\u2013phage interrelationship. Temperate phage development that may occur through the whole life cycle could be categorized into five major phases. (1) Diffusion: the phage gets through the biofilm to approach the host bacteria. (2) Absorption and injection: the phage binds with receptor proteins on the surface of the bacteria and injects the phage genome. (3) Integration and replication: the phage genome inserts into the bacteria genome and becomes a prophage, or persists independently as plasmid. (4) Induction and packaging: the prophage becomes activated and enters the lytic cycle. (5) Lysis of host bacterium: the progeny phage is released from the bacteria ,44,45,46There is expansive diversity in each step to help the phage adapt to multiple conditions . Upon eaHere, we introduce the mechanism and interaction in the order of the temperate phage life cycle. Understanding the close relationship between the phage and host in a life-cycle order will help us to build a more holistic and intuitive perception towards phage antibacterial activity .In this initial phase, phages diffuse and penetrate through the biofilm to locate and target bacteria. Composed of polymeric substances and bacteria-secreted enzymes and proteins, the biofilm matrix wraps around the microbial community and serves as a powerful physical barrier against not only phages but also immune system and antimicrobial agents ,49. BactThere is growing evidence supporting the theory that phages can promote biofilm formation ,54. HoweWhen initiating infection, phages first adsorb to specific receptors on the surface of bacteria . BacteriHowever, most of the temperate phages are limited in their receptor binding proteins (RBPs) and are obligated to specific hosts ,62. EvolListeria monocytogenes is key in escaping cell phagosomes [Many phages encode an integrase that integrates phage DNA into the host chromosome . Phages agosomes . Lysogenic conversion is the process of prophage gene expressing as a part of host genome and is reciprocal for the prophage and host . ProphagTo make sure of their chance of survival, phages develop a number of defense mechanisms to eliminate unwanted host-sharing. The known mechanisms are divided into three groups: blocking genome injection, expressing repressor protein, and binding inhibition . In addiL. monocytogenes strain 10403S, the two coexisting prophages regulate simultaneous induction and lytic activity under SOS conditions. Moreover, the host can also benefit from the cooperation of its habitants. To maintain harmonious coexistence of two prophages, AriS is discovered as a conserved phage protein and is demonstrated to be capable of avoiding SOS response and phage induction by inhibiting RecA [With regard to phage\u2013phage interaction within one same host, intricate coordination can be achieved upon triggering SOS responses by two phages sharing one host. In ing RecA .Pseudomonas aeruginosa exhibits a selective induction to phage phiMBL3 [Prophages can be induced into the lytic cycle under a series of stressors, such as antibiotics and UV rays . Some of phiMBL3 . In addi phiMBL3 . Separat phiMBL3 .Normally, the progeny phage is packaged, released, and keeps infecting the next host . HoweverSingle-stranded DNA phages cause hydrolysis of the host bacterial cell wall by synthesizing enzymes that interfere with host bacterial peptidoglycan synthesis, whereas double-stranded DNA phages, such as phage lambda, hydrolyze the host bacterial peptidoglycan structure by lysin or endolysin, which are synthesized late in replication .In a nutshell, the above steps illustrate the life cycle of temperate phages, using the lambda phage as an example. The interactions between temperate phages and their bacterial hosts are complex and intimate. While new mechanisms are being discovered, we should also look at how we can better exploit the intimate relationship between phage and bacteria to help in the fight against pathogenic bacteria.Pseudomonas, and may affect the entire lung ecosystem [Pseudomonas strains that are deleted of Pf4 prophage survive significantly longer, which indicates that the presence of prophage Pf4 is a virulence contributor [As we discussed in the temperate phage life cycle, the transduction and lysogenic conversion may cause undesirable outcome such as virulence promotion . For inscosystem . Mice intributor . Furthertributor . A changtributor . In the tributor .Nevertheless, temperate phages also have their advantages that cannot be ignored. A wide variety of temperate phages are found in nature and can be easily induced in the laboratory . TemperaTemperate phages have been widely demonstrated to promote bacterial virulence, which is the most important factor in causing infections . HoweverHosts prevent invasion by reducing virulence. The presence of prophage in the form of plasmid can provide host bacteria with resistance to other foreign DNA at the cost of host virulence. p2 is proposed to be an intact plasmid prophage in Klebsiella pneumoniae. Mutant Kp1604\u0394p2 exhibits an increase in host virulence which is determined by mouse infection models. The mutant p2 minus strain leads to 100% mortality compared to the 70% mortality of the p2 carrying strain, indicating that the presence of p2 decreases the virulence of its host [its host .L. monocytogenes, there is a classic trade-off situation. The cell wall of Listeria and its associated proteins are responsible for most of the interactions with the mammalian host [Listeria serotype 4b host range. In order to prevent phage adsorption, L. monocytogenes 4b underwent a mutation associated with phosphoribonate glycosylation, resulting in the loss of galactose from the phosphoribonate molecule. This loss of galactose not only prevented phage adsorption, but also led to a reduction in bacterial virulence [In ian host . The temirulence .Virulence shrinks under massive phage predation: A phage mixture targeting different extracellular structures such as receptors causes a huge impact on the bacterial fitness, virulence, and pathogenicity of P. aeruginosa. Secretory virulence factors, such as elastase, pyocyanin, and pyoverdine, significantly facilitate the P. aeruginosa colonization of new niches but are not directly related to the cell response to phage infection. The change in P. aeruginosa PAO1 biology is related to the number of phages that cause selection pressure on the population. The more phages appear in the environment, the deeper and more noticeable are the phenotypic changes involving a reduction of various virulence factors\u2019 production levels [n levels .B. bronchiseptica Bb01 has been elucidated in detail. Isolated from sewage water, temperate phage PHB09 is reported to attenuate host virulence by lysogenization. Not only does the temperate phage reduce the virulence of its host both in vivo and in vitro, most likely by inserting and thus disrupting the pilin protein gene, but the vaccine made of lysogenic B. bronchiseptica strain Bb01+ also showed effective protection of mice challenged with virulent B. bronchiseptica. Moreover, in the sight of possible risks rising from prophage induction and phage releasing, neither antibiotic resistance genes nor reversion of bacteria virulence are observed. All induced bacteria are lysed eventually. Thus, the successful virulence attenuation of prophage PHB09 proposes a promising frontier of temperate phages being developed as vaccines [To further investigate temperate phages\u2019 potential to benefit clinical treatment, temperate phage PHB09\u2032s interaction with its host vaccines .Conducted by Bao\u2019s team, an alleviating effect of temperate phage pre-treatment is observed on intestinal dysbiosis and inflammation in challenged mice. As opposed to the streptomycin treatment, the pre-treatment of mice with temperate phages safeguarded a stable and more diverse gut ecosystem and protected the intestinal system of mice against the pathogen challenge .Biofilms can inhibit drug penetration, and thereby significantly reduce the killing efficiency of antimicrobials . BiofilmTemperate phage cocktails enhanced with ions. Biofilm can provide a sanctuary for bacteria being hunted by antibiotics. The thick matrix of biofilm formed by bacteria is a special shield against antibiotics because of the reduced drug penetration and the accessibility [Siphovirdae family, administered with metal ions Ca2+ and Zn2+, shows the enhanced bactericidal impact both in vitro and in vivo compared to the phage cocktail alone. In this experiment, the reason ions such as Ca2+ and Zn2+ can offer an advantage to the phage cocktail is likely because of the promoted fluidity and stability of cocktail phages in the biofilm. The possible gene transfer through these temperate phages is avoided thanks to the lack of virulence gene in the studied four phages. In addition, by decreasing host virulence, prophage can also be a good helper in outcompeting other bacteria. The antibacterial effect of this recipe is determined by the biofilm removal efficiency, where added ions proved a higher bacterial CFU reduction ability. Moreover, using Galleria mellonella larvae as animal model against MRSA S-18 infection, the survival rate resulting from ions\u2013phages therapy is 10% higher than the phage cocktail alone [sibility . A phageil alone .Temperate phage encoded enzyme to eradicate biofilm. A temperate phage of Pseudomonas has been proved to be able to produce a lyase, LKA1gp49, to degrade LPS. LKA1gp49 is a lyase that degrades the O5-serotype specific polysaccharide. This enzyme degrades LPS molecules embedded in the cell envelope and disperses the biofilm matrix, resulting in an increased diffusion rate for small molecules. LKA1gp49 lyase efficiently reduces P. aeruginosa virulence in the in vivo G. mellonella infection model and sensitizes bacterial cells to the lytic activity of the serum complement. LKA1gp49 could also be a potential additive for antimicrobials, as it does not interrupt the efficacy of ciprofloxacin and gentamicin [ntamicin .Combined with antibiotics: Temperate phage can provide enhancement efficacy in killing bacteria with antibiotics [Burkholderia cenocepacia is one of the most important opportunistic pathogens in causing high mortality rates in cystic fibrosis (hereafter CF) patients [ibiotics ,123. Theibiotics . For inspatients . CF is apatients ,127. Thepatients . With unBurkholderia phage AP3 combined with antibiotics demonstrates increased bactericidal effects in in vivo experiments with moth larvae. This finding could be considered as a potent lead against bacterial strains belonging to B. cenocepacia IIIA lineage, which are commonly isolated from CF patients [P. aeruginosa is the cause of a typically challenging infection endocarditis. Increased resistance to antibiotics and a broadened host range of P. aeruginosa is observed, along with disease progression [Temperate patients . Attentigression .E. coli temperate phage HK97 and antibiotic ciprofloxacin. This synergy works in line with the depletion of lysogens which ciprofloxacin specially targets [For temperate phages, the prophage induction through the SOS response and resensitization to antibiotics are the two main synergy mechanisms . In the  targets . The res targets .In addition to functioning as a killing machine, a temperate phage can be a powerful and stable vector, for instance, carrying vaccines into bacteria and acting as a medium for protein expression. The engineering of a phage integration site or the inhibition of the toxins\u2019 gene expression can also take a toll on host virulence. Temperate phages are a robust platform for genetic engineering and modification .Phage vaccine: Temperate phage lambda\u2019s potential for delivering a DNA vaccine has been exploited and substantiated [antiated . It is eantiated . With alantiated . A vacciantiated . Using tantiated . Vaccineantiated .Lambda PLP: Phage-like particles (PLPs) derived from phage lambda have physicochemical properties compatible with drug standards, and in vitro particle tracking and cellular targeting is achieved by displaying fluorescein-5-carboximide (F5M) and trastuzumab (Trz), respectively. Phage-derived nanodrugs are modular systems that can be easily adapted to combined approaches, including imaging, biomarker targeting and the intracellular delivery of therapeutics. A \u2018designer nanoparticle\u2019 system that can be rapidly engineered in a tunable and unambiguous manner, trz-PLP binds to oncogenically active human epidermal growth factor receptor 2 (HER2) and is internalized by HER2 overexpressing subtypes of breast cancer cells, but not by breast cancers lacking HER2 amplified breast cancers. The robust internalization of Trz PLPs resulted in increased intracellular Trz concentrations, prolonged cell growth inhibition and the regulation of cellular programs associated with HER2 signaling, proliferation, metabolism and protein synthesis compared to Trz treatment. The robustness and flexibility of lambda PLP provides a platform that adapts to a wide range of utility and customized features [features .Engineered endolysins: Bacteriophage-derived endolysins are cell wall hydrolases which could hydrolyze the peptidoglycan layer from inside and outside bacterial pathogens [Gardnerella phage exhibits the ability to completely disrupt bacterial biofilms of Gardnerella vaginalis. G. vaginalis is a common vaginal bacterium, but can cause bacterial vaginosis under abnormal growth. This engineered endolysin PM-477 has a strong specificity and efficiency against Gardnerella strains, and has no effect on beneficial Lactobacillus or other species of vaginal bacteria [athogens . A genetbacteria .Temperate phage display: The temperate phage M13 phage has a wide range of applications in biomedical materials, and is used for different therapeutic applications [Chlamydia trachomatis, a globally prevalent human pathogen for which there is no effective approved vaccine [C. trachomatis infection alone, engineered phages stably express RGD motifs and C. trachomatis peptides and significantly reduce C. trachomatis infection in HeLa and primary cervical cells [ications . This is vaccine . Compareal cells .Gene insertion led to attenuated phenotype: The Rickettsia parkeri mutant strain is genetically modified by inserting a transposon into the gene encoding the phage integrase in the bacterial genome. Such a mutant exhibits significantly reduced virulence, significantly smaller phage plaques and improved histopathological alterations in intravenously infected mice compared to the parental wild type. Furthermore, single-dose intradermal immunization of this mutant strain provided mice with complete protection against the lethal R. parkeri rickettsioses in mice. Such a live attenuated rickettsial mutant strain could be used as a novel potential vaccine candidate for the treatment of spotted fever rickettsial disease [ disease .Modification of phage genes to inhibit toxin production: Produced by some E. coli, Shiga toxin (Stx) is causative of gastrointestinal diseases and hemolytic uremic syndrome with high incidence and lethality [E. coli is one of the four pathogens among the mostly benign intestinal commensal E. coli strains [ethality . Shiga-p strains . NotablyE. coli, and the genetic mosaicism of the \u03bb phage was exploited to create a hybrid phage capable of overcoming the phage resistance mechanism. The phage demonstrated superior toxin inhibition in both in vivo and in vitro infections. In the foodborne pathogen EHEC, the \u03bb prophage 933W both produces Stx2 and inhibits phage overlap infection of other \u03bb phages [To curb toxin release, the temperate phage \u03bb was genetically engineered to express a deterrent that neutralizes Stx production in \u03bb phages .Gene-modified phage with CRISPR-Cas3 system: CRISPR -Cas (CRISPR associated) system is a defense system in bacteria, possessed by around 40% of bacteria [bacteria . To exclbacteria . Temperabacteria . In typebacteria .E. coli infection and validates the superior performance over wild-type phages through in vitro and in vivo experiments. In addition, there is no evidence in this study showing that EHEC developed resistance to an engineered lambda phage [A genetically engineered lambda phage exhibits enhanced killing ability and host specificity when incorporated with the CRISPR-Cas3 system and knockdown of the lytic gene cro. This engineered phage specifically and effectively eliminates enterohemorrhagic da phage .Encode proteins that block the QS system: The Quorum Sensing (QS) system is a communication system amongst bacteria to modulate community behaviors, a regulatory system that controls the expression of virulence factors and secreted public goods [vibriophage VP882, through spying on the host-produced anti inducers during QS process, VP882 is capable of manipulating its own cycle switch [ic goods . These cic goods . Moreovee switch .P. aeruginosa phage DMS3 can protect bacteria from the attack of other phages by inhibiting bacterial quorum sensing. DMS3 encodes a QS anti-activator protein aqs1 that is expressed immediately after phage infection. aqs1 inhibits the activity of LasR, a major QS regulator, and restrains twitching motility and superinfection. Although there is a 100-fold increase in the number of cells killed by DMS3aqs1 infection compared to wild-type DMS3 infection, no more phages were produced. This suggests a role for anti-phage mechanisms. Aqs 1 offers a counterstrategy through which phages might simultaneously silence multiple antiphage defenses [defenses .Converted into the stable lytic phage: A virulent mutant SA13m obtained through the random deletion of temperate phage SA13 exhibits active lytic activity and no sign of lysogenicity. The application of SA13m in sterilized milk showed that S. aureus was reduced a non-detectable levels, suggesting that SA13m can efficiently control the growth of S. aureus in food [Enterococcus faecalis temperate phage is converted to a lytic phage for therapeutical purposes. By the deletion of the putative lysogeny gene module and replacement of the putative cro promoter from the recombinant phage genome with a 50 nisin-inducible promoter, the temperate phage is rendered virulent and with expanded host range [ in food . Two tem in food . An Entest range .Temperate phages, because of their natural biological properties, play an integral and indispensable role in the war between phage and bacterium. Temperate phages have contributed a variety of new genetic resources to the bacterial gene pool. Found in half of the bacteria, temperate phages have more accessibility than virulent phages . With prHowever, there is also room for improvement, for instance in the underutilization of the induction of hidden lysogenic phages. The actual employment of induced temperate phages can be more technically demanding, considering the need to remove integrase and integrate related genes. Targeted embedding of phages to disrupt virulence genes is a promising direction for research, of course making sure that the phages do not carry any virulence genes or integrases.In summary, this paper focuses on the life cycle of temperate phages and the interactions they have with their host bacteria. Most importantly, this paper illustrates an array of temperate phage applications we could employ in order to combat bacterial infection and benefit clinical treatment. Many experiments have demonstrated the great efficacy and usefulness of temperate phages in the treatment of bacterial diseases, but more in-depth studies are yet to be discovered."}
{"text": "All 13 modern trials concluded that phage therapy was safe. Six of the 13 modern trials were exclusively safety trials. Seven modern trials investigated both safety and efficacy; efficacy was observed in two. Two of three historical trials did not comment on safety, while adverse effects in the third likely reflected the use of phage preparations contaminated with bacterial debris. None of the historical trials contained evidence of efficacy. The evidence from trials is that phage therapy is safe. For efficacy to be observed a therapeutic amount of the right phage(s) must be delivered to the right place to treat infections containing enough susceptible bacterial cells. Trials that have not demonstrated efficacy have not fulfilled one or more elements of this principle.Trials of phage therapy have not consistently reported efficacy. This contrasts with promising efficacy rates from a sizeable and compelling body of observational literature. This systematic review explores the reasons why many phage trials have not demonstrated efficacy. Four electronic databases were systematically searched for safety and/or efficacy trials of phage therapy. Sixteen trials of phage therapy were included, in which 378 patients received phage. These were divided into historical (pre-2000; N = 3;  Phages ns alone . We therThe use of carefully selected naturally occurring lytic phage(s) to treat bacterial infections is known as phage therapy. The first recorded instance of phage therapy occurred in Paris in 1919 . The widThere is a compelling body of observational evidence supporting the safety and efficacy of phage therapy ,11,12,13This systematic review will collate and critically appraise the results of all available clinical and safety trials of phage therapy. The objective of this review is not to derive summary statistics about the safety and efficacy of phage therapy. Instead, this review will explore the question of why trials may not have yielded efficacy data in line with compelling observational data. This analysis will inform a subsequent discussion of the challenges presented by clinical trials of phage therapy. \u00ae Epub Ahead of Print, In-Process & Other Non-Indexed Citations, Ovid MEDLINE\u00ae Daily, Ovid MEDLINE and Versions\u00ae (1946\u20132021) and Web of Science (1900\u20132021). The Web of Science Core Collection Citation Indexes searched were: Science Citation Index Expanded (1900\u20132021), Book Citation Index\u2013 Science (2005\u20132021) and the Emerging Sources Citation Index (2015\u20132021). The search was performed using the following terms: (phage OR bacteriophage) AND . In Ovid these terms were followed by the suffix \u2018.mp.\u2019 and they were searched as topics in Web of Science. A study protocol was not prepared or published prior to this study.Three electronic databases were searched for articles published up to and including 18 October 2021: EMBASE (1980\u20132021), Ovid MEDLINEThe raw search results were deduplicated using Endnote (version X8.0.1). The remaining studies underwent title and abstract screening. Eligible studies were explicitly clinical or safety trials of phage therapy and were published in the English language. Trial protocols and reports of pre-trial pilot studies were excluded. There were no limitations on study date or location. Studies identified as eligible by title and abstract screening were subsequently accessed in full to ensure they fulfilled the inclusion criteria. Studies which could not be accessed in full, including presentation abstracts, were excluded; their authors were not contacted. Title and abstract and full-text screening were performed independently by the authors (SDS or HJS and JDJ), with discrepancies resolved by agreement or, where necessary, a third author (SDS or HJS). This review was conducted in accordance with the PRISMA  guidelines , and a PThe following information was extracted from each eligible study into a spreadsheet: publication year; author(s); study location; study type; number of patients; pathogen(s); condition details; bacterial sensitivity to phage(s); details of the phage(s) used; trial treatment schedule and route(s), assays used to monitor safety and efficacy and details of other ongoing therapies  where reported; efficacy findings; comments or data regarding safety and adverse effects. All eligible studies were critically assessed using a Joanna Briggs Institute checklist for clinical trials . Data exn = 5), not published in English and/or not available in full (n = 2), a trial protocol (n = 1) or deemed to be compassionate use and not a clinical trial (n = 1). Patients in the latter report were treated with intravenous phage for severe staphylococcal infection. Nine patients were treated under an Australian \u2018compassionate-use special access scheme\u2019 [Systematic searching of three databases yielded 2485 articles, from which deduplication removed 583. The titles and abstracts of the remaining 1902 articles were screened against the selection criteria. Title and abstract screening identified 26 eligible articles, nine of which were excluded after full-text screening. Articles were excluded at the full-text stage because they were: not a trial of the safety or efficacy of phage therapy , the United States (n = 2), France and/or Belgium (n = 2), Australia (n = 1), Georgia (n = 1) and the UK (n = 1). Of the 13 trials, four were designated as \u2018phase I\u2019, two as \u2018phase I/II\u2019 and seven did not contain any explicit designation. The trials covered the administration of phage orally (n = 6), topically (n = 3), both orally and intra-nasally (n = 1), intra-aurally (n = 1), intra-nasally (n = 1) and by intra-vesicular washing (n = 1). No pre-phage gastric neutralisation was employed in any of the seven studies which administered oral phage. However, phage was suspended in weakly alkaline mineral water in five of the seven trials which used orally administered phage. The diverse routes of administration reflect the diverse range of pathogens and clinical conditions to which phages have been applied. Phages were applied as cocktails (n = 11), single-phage preparations (n = 1) or both cocktails and single-phage preparations (n = 1). Six of the 13 trials were safety trials among healthy adults and children. The remaining seven studies examined the efficacy of phage therapy for burn wound infections caused by P. aeruginosa and/or S. aureus (n = 2); diarrhoea caused by E. coli (n = 1); chronic otitis caused by P. aeruginosa (n = 1); staphylococcal chronic rhinosinusitis (n = 1); complex urinary tract infection caused by various pathogens (n = 1); or chronic venous leg ulcer patients in whom the presence of infection was not a criterion for inclusion (n = 1). Patients received, or were able to receive, simultaneous antibiotic therapy in three of the seven efficacy trials. Among the seven efficacy trials, prospective investigation of the bacterial sensitivity to phage therapy was undertaken in three trials, retrospective investigation in two and no investigations in two trials. Together, excluding control groups, the modern trials represented the application of phages to 302 patients from the ages of six months old, of which 156 were healthy/uninfected and 146 had a relevant bacterial infection.The 13 modern clinical or safety trials identified were published between 2005 and 2021 ,28,29,309 phages per gram of human faeces [Given the diverse array of phages, treatment regimens and routes of administration, the 13 modern clinical and safety trials included in this review provide a broad overview of the safety of phage therapy. Representing the administration of a range of phages in high concentrations by a variety of routes and, having used a comprehensive array of appropriate safety monitoring assays, all 13 of the modern trials concluded that phage therapy was safe and without phage-related adverse effects. This will be reassuring to readers less familiar with phage therapy. However, that trials have consistently demonstrated the safety of phage administration by a variety of routes is perhaps unsurprising when the evolutionary context is considered. Phages are ubiquitous in the environment and form a significant part of commensal human flora. For example, there are an estimated > 10n faeces . Phage gn faeces ,33. TogePseudomonas with one dose of a six-phage cocktail and observed significant clinical improvements from baseline and lower bacterial counts in the phage-treated group compared to the control group [S. aureus and in 2/9 bacterial eradication was achieved [Six of the 13 modern trials included in this review were safety trials and did not evaluate efficacy. Of the remaining seven trials, two demonstrated the efficacy of phage therapy in the context of chronic antibiotic-refractory infections ,29. In 2ol group . More reThe remaining five clinical trials were published over the last seven years and did not adequately demonstrate the efficacy of phage therapy. Examination of these trials shows that in hindsight these results are not a comment on the mechanistic efficacy of phages in killing bacteria, but instead reflect ongoing clinical and technical challenges facing phage therapy trials. In 2009, Rhoads and colleagues undertook a phase I safety trial of topical phage therapy among patients with chronic venous leg ulcers . The rep2 of phage cocktail was administered to half of a patient\u2019s wound area. However, the patients experienced a delay of up to seven days in admission to the trial. In that time all the patients were receiving appropriate antibiotic therapy. Consequently, comparison of the bacterial loads between the phage treated and standard care halves of the wounds did not reveal any notable difference. Moreover, the authors noted that it was not expected that a single dose of topical phage therapy would elicit any conclusive proof of phage efficacy and rather the significance of the trial lay in its very undertaking. In 2014, Rose and colleagues undertook a small-scale clinical trial of topical phage therapy for burn-wound infection . A singlE. coli. No intestinal amplification of phages was observed, including in patients with phage-sensitive strains of E. coli, and the diarrhoea was attributed to streptococcal overgrowth in some patients. The authors considered that for those patients with sensitive E. coli there were likely too few E. coli organisms to sustain sufficient phage replication. Moreover, it is considered to be advantageous to administer gastric neutralisation prior to oral phage therapy [In 2016, Sarker and colleagues treated 78 children with diarrhoea, aged 6\u201324 months, with one of two oral phage cocktails . The tre therapy . However6 PFU/mL to a subtherapeutic 102 PFU/mL. This rendered PhagoBurn at best a safety trial as, although many phage particles were inactive, the equivalent of 106 PFU/mL of phage proteins were nonetheless administered topically. Aside from the phage concentration, it is notable that the infecting P. aeruginosa strains were of varying antibiotic sensitivity which could have complicated delineation of the relative antimicrobial effects of phage and antibiotics. Moreover, during the trial, it was found that three of the 10 participants in the phage group had P. aeruginosa colonies resistant to phage on day 0 of the trial. PhagoBurn therefore also illustrates the microbiological complexities associated with phage therapy and varying levels of resistance to antibiotics and phages that have the potential to complicate clinical trial results. In 2019, the much-anticipated results of the PhagoBurn trial were published, however the trial did not yield evidence of efficacy for several reasons . CrucialMost recently, in 2021, Leitner and colleagues published a clinical trial of intra-vesicular phage therapy for patients with complex urinary tract infection . DespiteIn summary, the four recent clinical trials that have failed to demonstrate the efficacy of phage therapy have done so because they have not achieved the \u2018Goldilocks\u2019 constellation of getting the right amount of the right phage(s) to the right place to treat infections containing enough susceptible bacterial cells. If just one of these factors is sub-optimal the efficacy of phage therapy will be dramatically reduced. However, the success of other clinical trials shows that it is possible to achieve a favourable constellation of these factors. However, such can be the variation between infections that achieving the appropriate constellation of these factors is arguably simpler on a case-by-case basis. This explains the notably greater success achieved by personalised clinical applications of phage therapy, in which both the phage(s) and clinical approach are tailored precisely to a patient\u2019s individual clinical needs.Three historical trials of phage were identified by the systematic search ,37,38. TTwo of the three historical trials, published in 1966 and 1971, did not comment on safety or adverse effects ,37. The Historical users of phage were unable to purify their phage preparations. Impure phage preparations, such as raw staphylococcal phage lysate, contain a mixture of dead (lysed) bacterial cells, phage particles and bacterial growth media. Although a reaction to phage cannot be ruled out it is unlikely given the absence of adverse effects from modern clinical trials. Instead, the reactions reported most likely reflect a combination of an injection-site reaction and immune response to the substantial quantity of staphylococcal debris. Similar reactions have been observed in other 20th Century clinical reports of the use of subcutaneously injected raw phage lysates ,40. TherS. aureus. There is also insufficient information in the report to confirm the nature of the pathologies in question; for example, we now consider acne to be a multifactorial complex pathology, rather than a straightforward bacterial infection [None of the three historical trials reported evidence of efficacy. Bryant and colleagues evaluated the efficacy of intramuscular phage injections for the treatment of chronic furunculosis among children . It is nnfection . Third, S. aureus between the two groups reflects the lack of impact that phage administered by IM injection could reasonably be expected to have on nasopharyngeal colonisation, without considering the prior absence of bacterial culture and phage sensitivity analysis. Wittig and colleagues also evaluated the efficacy of injected staphylococcal phage lysate, in this case for the treatment of infective childhood asthma . The outV. cholerae. Although phage sensitivity testing was undertaken it was not clear whether it was pro- or retrospective. Nonetheless, the results of the phage sensitivity testing showed that while all classical strain isolates were susceptible to the phages used, the El Tor isolates exhibited varying degrees of substantial resistance. The authors noted that it is difficult to predict the phage sensitivity of Vibrio strains and suggested incompatibility between the phage and bacterial strain as an explanation for the El Tor results. Of greater importance however, and neatly accounting for the failure of phage therapy among the patients with classical Vibrio, was the authors observation of the temporal incompatibility of phages with acute cholera. The authors noted that only small numbers of phages were used relative to the high number of Vibrio in the intestine. The authors further noted that the transit time in an \u2018actively purging\u2019 cholera patient is sometimes as short as 20 min. Meanwhile, at least 30 min is required to complete a phage replication cycle. Effective phage-mediated elimination of Vibrio would therefore have required many rounds of phage replication over several hours, time not afforded by the action of cholera toxin. It\u2019s therefore likely that many phages were simply excreted before they were able to undergo replication. Moreover, it was not reported whether pre-phage gastric neutralisation was performed and it is therefore unclear the extent to which gastric pH may have reduced the amount of phage progressing beyond the stomach.In 1971 Marcuk and colleagues evaluated the efficacy of oral phage therapy for the treatment of acute cholera . This trTogether the trials of phage therapy identified in this review provide reassuring data about the safety of phage therapy, affirming the findings of other recent reviews ,13. HoweNotably, despite over 100 years of clinical use, this review identified only 16 clinical or safety trials. This partly reflects the limitations of a systematic review approach: to be eligible for inclusion in this review, trials must have been indexed in full-text form in one of three online databases and published in English. This method, although thorough and appropriate for a systematic review, does not adequately account for the history of phage therapy. Many historical trials will not be indexed in online databases. Moreover, Russian and Georgian clinical trial data is generally not available to Western audiences. This creates a bias in the impression created by examination solely of reports published in Western English-speaking contexts.The evidence presented by the trials included in this review must also be viewed in the wider context of available clinical evidence, most notably observational clinical data ,12. MostNevertheless, the available clinical results paint a reassuring picture of the safety of phage therapy; in keeping with human co-evolution with phages. To the best of our knowledge there have only been 11 instances of possible clinical adverse effects to modern phage therapy, none of which were believed by the report authors to be directly attributable to phage ,52,53,54In terms of efficacy, most patients that receive phage therapy do so because they have failed antibiotic therapy. Although it is usual for such patients to continue to receive antibiotics during phage therapy, resolution of infection in the context of antibiotic resistance or tolerance in a manner that coincides with phage therapy is both compelling and consistently observed . MoreoveClinical trial evidence has become a central tenet of Western medicine, providing broad reassurance. However, it is important to acknowledge that clinical trial data is not the only form of evidence. As we have observed among the trials discussed above, phage therapy, by its dynamic and biological nature, presents inherent difficulties to the clinical trial model. First, phage preparations will require ongoing adjustment and reformulation to adapt to changing bacterial populations and resistance patterns. For example, ongoing reformulation is performed by the Eliava Institute for their pre-formulated phage cocktails. A clinical trial of a single phage is therefore likely to be uninformative, as, notwithstanding some exceptions, it will likely need to be used in combination with other phages to mitigate bacterial resistance. Likewise, a cocktail of phages subjected to a clinical trial will likely require ongoing reformulation, fundamentally altering the \u2018active ingredients\u2019. Notably, personalised phage cocktails present similarly unique challenges to the existing clinical trial model. Second, there are potentially thousands of clinically useful phages in the environment. It would be impossible to put every phage, or combination of phages, through clinical trials for just one type of infection, let alone the breadth of bacterial infections that phage could be applied to. Third, as we have seen with the trials included in this review, successful phage therapy involves the \u2018Goldilocks\u2019 constellation of getting the right amount of the right phage(s) to the right place to treat infections containing enough susceptible bacterial cells. This will be easier for some clinical indications than others, which may require a more tailored clinical approach. Consequently, demonstrating efficacy through one-size fits all clinical trial protocols may be more difficult for certain infections. Fourth, as is also evident in this review, there is much variation between existing treatment protocols. This reflects both variation in clinical phage practice and in some circumstances the tailoring of clinical phage protocols to individual patients. For example, joint infections have been successfully treated by daily administration of phage via wound catheter, a single intra-operative administration of phage or simultaneous courses of intra-articular and intravenous phage ,45,50. TRecommendations for successful phage therapy clinical trials have been published elsewhere ,62. ThisClinical and safety trials consistently demonstrate that the use of naturally occurring phage for therapy, by various routes of administration, is safe. Clinical trials also show that phage is efficacious when the right amount of the right phage(s) are delivered to the right place to treat infections containing enough susceptible bacterial cells. However, the alignment of this constellation of factors has proved tricky for previous clinical trials and, despite this and other challenges, it is hoped that future trials will deliver the compelling results much anticipated by the field. In the meantime, the evidence around the safety and efficacy of phage therapy is considered sufficiently reassuring for ongoing compassionate use when antibiotics are unable to meet clinical needs."}
{"text": "Increasing antibiotic resistance numbers force both scientists and politicians to tackle the problem, and preferably without any delay. The application of bacteriophages as precision therapy to treat bacterial infections, phage therapy, has received increasing attention during the last two decades. While it looks like phage therapy is here to stay, there is still a lot to do. Medicine regulatory authorities are working to deliver clear instructions to carry out phage therapy. Physicians need to get more practical experience on treatments with phages. In this opinion article I try to place phage therapy in the context of the health care system and state that the use phages for precision treatments will require a seamless chain of events from the patient to the phage therapy laboratory to allow for the immediate application of phages therapeutically. It is not likely that phages will replace antibiotics, however, they will be valuable in the treatment of infections caused by multidrug resistant bacteria. Antibiotics will nevertheless remain the main treatment for a majority of infections. Yersinia pestis [Bubonic plague, pertussis, diphtheria, pulmonary consumption or tuberculosis, typhoid and cholera are still ominous names of lethal infectious diseases caused by diverse bacterial pathogens. Prior to the invention of antibiotics, bacterial infections killed humans irrespective of age or sex . Often ta pestis ,3,4. Sina pestis . This tra pestis . The avaa pestis .Already in the 1950s, more and more bacteria resistant to the earliest used antibiotics were isolated from patient samples. The inventor of penicillin and Nobel prize laureate Sir Alexander Fleming warned in his Nobel-seminar in 1945 that bacteria will develop resistance to antibiotics, especially if the treatment regime is undersized . Thus, vHumans live in a world where there are bacteria everywhere, and it is impossible to avoid encountering them. Most of the bacteria cause no harm to humans, while some of the bacteria are symbiotic and thereby even beneficial and necessary for the wellbeing of them. On the other hand, some bacteria have specialized to use the human body as their source of vital nutrients or growth media. This has become possible for the bacteria during the evolution through the acquisition of special virulence or pathogenicity factors that enable the specialized pathogens to defend against or evade the immune defense mechanisms of the human body, and thereby the pathogens can thus colonize the humans . DiffereYersinia pestis and Shigellae, respectively, are contagious in very small numbers. The pathogens also differ based on their infective doses. For some bacteria it is very low, even one single bacterium may be enough to start the infection; however, for other bacteria the dose may be high, even millions of bacteria . For exaSince the causative agents of bacterial infections are highly diverse, their prevention and treatment also requires diverse actions. The spreading of some bacterial infections can be prevented by improved hygiene and better living standards, therefore, a functional waste management system and the availability of clean water plays a central role. The improvement of these two factors may specifically decrease the number of intestinal infections. In addition, malnourishment of children poses an increased risk to infections. Corynebacterium diphtheria or Clostridium tetani provide protection against diphtheria or tetanus, respectively, or the virulence factors of Bordetella pertussis protect against pertussis.Vaccination is an efficient measure to prevent bacterial infections caused by pathogens that demonstrate little antigenic variability between the bacterial isolates. Such pathogens are, for example, those included in the current vaccination programs. The vaccine containing the weakened toxins produced by Haemophilus influenza bacteria that causes respiratory and other severe infections. There are also vaccines against the causative agents of tuberculosis, bubonic plague, cholera, typhoid, tularemia and anthrax, to mention but a few. On the other hand, the production of vaccines against many other pathogens has not been successful. This may be due to the nature of the infection or the fact that the bacterial species in question has a vast number, even hundreds, of antigenic variants, making the production of vaccines not feasible.The vaccination program also includes a vaccine against pneumococci that is a result of a compromise, as there are over 100 known capsule types among pneumococcus strains. Among those the 23 most common capsule types that cause human infections have been chosen . To prevClostridioides difficile, and can cause very severe symptoms. FMT may revert the normal balance of the gut microbiome and eradicate C. difficile. It has been reported that FMT may also be a viable alternative to treat disturbations of the gut caused by other bacteria. Analogously, vaginal infections have been addressed by vaginal microbiota transplantations [Prebiotics and synbiotics have been proposed as a possible solution to manage intestinal infections . Probiotntations . An intentations . This im31) of phages in the world, a 10-fold excess over the estimated number of bacteria [Bacteriophages (phages) are viruses that are specialized to infect bacteria. It has been estimated that there is an unbelievable number , they will be released from the bacterial cell by a specifically dedicated lysis system composed of lysins and holins. After receiving an exactly timed activation signal, the lysis system breaks the cell membranes and the cell wall of the bacterium, thereby releasing the phage progeny to the environment . This al\u221210 to 10\u22129 per nucleotide per generation for bacteria [A continuous arms race between bacteria and phages is going on in nature . The bacbacteria . Thus, abacteria . The sitIt is possible that during phage therapy, virulent phage-resistant mutants are selected, as was the situation in the Tom Patterson case . In such23 phage infections occur every second [4/mL or g [In nature, phages respond to bacterial defense strategies flexibly by changes that retain their infectivity. There is a consensus that one role of the phages in nature is to keep a balance between bacterial populations to prevent uncontrolled increases in the bacterial numbers. It has been estimated that 10y second . It has /mL or g ,29. This/mL or g . This wa/mL or g . This ma/mL or g ,31.As a consequence of the arms race between the bacteria and phages, the phage populations have been divided into subtypes that are specialized to certain subtypes of the bacteria. Therefore, the bacterial strains are classified as phage-sensitive and phage-resistant. This emergence of subtypes has proceeded such that within a single bacterial species there are only a few strains that are sensitive to a certain phage. Because of this, phages are typically regarded to possess a narrow host range. In practice, this means that in phage therapy each patient isolate has to be tested for suitable phages hopefully present in the laboratory phage collection, and that phage therapy is always precision medicine where the phages eliminate from the patients only those bacteria that are sensitive to the phage. The beneficial microbiome of the body will not be affected at all by the phages .Vibrio cholera [Bacteriophages have been known of for 100 years. The first observations of phage activity were reported by British researcher Ernest Hanbury Hankin in 1896 when he studied the influence of the water of the River Ganges on  cholera . The wat cholera ,35. d\u2019HeShigella, Salmonella, Escherichia, Enterococcus, Proteus, Staphylococcus and Pseudomonas. In Georgia and Russia, physicians can use phages in routine treatment of bacterial infections. This takes place specifically when the infection is not responsive to antibiotic treatment or if the patient is allergic to antibiotics.d\u2019Herelle and Georgi Eliava founded a phage-institute in Tbilisi, Georgia in 1923. The institute started to produce phage preparates for diverse indications, and that activity still continues in Tbilisi . Phage pWhile phage therapy is experiencing a rebirth in Western countries, the present legal issues on medicinal products places special requirements on phage therapy and phage products. This regulatory framework is not completely clear yet ,39. The Phage therapy as precision medicine implies that the causative agent of the infection is identified and cultured so that it can be tested for phage susceptibility.The interaction of a phage with the target pathogen may differ dramatically in vitro and in vivo in the body fluids, and that can only be tested using surrogate systems.To meet the demand, the phage therapy laboratory needs to possess a phage collection that covers all the most common pathogens. This could involve several thousand phages, the management of which requires skilled personnel and dedicated laboratory space. All the phages need to go through a thorough characterization before they can be approved for phage therapy Production of safe phage products, even at a small personalized treatment scale, requires that approved quality control measures are in place. The sterility, stability, and the absence of endo- and exotoxins or other harmful compounds has to be ensured. In future, it is likely that phage products will be, at least partly, produced in dedicated GMP-facilitiesWhile natural phages are relatively easy to isolate against many pathogens, there are still many bacterial species for which phages are rare or even non-existentWork on phages against anaerobic bacteria or BSL class 3 pathogens presents additional challengesA realistic view is that phage therapy is not a magic bullet that would be \u201cThe Cure of Everything\u201d. It has its inherent limitations:In the context of phages, we also need to discuss (i) the (in)direct utilization of basic phage products as potential actors in therapeutic purposes, and (ii) the engineering of wild phages to better meet the need of therapeutic phages. With respect to the use of the phage products, endolysins have received most attention. Endolysins, with the help of holins, break down the bacterial cell wall and release the phage progeny to the environment at the end of the phage life cycle ,42. As eMost phages encode products that take over the bacterial systems immediately after the injection of the phage genome into the bacterial cytoplasm. A detailed understanding of these effectors would likely open possibilities to develop drugs to their targets .7 variants [In many cases, suitable phages for patient isolates are not found, and then the question of the generation and production of recombinant or even synthetic phages becomes actual. A scenario for the year 2035 on this was recently presented . It is cvariants .Mycobacterium phages converted the phages into lytic ones that were subsequently used in the treatment of a lung transplantation patient who suffered from a Mycobacterium abscessus infection [Finally, in cases where suitable lytic phages are not available for a specific pathogen but temperate ones exist, DNA engineering can be utilized the delete the gene(s) involved in directing the phage to the lysogenic cycle. Thus, for example, the deletion of the repressor gene of two temperate nfection .While phage therapy is in active use to treat bacterial infections under diverse indications in Russia and in Georgia strongly reflecting the historical situation, it has also been actively applied in Poland at the Hirzfeld Institute of Immunology and Experimental Therapy located in Wroclaw. There, starting in the 1950s, hundreds of patients have been treated successfully, with a cure rate close to 40% ,50,51. AAfter 2000, increasing numbers of scientific reports have been published on experimental therapy trials that have used phages to cure infections caused by multidrug resistant (MDR) bacteria when the regular treatments have been unsuccessful. Typically, these treatments have not been conducted singly with phages, but phages have been combined with conventional treatments ,56,57,58At the same time a few phase I and II clinical double-blind trials have been carried out with variable success ,59. Due Increasing the therapeutic phage collection. It is recognized that the most dangerous bacterial pathogens for humans belong to the so called ESKAPEE-bacteria  that oftentimes show resistance towards most antibiotics, which makes the infections caused by them difficult to cure. Therefore, our primary aim is to isolate phages against these bacterial species. Phages are relatively easy to isolate; they are especially abundant and diverse in the sewage of cities and hospitals. Our phage collection at present comprises ca. 600 different phages. We have estimated that a collection of 1500\u20132000 different phages could cover 70\u201380% of the multidrug-resistant patient isolates. The isolated phages need to be thoroughly characterized before they can be included in the therapeutic phage collection [llection . This reRapid phage typing method. Phage therapy in the future will be precision medicine for which there are selected phages that are able to infect and kill the pathogen that has caused the infection. In practice, we screen our phage collection for suitable phages using a rapid method that also allows us to react to acute bacterial infections. To this end, we have set up a method based on liquid culture that can detect phage sensitivity in 3\u20134 h (unpublished results).Production of phage preparates. One of the important goals has been to set up the processes to produce and purify phages for treatment. The purity requirements of the phage products are the same as those for other medical drugs, and there are border-values for possible contaminating impurities that should not be exceeded in the final preparate. We have optimized a purification process that requires no harmful chemicals and that can be scaled up upon request. For that we systematically tested several purification protocols in different combinations and the results were published in an article that raised wide interest [interest . In the In my laboratory at the University of Helsinki, we have worked since 2013 with an aim to make phage therapy possible in Finland. The work has been carried out mainly via external funding but also with support from the Faculty of Medicine, University of Helsinki, and from the Helsinki University Hospital laboratory diagnostics. We are close to being ready to offer safe and efficient phage products to treat severe bacterial infections. The following subprojects have paved the way for this:With our expertise and experience, we have prepared phage products for the treatments of ten patients suffering from different chronic infections caused by MDR pathogens. In all cases, the phage therapy has been safe, and for most patients the symptoms have been at least temporarily relieved (unpublished).While it looks like phage therapy is here to stay, there is still a lot to do. Clear instructions for actions and permissions for the treatments should be delivered by the medical authorities, and not just for experimental therapies. More practical experience on treatments should be collected, especially by clinicians that carry out the treatments. The curriculum of medical students does not include phage therapy yet, although it should be implemented there. Therefore, it is not imminent that phage therapy would become a common practice in the near future. Upon the accumulation of experience, knowledge of phage therapy will spread among physicians and among patients, and this will promote the position of phage therapy as a seriously considered alternative in the treatment of bacterial infections. Phages most likely will never replace antibiotics completely; however, they will be valuable in the treatment of infections caused by multidrug resistant bacteria. Antibiotics will still remain the main treatment for the majority of infections, especially the acute ones, for a long time.In principle, phage therapy could in the future target any bacterial infection irrespective of whether the causative pathogen is sensitive or resistant to antibiotics as long as suitable phages are found for the pathogen. In practice, the use of precision treatments will require a seamless chain of events where the bacterial pathogen isolated from a patient sample, especially if it is MDR, will be transferred to a phage laboratory that screens the phage collection for suitable phages. In the best case, suitable phages can be ready-made products on the storage shelves. In such a case, a phage cocktail can be delivered to the clinician within a single working day. If the phage is not ready and immediately available, production and purification of the phage will take at least three to four days, to which a few days needs to be added for quality control. In the case that no phages are found in our collection, there is a possibility to get such from other phage laboratories around the world. The patient isolate can be shipped abroad in a couple of days and tested for suitable phages in the other collections. Finally, if this also fails, there is always a possibility to isolate phages from sewage samples. These last possibilities take from several days to weeks, so for the treatment of acute infections, the only possibility is that a suitable phage is already available."}
{"text": "Pseudomonas aeruginosa or Escherichia coli\u2014two VAP-associated pathogens\u2014in na\u00efve mice without the confounding effects of a bacterial infection. Active or UV-inactivated phage cocktails or buffers were injected intraperitoneally daily for 7 days in C57BL/6J wild-type mice. Blood cell analysis, flow cytometry analysis, assessment of phage distribution and histopathological analysis of spleens were performed at 6 h, 10 days and 21 days after treatment start. Phages reached the lungs and although the phage cocktails were slightly immunogenic, phage injections were well tolerated without obvious adverse effects. No signs of activation of innate or adaptive immune cells were observed; however, both active phage cocktails elicited a minimal humoral response with secretion of phage-specific antibodies. Our findings show that even repetitive injections lead only to a minimal innate and adaptive immune response in na\u00efve mice and suggest that systemic phage treatment is thus potentially suitable for treating bacterial lung infections.Phage therapy of ventilator-associated pneumonia (VAP) is of great interest due to the rising incidence of multidrug-resistant bacterial pathogens. However, natural or therapy-induced immunity against therapeutic phages remains a potential concern. In this study, we investigated the innate and adaptive immune responses to two different phage cocktails targeting either  Pseudomonas aeruginosa (P. aeruginosa) or Escherichia coli (E. coli), which are on the WHO list of \u201cglobal priority pathogens\u201d continuously developing antibiotic resistance [Given the rising incidence of multidrug-resistant (MDR) bacterial pathogens, bacteriophages (phages) are becoming a focus of interest for treating infectious diseases ,2. Ventisistance ,6.An increasing number of in vivo studies in animal models ,8,9, as Although the interaction of phages with the innate immune system is not fully understood yet, natural or therapy-induced immunity against therapeutic phages remains a potential concern. Since phages are, among others, an ubiquitous part of the microbiome ,26, theyP. aeruginosa, highlighting another potential advantage of phage therapy against MDR-bacteria [In addition to a phage-specific immune response, a significant risk of phage therapy is the development of bacterial phage resistance . To redubacteria .P. aeruginosa or E. coli, two clinically highly relevant MDR-pathogens responsible for a large number of VAP cases. To avoid the confounding effects of phages infecting bacteria and pre-existing activation of the immune system by bacterial breakdown products or the bacterial infection itself, we assessed intraperitoneal phage administration in uninfected, na\u00efve mice. Although the two phage cocktails were slightly immunogenic, even repetitive injections over 7 days led only to a minimal innate and adaptive immune response in na\u00efve mice at either 6 h or up to 21 days after treatment start. The two phage cocktails were well-tolerated and disseminated throughout the whole organism, including the lungs. These data potentially open the way for further investigations towards the efficacy of the systemic administration of phages to treat MDR-bacteria-induced VAP.Although it was recently shown that the success of pulmonary phage therapy relies on the synergistic action between phages and neutrophils , the immAll animal studies were approved by the institutional and local governmental authorities at the Charit\u00e9\u2013Universit\u00e4tsmedizin Berlin and Landesamt f\u00fcr Gesundheit und Soziales (LaGeSo) Berlin.Mice were housed under specific pathogen-free conditions with free access to food and water and a 12 h (h) light/dark cycle. Animal housing and experimental procedures complied with the Federation of European Laboratory Animal Science Associations (FELASA) guidelines and recommendations for the care and use of laboratory animals.P. aeruginosa cocktail 5 \u00d7 107 PFU/injection per phage, anti-E. coli cocktail 1 \u00d7 108 PFU/injection per phage), UV-inactivated phage cocktail or buffer  buffer ) once or daily for 7 days (d). The mice were monitored every 24 h for body temperature, body weight and general condition according to pre-defined score sheets. At 6 h (n = 9 per group), 10 d (n = 9 per group) or 21 d (n = 5\u20136 per group), mice were euthanized with ketamine (200 mg/kg body weight) and xylazine (20 mg/kg body weight). At the 6 h and 10 d time points, intraperitoneal lavage and bronchoalveolar lavage (BAL) were performed, blood samples collected and lungs and secondary lymphoid organs  removed for further analysis. Blood cell analysis, flow cytometry analysis and determination of plaque-forming units (PFU) were performed. At the 21 d time point, blood samples were collected and spleens were removed for histopathological analysis.The 8\u201310 week old female C57BL/6J WT mice , a common model organism in preclinical research, were used. Mice were injected intraperitoneally (i. p.) with 100 \u00b5L of either active phage cocktail . In addition, whole blood was centrifuged at 15,000\u00d7 TM, Mini Protease Inhibitor Cocktail; Roche, Basel, Switzerland). BAL and peritoneal lavage samples were stored on ice and analyzed for PFUs.Mice were subjected to deep anesthesia. Peritoneal lavage was performed by flushing the peritoneal cavity (PC) with 5 mL 1 \u00d7 phosphate-buffered saline (PBS). After exsanguination via the vena cava, the tracheas were cannulated and lungs lavaged twice with 0.8 mL 1 \u00d7 PBS protease inhibitor (PI) solution . The homogenates were stored on ice for PFU analysis.Lavaged lungs were perfused via the right heart chamber with 10 mL 1 \u00d7 PBS and homogenized in 1 mL 1 \u00d7 PBS PI-solution with gentleMACSSpleens were removed and stored in 1 \u00d7 PBS on ice. PFU determination was performed with the supernatant of the single-cell suspensions described below. Draining lymph nodes  were collected, pooled in 1 mL PBS and stored on ice for further use.g for 5 min at 4 \u00b0C), the supernatant discarded and the pellet resuspended in 2 mL 1 \u00d7 red blood cell lysis buffer  for 2 min (spleen only). Cells were washed, centrifuged, pellets resuspended in PBS/0.1% BSA buffer and analyzed by flow cytometry.For isolation of leukocytes from spleen and lymph nodes, the tissue was first forced through a 70-\u00b5m cell strainer  using a syringe plunger. After washing the cell strainer with 1 \u00d7 PBS, the resulting cell suspension was centrifuged  and stained with anti-CD45 , anti-CD11c , anti-CD11b , anti-F4/80 , anti-Ly6G , anti-MHCII , anti-CD80  and anti-CD86  monoclonal antibodies (mAbs). The gating strategy for the analysis of innate immune cells is shown in For analysis of adaptive immune cells, cells were blocked with anti-CD16/CD32 , surface stained with anti-CD3 , anti-CD4 , anti-CD8\u03b1 , anti-CD44  and anti-\u03b3\u03b4TCR , fixed and permeabilized with FoxP3/Transcription Factor Staining Buffer Set  and stained intranuclearly with anti-FoxP3 , anti-ROR\u03b3t , and anti-T-bet  mAbs. The gating strategy for the analysis of adaptive immune cells is shown in All stained cells were analyzed using the BD FACS Canto II with the BD FACSDiva software. CountBright Absolute Counting Beads  were used for calculation of total cell numbers.The spleens of mice at 21 d were carefully removed and fixed in 4% buffered formaldehyde solution for 24\u201348 h, embedded in paraffin and cut into 2 \u03bcm sections. After routine dewaxing and dehydration, sections were stained with GL7  mAb and hematoxylin. Histopathological analysis of the germinal center B cells was performed by the Institute of Veterinary Pathology, Faculty of Veterinary Medicine, Freie Universit\u00e4t Berlin, Germany. The forming of germinal centers was scored into non-existent (0), minimal (1), low-grade (2), moderate (3) and intense (4). Section analysis was performed in a blinded manner.P. aeruginosa phage cocktail used for these experiments were prepared from plates with confluent lysis for each individual phage. After resuspension in phage buffer, centrifugation at 1254\u00d7 g for 15 min w/o break, phage suspensions were filtered consecutively with 0.45 \u00b5m and 0.22 \u00b5m and stored at 4 \u00b0C until further use. Flat-bottom 96-well plates (Sarstedt) were coated with the anti-E. coli phage cocktail (1 \u00d7 108 PFU/phage) in 100 \u03bcL/well or with the anti-P. aeruginosa cocktail (5 \u00d7 107 PFU/phage in 100 \u00b5L) overnight at 4 \u00b0C. Wells were washed 5 times with PBS with 0.05% Tween 20  and blocked with 1% bovine serum albumin  in PBS (100 \u00b5L/well) at room temperature (RT) for 45 min. Plates were washed 5 times with PBS/0.05% Tween 20. Diluted plasma samples in duplicates were added to the wells (100 \u03bcL/well) and incubated at 37 \u00b0C for 2 h. Samples were diluted as follows: 1:10 for plasma IgM, IgA and 1:500 for plasma IgG testing for the active phage-cocktail treated mice, while 1:10 each for the UV-inactive cocktail groups and the buffer controls. After washing the plates 5 times with PBS/0.05% Tween 20, 100 \u00b5L/well of the corresponding detection antibodies were added and the plates were incubated for 1 h at RT in the dark. The following secondary antibodies were used: biotinylated goat anti-mouse IgG , biotinylated rabbit anti-mouse IgM , biotinylated rabbit anti-mouse IgA . The antibody solution was removed and the plates were washed 5 times with PBS/0.05% Tween 20. HRP-conjugated streptavidin solution  was incubated at RT in the dark for 1 h (100 \u00b5L/well). As a substrate for peroxidase, we used TMB . After incubating the plates for 15 min at RT in the dark, 50 \u00b5L of 2N H2SO4 (Carl Roth) was added to stop the reaction. The absorbance was measured at 450 nm using a MultiskanTM FC photometer  and analyzed with SkanIt Software 4.1 for Microplate Readers RE, ver. 4.1.0.43 and normalized by subtracting the blanks (PBS instead of plasma).Specific anti-phage antibodies  in plasma were measured by ELISA. The anti-Soluble cytokines and chemokines in plasma samples of the 6 h, 10 d and 21 d mice were measured using the Mouse Anti-Virus Response Panel  and the corresponding software provided by BioLegend. Samples were analyzed once, according to the manufacturer\u2019s recommendations.P. aeruginosa or E. coli were used (P. aeruginosa phage cocktail includes the two Pseudomonas phages DSM 19872 (JG005) [7 PFU/phage in 100 \u00b5L) provided by the DSMZ  and ITEM Fraunhofer . The anti-E. coli phage cocktail includes the five E. coli phages 536_P3, CLB_P2 [8 PFU/phage in 100 \u00b5L) isolated, characterized and provided by Institut Pasteur . Single phage suspensions were highly purified and endotoxins were removed by the providing institutions. UV-inactivation was performed using a UVC 500 Ultraviolet Crosslinker  for 6 h at 95,000 \u00b5J/cm2. Phages were stored at 4 \u00b0C. PFU analysis was performed with the E. coli strain AN33  and the P. aeruginosa strain DSM 107574 (PA74)   and DSM  (JG005)  . The lyE. coli phages) or spot tests (anti-P. aeruginosa phages).Blood and organ samples were serially diluted (1:10) in phage buffer shortly before performing the plaque assays, either by double agar overlay assays (anti-600 = 0.05\u20130.08) and cultured with shaking  until reaching early logarithmic phase . Low melting agar (soft agar) (4 mL/glass tube) was melted in a heating block (110 \u00b0C for 10 min) and cooled down to 48 \u00b0C. For the double agar overlay assay, 100 \u00b5L of the sample dilution and 100 \u00b5L of bacterial suspension were added to the liquid soft agar (top), gently mixed and poured on agar plates (bottom). For the spot test, only 100 \u00b5L bacterial suspension was added to the liquid soft agar. Subsequently, sample dilutions were spotted (4 \u00b5L/spot) in triplicate directly on the plates. Agar plates were incubated overnight  before calculating PFU/mL. The detection limit of the overlay assay for the E. coli phages against AN33 was 10 PFU/mL and the detection limit of the triplicates from the spot test for the P. aeruginosa phages against PA74 was 83 PFU/mL. Bacteria cryostocks were streaked on blood agar plates (PA74) or lysogeny broth (LB) agar plates (AN33) one day before. Bacterial cultures (tryptic soy broth (TSB), PA74; LB, AN33) were prepared (20 mL) with single colonies .The two phage cocktails  were analyzed via transmission electron microscopy by the Core Facility for Electron Microscopy of the Charit\u00e9 Berlin. For the negative staining, carbon-coated mesh grids were hydrophilized with Alcian blue solution (1% in 1% acetic acid) followed by washing steps in dHData are expressed as mean \u00b1 SD. For grouped analyses, two-way analysis of variance (ANOVA) with Tukey\u2019s multiple comparisons test was performed. Results were considered significant if P was less than 0.05. Significance levels are indicated in the figures. Statistical analysis was performed using GraphPad Prism 9 . Sample sizes of individual groups are indicated in the figure legends.P. aeruginosa (5 \u00d7 107 PFU/phage in 100 \u00b5L) and the second includes five phages targeting E. coli (1 \u00d7 108 PFU/phage in 100 \u00b5L).In this study, we evaluated the immunogenicity of two intraperitoneally (i. p.) injected phage cocktails in na\u00efve mice. The first cocktail includes two phages targeting P. aeruginosa and anti-E. coli phages in all analyzed organs and compartments , except for the alveolar spaces (BAL). Phages in the BAL were only detected in one of nine (anti-P. aeruginosa phages) or five of nine (anti-E. coli phages) mice at this time point , BAL (n = 1) and lungs (n = 3) 10 days following treatment initiation  and i. p. lavage fluid ) were collected at either 6 h after the first injection or 72 h after the last injection (10 d) a and plame point b. On dayitiation b.To check whether mice showed clinical signs of illness related to the phage treatment, we monitored them for alteration in body temperature c, left aTaken together, these data indicate that i. p. injection allows for phage dissemination via the bloodstream as early as 6 h after application. Most importantly, phage treatment was well-tolerated without obvious adverse effects.To evaluate the systemic activation and sustainment of innate immunity triggered by the phage cocktails, we carried out complete blood counts at 6 h, 10 d and 21 d after start of treatment .P. aeruginosa and anti-E. coli phage cocktail treatments at this time point  and hemoglobin (HGB), were inconspicuous and did not differ between treatment groups and controls . Specifime point a. Howevet change b. At thet change a,b.To examine the adaptive immunity in lymphoid organs, which is initiated by activated antigen-presenting cells (APCs) loaded with antigen, we first probed spleens and draining lymph nodes for the presence of activated APCs in response to active phage cocktail encounter. Using flow cytometry for expression of costimulatory molecules and MHCII, we did not observe a significant increase in either PMNs, macrophages or dendritic cells (DCs) in the spleen at 6 h and 10 d a\u2013c. PMN As DCs are the most competent APCs ,51, we tMatching measurements of draining lymph nodes confirmed the absence of innate immune activation of DCs in secondary lymphoid organs at all time points analyzed .P. aeruginosa phage cocktail, the proportions of CD4 T cells were moderately yet significantly increased at the expense of CD8 T cells , 40%  and 1.3%  of all T cells (CD3+) a\u2013c. At 6 T cells a,b. We dOur analysis of the same T-cell populations in the spleen also failed to reveal evidence of T-cell proliferation . They di+), high expression levels of the activation marker CD44 (CD44hi) and intranuclear staining for the Th1-transcription factor T-bet. Only 1% of all CD4 T cells were of Th1 type and no increase in frequencies was observed in response to phage treatment at either time point  were identified by the surface expression of CD4 , soluble cytokines and chemokines were analyzed in plasma samples at 6 h, 10 d and 21 d after the first injection  or UV-inactivated anti-E. coli phage cocktail (III) did not show any specific positive signals. In contrast, both the active phage cocktail groups  showed minimal to minor positive signals for germinal center B cells stained by GL7 , UV-inactivated anti-d by GL7 . These tThe observed formation of minimal splenic germinal centers prompted us to further investigate the humoral immune response against the two phage cocktails. To this end, we measured phage-specific antibodies  in plasma by ELISA .E. coli phage cocktail induced an increase in IgG, IgM as well as IgA by 21 days compared to the buffer or UV-inactivated control, which for IgG was already evident by 10 d. In contrast, the treatment with the active anti-P. aeruginosa phage cocktail only induced a marked increase in IgG and only at the 21 d time point, but this did not reach significance. A small increase in IgG was also induced by the UV-inactivated phage cocktails but may be reflective of individual outliers. These findings suggest that the repetitive injection of each of the two phage cocktails initiated a minimal humoral B-cell response with secretion of phage-specific antibodies.In congruence with the presence of germinal centers, the active anti-For evaluating the immunogenicity of the two phage cocktails, we assumed that the immune response against the UV-inactivated and the active phages would be comparable, as we expected the UV treatment to merely cross-link phage DNA . To undeP. aeruginosa or E. coli in na\u00efve mice without the confounding effects of a bacterial infection. Our results show that phages reached the lungs after systemic injection. Even repetitive exposure to the phage cocktails led to only a minimal innate and adaptive immune response, while all mice remained healthy without evidence of any adverse effects.Phage treatment of bacterial lung infections may be developing into an effective resource in the fight against the increasing antibiotic resistance of clinically relevant pathogens. As natural or therapy-induced immunity against therapeutic phages remains a safety and efficacy concern, in this study we assessed the immunogenicity of two phage cocktails targeting either P. aeruginosa phage cocktail. There was no upregulation of MHCII or co-stimulatory molecules on DCs that would indicate maturation into APCs and only a minimal pro-inflammatory cytokine profile at this time point. Despite the lack of evidence for an early innate response, we did observe a minimal adaptive immune response. Although we failed to observe the activation of main T cells or sub-populations at 10 days post-treatment, the histopathology analysis at 21 days nonetheless revealed a minimal to low-grade germinal center formation with increasing levels of phage-specific antibodies in plasma. The active anti-E. coli cocktail induced IgG and lower levels of IgA already at 10 days and IgM at 21 days, while the anti-P. aeruginosa phages induced only IgG and only at the late point of 21 days.Assessing the possible immune responses towards the two different phage cocktails, we did not observe a significant activation of innate immune cells in blood or lymphoid organs at 6 h after treatment, except for splenic PMNs in response to the active anti-A. baumannii infection study [A. baumannii phages, where intraperitoneal application resulted in the most pronounced, yet still low, immune response compared to oral or intranasal application [Phages can induce pro- and anti-inflammatory responses, depending on phage type, the route and duration of administration and the overall amount of phage virions present in the organism ,59. Conson study  or afteron study . Differelication .A. baumannii phage cocktail [Our results of increasing IgG titers and decreasing IgM titers over time are consistent with an antibody class switch. Similar findings, as well as weaker systemic IgA titers at later time points, have been shown by other studies ,33. IgA cocktail .A. baumannii-specific phage cocktail in na\u00efve mice, this did in fact not negatively affect the efficiency of the phages in a wound infection model [Pseudomonas phages [P. aeruginosa phages belonged to the myovirus morphology, as well as four E. coli phages, the fifth belonging to the podovirus morphotype. In addition, our findings, as well as those of other groups [Although we did find some evidence of a weak antibody response to our phage cocktails, antibodies do not necessarily have neutralizing capacity\u2014the main concern regarding the immune response to therapeutic phages\u2014since phage-specific neutralizing antibodies could diminish therapeutic success. However, although the presence of phage-specific antibodies with neutralizing capacity, in particular IgG2a and IgG2b, was detected after repeated intraperitoneal applications of an on model . Antibodon model ,59,67. Ion model , the T4 on model ,68, as ws phages  as highlr groups ,37 alignr groups . Thus, tr groups . Whetherr groups ,33,34 anr groups ,37,38,70Pseudomonas phages [Staphylococcus phages [E. coli phage cocktail we observed in the present study.Natural antibodies result from continuous contact with endogenous phages, which make up a significant part of the mammalian virome ,71. If ss phages  as well s phages  in the ss phages . Converss phages . PrimingE. coli cocktail consisted of five different phages each at 1 \u00d7 108 PFU/phage in 100 \u00b5L, while the P. aeruginosa phage cocktail contained only two each at 5 \u00d7 107 PFU/phage in 100 \u00b5L, resulting in a higher total phage sum applied for the E. coli cocktail. Endotoxins and other bacterial components that remain in the solution during phage production may elicit immune responses by themselves [E. coli phages had a lower endotoxin level compared to the anti-P. aeruginosa phage cocktail, indicating that in our study endotoxin concentration did not correlate with immunogenicity. Despite the two different protocols, the immune responses to each phage cocktail remain globally weak showing that different processes can lead to similar outcomes. The absence of humoral immunity observed with UV-inactivated phages is elucidated by the EM analysis. It revealed that the UV-inactivation protocol led to major damage to viral particles. Thus, intact phages seem to elicit minimal humoral-immune responses, while phage fragments alone seem non-immunogenic.However, the different responses to the different phage cocktails may not root in the immunogenicity of the phages themselves but rather in technical aspects: the emselves . NotablyE. coli strains, the rapid lysis of bacteria by phages did not increase the innate inflammatory response compared to that observed after antibiotic treatment [Our study has some limitations. Since we chose three different time points to study the possible early innate and late adaptive immune responses, we might have missed some responses that occurred in between. We also did not extend our analysis beyond 3 weeks, which may have provided insight into the later humoral response. In addition, we did not investigate B- and T-cell memory, which are the main parameters of rapid induction of phage-specific antibodies following phage treatment after a second interval. Moreover, our study is limited to the two phage cocktails investigated here with their corresponding phage concentration. Preparations with lower or higher phage content might induce differential immune responses. In our study, we used na\u00efve mice under SPF conditions without a bacterial infection, to avoid any confounding immune effects by the infection itself. However, the presence of bacteria may indirectly affect the immunogenicity of phages as well. Phage-induced bacterial lysis can further boost inflammation via bacterial particles and PAMPs, as well as cell-free phage DNA and higher numbers of phage virions released locally , potentireatment . Neverthreatment .In the context of lung infections with MDR-bacteria, phage therapy is of great interest for treating VAP patients. While recent studies used intratracheal applications  or nebul"}
{"text": "Pharmacodynamics of phages mainly focuses on the phage titer used to kill the infection-causing bacterial cells; thus, a number of phages or phage cocktail standardization are very important before administration or initiation of phage therapy. In addition, the phage solution must be free from any type of pyrogenic substances that could cause an altered immune response. Pharmacologically, phages can be treated as very selective and specific toxic antibacterial agents  epithelial barrier circumvention via a Trojan horse mechanism in which phages hide inside bacteria, 3) direct sampling of luminal material by intestinal dendritic cells, and 4) phage translocation across a damaged epithelial barrier (Antigen-presenting cells (APCs) must process and present phages to T cells to trigger the generation of antibodies (Abs) and produce a long-lasting memory response. Several mechanistic theories have been proposed to explain how phages manage to get past epithelial barriers and into the generally sterile lymphoid organs: 1) phage particle transcytosis  barrier . The pre barrier . Phage-mThe present research topic focused on phages, which has highlighted the current immunology and pharmacology of phage therapy along with its related advancements. This issue comprised a detailed case report, opinion, and a review article on bacteriophages.Gondil and Chhibber, 2021 focused on the encapsulation system of bacteriophage and endolysin, which is found to be a promising strategy to improve therapeutic outcomes of phage therapy. Phage and endolysins\u2019 encapsulation can improve various pharmacokinetic parameters with improved host immune response. The authors systematically discussed the role and advantages of endolysins (phage-borne lytic proteins) such as improved specificity, modular structure, rapid host lysis, and reduced chances of resistance. The authors mentioned various advanced encapsulation systems such as natural polymers, synthetic polymers, liposomes, nanosphere, and electrospun fibers. Thus, this type of encapsulation for phages protects from digestive enzymes and bile juices and provides permeability to the mucous lining. However, the endolysin delivery system of phages faced more challenges than phage delivery due to its proteinaceous nature and labile enzymatic activity. Thus, organic solvents play a major role in delivering the encapsulation of phages. The authors also mentioned the challenges and benefits of the endolysin delivery system; however, phage encapsulation system has been successfully reported in many clinical applications. As reported worldwide, these novel phages and endolysin delivery systems would be a promising method of drug delivery to combat multidrug-resistant infections.Johri et al. investigated a case report of successful phage therapy against Chronic Bacterial Prostatitis (CBP) in a 33-year-old male. Before the start of the phage therapy, the patient underwent multiple courses of antibiotics treatment without any long-term clinical benefits. The case report stated the culture of prostatic secretion and semen samples with the presence of Staphylococcal species such as Methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus haemolyticus, Enterococcus faecalis, and Streptococcus mitis. Thus, specific phage preparations were finalized from Eliava Institute and were administered as oral liquid, rectal suppositories, and urethral instillations. The case defined significantly decreased symptoms after the treatment period (approximately 2\u00a0months) such as high body temperature, weakness, night sweating, and chills. Thus, phage therapy can be proven to be a better alternative in CBP cases with improved clinical manifestations.Kaur et al. systematically reported the role of nanotechnology in phage encapsulation, which can be an ideal pharmaceutical formulation approach in overcoming the pharmacological barriers such as less bioavailability, low stability, targeted delivery, inactivation of active phages, poor in-vivo retention, maintenance of viability, neutralization by the immune system, and poor penetration. Instead of focusing on nano-encapsulation, the authors mentioned a detailed insight about the recent nanotechnologies such as lipid-based nano-carriers, microfluidic, surface modification, nano-emulsions with integrated microfluidics for phage-cocktails, phage-loaded nanofibers using electrospinning method, and smart drug delivery platforms to control the high phage counts as required.Thus, overall, in this alarming crisis due to multiple drug resistance microbes, the development of newer antibiotics alternatives with improved efficacy is the need of the hour. Phage therapy is one of the new rays of hope among the existing alternatives. The major limitations, such as immunological and pharmacological barriers, in front of phage therapy can be overcome by using these advanced formulation and drug targeting approaches. The immune system is one of the major challenges for scientists working on phage therapy, however lack of social acceptance due to minimal public awareness and unacceptable regulatory guidelines are the major hurdles to addressing and establishing phage therapy as one of the safest alternatives for MDR microbes and their associated infections. The aim of this research topic was to address the unique mechanism and pharmacological challenges in phage therapy. Finally, this topic offers an in-depth opinion and case report of phage therapy along with the successful delivery approaches of phages, which may inspire many researchers worldwide to continue working in or enter the science of phage preparation, formulations, and targeted delivery against various uncurable infections."}
{"text": "Staphylococcus aureus (S. aureus) represent a major challenge to successful treatment. Further, although bacteriophages (phages) could be an alternative to antibiotics, there exists a lack of correlation in phage susceptibility results between conventional in vitro and in vivo assays. This discrepancy may hinder the potential implementation of bacteriophage therapy. In this study, the susceptibility of twelve S. aureus strains to three commercial phage cocktails and two single phages was assessed. These S. aureus strains  were compared using four assays: the spot test, efficiency of plating (EOP), the optical density assay  and microcalorimetry in human serum. In the spot test, EOP and optical density assay, all cocktails and single phages lysed both methicillin susceptible and methicillin resistant S. aureus strains. However, there was an absence of phage-mediated lysis in high concentrations of human serum as measured using microcalorimetry. As this microcalorimetry-based assay more closely resembles in vivo conditions, we propose that microcalorimetry could be included as a useful addition to conventional assays, thereby facilitating more accurate predictions of the in vivo susceptibility of S. aureus to phages during phage selection for therapeutic purposes.Infections involving antibiotic resistant  Staphylococcus aureus (S. aureus) causes many types of infections, ranging from relatively harmless skin infections to life-threatening endocarditis. In addition, it is the leading cause of bloodstream infections in industrialized countries .,63.S. auS. aureus but also multiple other bacterial species. To examine the content of the three phage cocktails, next generation sequencing was used. Firstly, a general overview of phage diversity in the cocktails was obtained using BLASTn. This confirmed the presence of multiple bacteriophage genera that infect various bacterial hosts, including S. aureus  plate-based assays such as \u2018efficiency of plating\u2019 (EOP) [Even though conventional assays showed lysis of most of the g\u2019 (EOP) ,64. TherS. aureus to clump together in aggregates [S. aureus aggregation. However, S. aureus grows less efficiently in human serum compared to TSB, meaning that higher starting concentrations of S. aureus were required in order to measure sufficient metabolic activity of bacteria in human serum. With a higher starting concentration of bacteria, but not a higher concentration of the phage cocktails, the MOI was automatically reduced and therefore lower in the MC compare to the OD assay. For this protocol, we assessed the susceptibility of the two most phage-sensitive clinical S. aureus strains (Mup15 and Mup2723) to the phage cocktails in heat-inactivated human serum. As a control for bacterial cell death, the antibiotics rifampicin and flucloxacillin were used. While there was a clear effect of the antibiotics, the addition of the phage cocktails did not result in a decrease of metabolic activity at an MOI of 0.03  cause gregates . Microca of 0.03 . FurtherS. aureus phages, a total of eighteen phages were isolated from the RPC. The host range of these phages was determined using the spot test (data not shown). Two phages with a broad, but not identical, host range were selected: namely, RPCSa1 and RPCSa2 , Kayvirus phages bind to the backbone of WTA and are therefore not affected by these modifications [In both the OD assay and spot test, three commercial phage cocktails and the single phages used, there was a broad host range against clinical ocktails ,68. Thes-systems ,69,70. Iications ,69,70,71S. aureus strains were lysed in our experiments. Interestingly, the genomes of the S. aureus strains that showed low phage susceptibility were not genetically closely related to each other, as shown by the phylogenetic analysis. Therefore, no clear link between phage susceptibility and the genetic background of our S. aureus strains could be made. Moreover, the evaluation of known phage-resistance genes in the genome could not explain the differences of phage susceptibility of the isolates either. For example, although Mup3199 was resistant to the phage cocktails tested, no known phage-resistance genes were found. In contrast, R5 is very phage-sensitive despite the presence of multiple phage-resistant genes. This is consistent with the observation of Moller et al. that phage susceptibility relies on both host factors, most of which are still unknown, and phage-specific factors. The lack of a clear relation between the genetic background of isolates and their phage susceptibility highlights the importance of susceptibility testing prior to treatment with phages [Despite these phage characteristics, not all h phages .S. aureus strains tested. For example, strain M116 was susceptible to GPC and RPC in the spot test but not in the OD assay. This supports previous observations that the spot test might give an overestimation of phage susceptibility [S. aureus strains that were not susceptible in the spot test showed a decrease in growth in the OD assay. However, this high MOI might not be realistic for in vivo use because bacteriophages do not accumulate well in all tissues and are cleared from the blood both passively by the spleen and liver and actively by the immune system [Comparison of the spot test and OD assay did not reveal identical phage susceptibility patterns for the tibility . Discrepe system . Some ofe system ,64. HoweS. aureus in human serum, resulting in reduced phage propagation [In contrast, MC allowed the determination of phage susceptibility in human serum, despite the presence of bacterial aggregation, showing a drastic decrease in phage susceptibility to both the phage cocktails and single phages when compared to TSB. However, it should be noted that due to the unknown content of the commercial phage cocktails used, we were not able to concentrate them without the risk of selecting for specific phages . As a repagation ,74. Howepagation ,73,75,76S. aureus. The lack of phage susceptibility in human serum could explain the discrepancy between in vitro results obtained using conventional assays and in vivo data from clinical phage therapy trials. For example, the intravenous administration of AB-SA01 (a cocktail containing Kayvirus phages) resulted in a response rate of only 62%, despite the spot test indicating complete in vitro phage susceptibility of the infecting bacterial strain [In this study, we highlight the importance of experimental conditions on the phage susceptibility of l strain . So evenHere, we propose the use of MC testing in combination with current conventional assays, for more accurate in vitro phage selection for phage therapy. This is due to the assay\u2019s better resemblance to the microenvironment encountered by bacteria and phages in vivo. In the future, MC could be used to determine phage susceptibility in other media resembling in vivo environments. For example, it has already been shown to be a valuable tool for phage susceptibility determination in urine . MC migh"}
{"text": "Bacteria and their predators, bacteriophages, or phages are continuously engaged in an arms race for their survival using various defense strategies. Several studies indicated that the bacterial immune arsenal towards phage is quite diverse and uses different components of the host machinery. Most studied antiphage systems are associated with phages, whose genomic matter is double-stranded-DNA. These defense mechanisms are mainly related to either the host or phage-derived proteins and other associated structures and biomolecules. Some of these strategies include DNA restriction-modification (R-M), spontaneous mutations, blocking of phage receptors, production of competitive inhibitors and extracellular matrix which prevent the entry of phage DNA into the host cytoplasm, assembly interference, abortive infection, toxin\u2013antitoxin systems, bacterial retrons, and secondary metabolite-based replication interference. On the contrary, phages develop anti-phage resistance defense mechanisms in consortium with each of these bacterial phage resistance strategies with small fitness cost. These mechanisms allow phages to undergo their replication safely inside their bacterial host\u2019s cytoplasm and be able to produce viable, competent, and immunologically endured progeny virions for the next generation. In this review, we highlight the major bacterial defense systems developed against their predators and some of the phage counterstrategies and suggest potential research directions. CurrentBacteria and phages are seemingly involved in a continuous battle. It is part of continuous cycles of coexistence and evolution, resulting in phage-resistant hosts protecting bacterial lineages, while counter-resistant phages threaten such strains. Phages, by developing resistance, play a crucial role in controlling bacterial populations in most, if not in all, the milieus. Bacteria can evade the phage attack via several mechanisms and some of these strategies include the following: DNA restriction-modification (R-M), spontaneous mutations, blocking of phage receptors, production of competitive inhibitors, and extracellular matrix and acquired immunity via the clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) mechanism ,7,8. On In this review, we present an overview of the major anti-phage defense strategies of bacteria and the counterstrategies used by phages to evade these systems and suggest potential research directions.The mechanism of phage resistance could take place at various stages of phage replicative cycle. During the phage replication cycle, a phage introduces its DNA via translocation into the host\u2019s cytoplasm. This will lead to either the lysogenic cycle (prophage formation) or the lytic cycle. In the lytic cycle, phages pass via several steps and early and late gene expression, which leads to maturation and aggregation of newly produced virion, which are ultimately released via lysis. The phage resistance strategies can interfere with one of these steps as shows in Campylobacter fetus slime layer) or capsules  can cover the bacterial surface and makes the phage receptors inaccessible for phage binding so that protecting bacteria from phage attack [Campylobacter jejuni (strain NCTC11168) against phage F336. The authors proven that the successful adsorption of the phage F336 to the bacterial surface depends on the hypervariable O-methyl phosphoramidate (MeOPN) modification of capsular polysaccharides (CPS). However, phage resistance has been acquired by loss of MeOPN receptor on the surface of the organism because of cj1421 gene phase variation, which encodesd the MeOPN-GalfNAc transferase [Phage adsorption to the bacterial cell surface is performed through specific receptors as the first step of phage infection cycle. Several bacterial cell surface proteins, lipopolysaccharides, and other surface polysaccharide and carbohydrate moieties can serve as receptors for phages . Bacterie attack . Some bae attack  . In the case of Sie defense systems phage encoded anti-phage proteins can be utilized by bacterial cells to prevent the translocation of DNA of lytic phage into the cytoplasm of host cells, thereby acquiring protection against virulent phages  Figure . These pgp5, downregulate the activity of the T4 lysozyme. The activity of the T4 lysozyme encoded by gp5 has inhibited the membrane protein Sp, thereby likely preventing the peptidoglycan degradation and the consequent translocation of phage DNA [Virulent phages, such as Coliphage T4, have two Sie systems mediated by Sp and imm. These systems prevent the entry of phage DNA into the cytoplasm of bacterial cell, thereby affecting successive infection by other T-even-like phages . The Sp hage DNA ,19.Neisseria gonorrhoeae and others such as Helicobacter pylori, Streptococcus pneumonia and Haemophilus influenzae have abundant R\u2013M systems [Restriction-modification (R-M) systems are universal and tremendously varied in the bacterial primitive immune system. The R\u2013M system has two main components: a methyl transferase (MTase) and a restriction endonuclease (REase). The restriction endonuclease pathway detects short DNA segments, measuring between four to eight base pairs long, and they are chopped into pieces. The mis-recognition and cleavage of the host DNA is protected by the methyl transferase, which is hidden from being recognized by the restriction enzyme . The pha systems .There are four R\u2013M systems, so far identified, based on their subunit composition and mechanism of action . Type I The genome of some virulent phages may be modified by harboring a rare base hydroxymethylcytosine (HMC) in place of the base cytosine. This modification enables T4 phage DNA to be resistant to the classical R\u2013M-based degradation. In co-evolutionary warfare, some bacteria have developed modification-dependent systems (MDSs) that used to attack the modified DNA of phage .Staphylococcus aureus, where they are specifically located in the pathogenicity islands name as \u201cSaPIs\u201d. In Gram-positive bacteria, PICI expression has been downregulated by a transcription repressor. PICI is excised from the host chromosome by the anti-repressor that has been produced by helper phages. Proteins translated from PICI genome suppress the expression of late helper phage genes and also modify the size of capsid protein to be suitable to accommodate the PICI genome, which ultimately end up with proper packaging of PICI genomes and prevent the formation of helper phage virions [Phage-inducible chromosomal islands (PICIs) are one of phage resistance mechanism developed by bacteria, which involve the integration of small (\u223c15 kb) gene sequences and are excised with the help of a specific \u201chelper phage\u201d . The gen virions   is a strategy of bacterial cells halt the release of newly produced progeny virions at the expense of the life of infected cell, thereby preventing the uninfected cell from being infected. It is considered a self-sacrificing event, or apoptosis that averts the subsequent infection of the neighboring bacterial community . Each Abed cells . Prematued cells . Anotheron cycle . An Abi proteins . In gene. lactis   [E. coli can inhibit the infection process of T4 phage by inducing MazF\u2019s ribonuclease activity, which ultimately leads to complete cessation of the infection cycle [Phages may trigger the bacterial cells to produce toxins, which my attack the phage infection cycle. There are six major bacterial toxin\u2013antitoxin (TA) types, classified based on the toxin neutralization mechanisms, the biomolecular and functional characteristics of the TA and, the TA count  . This wion cycle . Table 1E. coli retron, Ec48, has 48 nt long reverse-transcribed DNAs [E. coli RecBCD, causing the retron (Ec48) to activate and kill the cell via Abi [Retrons are bacterial genomic materials composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). The ncRNA served as a template for RT, producing a chimeric DNA/RNA molecule in which the DNA and RNA are covalently connected . Retronsbed DNAs ). It has via Abi . The autStreptomyces spp. They reported that Streptomyces spp. synthesized two vital bioactive metabolites (doxorubicin and daunorubicin) that bind  into the DNA of phage and prevent the replication cycle. Interestingly, the bacterial growth pattern was not affected by these molecules. It has been proposed that daunorubicin exerted its action at an early stage in the phage replication cycle, after the translocation of the DNA, but ahead of replication. Doxorubicin forms free radicals , which can damage DNA and cause DNA oxidation  enzyme, which cleaves the phage DNA at specific recognition site(s). In response to this defense feature, phages developed a broad range of passive and active anti-restriction strategies ,64 Figu.Myoviridae coliphage P1 encodes two inti-restriction proteins, DarA and DarB, that are co-translocated into host cells, along with their genome, thereby hiding the type I R-M recognition sites and protecting phage DNA degradation [The radation . In addiradation ,67.In the case of passive mechanisms of phage evasion, MTase modifies the double-stranded DNA of phages rapidly within a host comprising a R\u2013M system before it is recognized by the host REase. Thus, the invading phage DNA will be protected from degradation by R\u2013M systems. Hence, the modified genome of phage can replicate safely in the R\u2013M-consisting host cell and can also infect and replicate in other cells which express identical R\u2013M system. Nevertheless, as the R\u2013M system is specific to specific host, the same DNA of a phage can be detected as foreign in a cell consisting of a different R\u2013M system, and it will, therefore, be degraded by a different REase. In yet another twist, some REases of bacterial cell even can detect and degrade modified DNA .Lactococcus phages are the best example for this counterstrategy. Mutations of one or more specific genes of the Lactococcus spp. can enable the phages to escape the Abi systems [Lactococcus spp., for instance, can bypass the AbiQ mechanism by mutating genes involved in nucleotide metabolism. A phage can encode a molecule (such as Dmd in coliphage T4) that can replace a bacterial antitoxin, thus inhibiting the activity of the bacterial toxin and protecting the cell from death [Pectobacterium atrosepticum phage, phiTE, produces pseudo-anti-toxin RNA or takes over the antitoxin, ToxI, during its infection to deactivate the ToxN toxin [Certain phages have developed anti-Abi mechanisms to undergo protected replication in the targeted host cell . Lactoco systems . Phages om death . It has xN toxin .An individual nucleotide substitution can enable phages to evade the CRISPR system in the protospacer site or in the conserved region adjacent to the protospacer motif . In contPseudomonas aeruginosa temperate phages [Current research findings indicated that phages encode anti-CRISPR genes, which are active towards the CRISPR of bacterial defense systems, as it is recognized in e phages . Some phe phages .Some examples of the phage\u2019s counterstrategies towards different phage resistance mechanisms that we discussed so far are summarized in In this review, we have reviewed a variety of phage resistance mechanisms. These antiviral defense systems involved several biomolecules, proteins, enzymes, cellular structures, and inter-cellular interactions that protect bacterial cells from lysis and death. The bacterial antiphage defense strategies, which are described in this review, reflect the tremendous diversity of phages, and thus some other resistance mechanisms could be discovered.The study of phage resistance should be scaled up beyond the discovery of the baseline mechanism towards the detailed understanding on the molecular root behind these antiviral systems. In this context, advancement in phage biology will certainly be required to fully understand the mechanism of phage resistance. Moreover, most studied antiphage systems were associated with the phages whose genomic matter is double-stranded-DNA. Resistance mechanisms linked to single-stranded DNA and RNA, or double-stranded RNA genomes, as well as other genetically unique phages, are waiting to be investigated. In addition, lysogenic phage mediated resistance mechanisms are less understood, and therefore this gap needs to be filled.Siphoviridae) are not continually evaluated.The above-mentioned phage resistance strategies are often investigated in a laboratory-controlled setting, in a single replica, and using a single host\u2013phage model. Nevertheless, bacterial pathogens usually comprised multi-lateral antiphage systems. The coexistence of these systems in one host cell has seldom been studied, and the outcome of such interactions on phage evolution and their community is often overlooked. Likewise, the effectiveness of phage resistance strategies towards some families of phages (non As bacteria and phages have ancient co-evolutionary history, phages can profoundly generate a counter-resistance, via several mechanisms and with small fitness cost. One phage may also possess multiple phage resistance systems that may generate strong defenses over individual systems, allowing specific clonal inhabitants to stay in phage-containing ecosystems. The phage counteracting strategies enable the phages to undergo their replication safely and to produce viable progeny virions for the next generation. However, there are many unexplored mechanisms, cascades of reactions, molecular interactions, and involvement of newly produced biomolecules that need additional in-depth investigation for better understanding of phage science. Hence, the limitation of this review is that we have presented limited information regarding the mechanisms of action of some phage resistance and counterstrategies that have not yet been fully explored."}
{"text": "Pantoea comprises species found in a variety of different environmental sources. Pantoea spp. are often recovered from plant material and are capable of both benefitting the plants and acting like phytopathogens. Some species of Pantoea (including P. agglomerans) are considered opportunistic human pathogens capable of causing various infections in immunocompromised subjects. In this study, a strain of P. agglomerans (identified by 16S rRNA gene sequencing) was isolated from a dead specimen of an unidentified Latvian grasshopper species. The retrieved strain of P. agglomerans was then used as a host for the potential retrieval of phages from the same source material. After rounds of plaque purification and propagation, three high-titer lysates corresponding to putatively distinct phages were acquired. Transmission electron microscopy revealed that one of the phages was a myophage with an unusual morphology, while the two others were typical podophages. Whole-genome sequencing (WGS) was performed for each of these isolated phages. Genome de novo assembly and subsequent functional annotation confirmed that three different strictly lytic phages were isolated. Elaborate genomic characterization of the acquired phages was performed to elucidate their place within the so-far-uncovered phage diversity.The bacterial genus  Pantoea includes multiple Gram-negative, yellow-pigmented rods that are frequently isolated from a variety of environmental and higher-organism-associated sources [The bacterial genus  sources , hence tPantoea spp. are still, arguably, mainly thought of as bacteria associated with plants, capable of both having positive effects on plants  a,4,5 aPans e.g., ,7,8,9). ,8,9. Pans \u2014are omnipresent in every environment where bacteria thrive. As natural predators of bacteria that have the ability to rapidly propagate themselves upon invading a susceptible host bacteria population, phages can be harnessed as biocontrol agents to eliminate unwanted bacteria. However, phages generally have a very limited host range, which might be as narrow as several strains of a particular bacterial species. Thus, isolation and characterization of new bacteriophages capable of infecting representatives of the bacterial genera or species even of modest economic or healthcare importance are of particular interest, especially in a case such as when a host has none or just a few bacteriophages yet known to infect it. In addition to the expansion of the knowledge on the uncovered phage diversity, which is of fundamental importance, such studies might potentially provide a good lead for further practical application evaluation of a given novel phage or its proteins.Pantoea-infecting phages that have their complete genomes publicly available. These phages represent all the tailed phage morphotypes , and their genomes range from 36,790 to 149,913 base pairs, showing a percentage GC content of 39\u201355.35%. Based on the complete genome-associated metadata, strains of Pantoea agglomerans are indicated as the specific host of these phages most commonly (n = 12) followed by P. deleyi (n = 3) and P. dispersa (n = 2). Eleven of the so far sequenced Pantoea phages have been isolated from plant sources . Additionally, five Pantoea phages were previously retrieved from water-associated sources and another three from soil samples. This makes the phages Nifs112, Nufs112, and Nafs113 described in this study the first three Pantoea phages to be isolated directly from an insect-associated source , isolate LS5-2, the isolation host of phages Nifs112, Nufs112, and Nafs113, formed beige colonies at all of the three incubation temperatures tested . The fastest growth was observed at +30 \u00b0C, and the slowest was observed at RT. After their appearance, the LS5-2 colonies were initially beige but turned yellow-orange during the subsequent incubation.Pantoea. More specifically, as LS5-2 shared a well-supported most recent common ancestor with P. agglomerans DSM 3493 in the phylogeny, and both sequences had only a few differences, the bacterial isolate LS5-2 was considered to be a strain of P. agglomerans  than that of Nufs112 (54.4 \u00b1 1.8 nm). With the opposite holding true for tail lengths, the tail of Nifs112 was measured to be around 11.2 \u00b1 1.6 nm in length, whereas Nufs112 had a tail of 12.8 \u00b1 2 nm. However, a more detailed TEM examination involving at least several tens of virions, additionally taking the planes at which the virions were seen into account, would be necessary when drawing conclusions on whether these capsid size and tail length differences were meaningful. What stood out in the micrographs, however, were the tail appearances, with Nifs112 having seemingly slimmer tails in contrast to the \u201cchunky stubs\u201d of Nufs112  that has been observed on numerous occasions for a plethora of different podophages . CombineAlthough having an unremarkable plaque appearance, Nafs113 virions were determined to have a less-common myovirus morphotype\u2014an elongated head with an approximately 3:1 length (121.4 \u00b1 6.8 nm)-to-width (43.4 \u00b1 3.8 nm) ratio, to which a 88.5 \u00b1 3.2 nm long contractile tail with a width of 15.4 \u00b1 0.6 nm (uncontracted state) was attached , Nufs112 , and Nafs113  complete genomes, respectively  were found to belong to the family lymerase ). HoweveAutographiviridae podophages. No protein, however, was found to be present in more than a single studied phage at the 95% identity threshold. Lowering the threshold to 50% only identified five proteins that were shared between Nifs112 and Nufs112\u2014a terminase large subunit, phosphatase, a HTH domain-containing protein, and two hypothetical proteins. Setting an identity threshold to 30% revealed that phosphatase was the only protein that could be considered to be shared by all three of the phages. As expected, the lowest threshold used (30%) also revealed that Nifs112 and Nufs112 might have up to 20 homologous proteins, i.e., the ones involved in either virion morphogenesis  or those responsible for nucleic acid metabolism, modification, and repair , and several other, mostly hypothetical, proteins. At this threshold, a homolog of HNH endonuclease from Nafs113 (ORF115 product) was also identified in the Nifs112 proteome (ORF30 product).Roary  pangenoAfter functional annotation of the studied phage genomes, termini types were also additionally verified using the TerL phylogeny reconstruction approach based on a dataset of Merrill and colleagues . The TerHamiltonella phage APSE-1 representing the same TerL sequence clade also has a terminally redundant and circularly permuted genome, with the sequence and electron microscopic analysis carried out indicating that \u201cmost likely the permutations are randomly distributed over the genome\u201d [Shigella phage Sf6 noted \u201clittle evidence for specific termini\u201d, but they also found out that Sf6 DNA appeared to be cleaved at many sites within a large region of about 1800 bp, including a possible pac site for the initiation of a packaging series from a DNA concatemer [Pantoea phage Nafs113\u2019s genome\u2019s circular permutation , however, was not further looked at experimentally at the time being.Yet, the TerL phylogeny reconstruction showed that the TerL of Nafs113 clustered together with Sf6-type headful-packaging-employing phages. This further supported our assumptions regarding the Nafs113 headful genome packaging, as, for example, the  genome\u201d . Researcncatemer . The extP. agglomerans . The genomes of Nifs112 (50.2%) and Nufs112 (47.7%) deviated way more in that regard, although a Pantoea-infecting phage with a GC% content as low as 39% was previously documented  similar to that of its host species, 70119.1; ).Pantoea agglomerans phages uncovered so far is noticeably lower than that of their host  possibly allows us to propose two of the studied phages (Nifs112 and Nafs113) as novel species within the already-recognized phage genera, whereas Nufs112 might even be a sole representative for a novel phage genus based on the ICTV adopted genome nucleotide sequence similarity phage genus and species demarcation criteria .Pantoea phage Nifs112 (OK570184.1), the highest-total-scoring hit was documented to a complete genome of the Erwinia phage vB_EamP-S2, representing an Eracentumvirus S2 species within the genus Eracentumvirus . This hit to Erwinia phage vB_EamP-S2 was followed by hits to other Erwinia phages either officially or tentatively belonging to the Eracentumvirus genus  among the top five highest-scoring hits.When querying the Pantoea phage Nufs112 (OK570185.1), the top hit to a cultured phage complete genome was noted to the Klebsiella phage 6939  unclassified at the genus and species level. This hit to Klebsiella phage 6939 was followed by hits to the phage Reminis, showing a similar intergenomic distance, and recognized or putative representatives of the Gajwadongvirus genus , showing a >60% intergenomic distance to Nufs112.In the case of the Pantoea phage Nafs113 (OK570186.1) had only a single meaningful hit\u2014to Pantoea phage vB_PagM_LIET2  representing a sole Lietduovirus LIET2 species within the genus Lietduovirus, with hits to other phage genomes having a \u22641 percent query coverage.Interestingly, the genome of the When the studied phage genomes were queried against the bacterial sequences found in GenBank (taxid: 2) using megablast, hits to bacterial genomes were limited to up to a few hundred nucleotides (less than a few percent query coverages) for each of the three phages, indicating a lack of highly similar prophages or the remnants of thereof in the so-far-sequenced bacterial isolates.Having gained a glimpse at the genome nucleic acid sequence-wise similarity to other phages, a reconstruction of the selected analogous protein phylogenies was performed within the context of their most closely related counterparts encoded by other phage genomes . For thiEracentumvirus. Although the Eracentumvirus clade with Nifs112 had a well-supported MRCA in three of the trees (except the endolysin tree), its closest neighbors differed across all four of the trees with regard to taxonomical placement.In all four of the trees, proteins from Nifs112 were most like their counterparts from the representatives of Pantoea phage vB_PagM_LIET2 belonging to the genus Lietduovirus, and in all of the trees save for an endolysin tree, there was a rather long branch leading to their two-sequence clade, indicating distinctness of their selected homologous proteins from the homologs found in other phages.Selected proteins from Nafs113 shared an MRCA with the Klebsiella phage 6939 and the Ralstonia phage Reminis. These two phages, however, shared a well-supported MRCA in all four of the trees, whereas Nufs112 reliably clustered with them only in the case of MCP and TerL. Interestingly, the phage Reminis and the Pantoea phage MR4 seemed to be classified in GenBank as tentative representatives of the genus Gajwadongvirus, but from the generated trees, only MR4, and not Reminis, seemed to form monophyletic clades together with the officially recognized Gajwadongviruses PP99 and ECBP5, which together boasted very short within-clade evolutionary distances. Thus, the designation of Reminis as a Gajwadongvirus representative did not really seem justified based on the selected protein phylogenies. While an evolutionary link to the recognized Gajwadongvirus proteins could also be established for Nufs112, their counterparts seemed to have diverged in a more distant past and were very unlikely to share an immediate ancestry.The situation with Nufs112 was revealed to be more ambiguous, as it did not reliably fall within the same monophyletic group, and the tree topologies surrounding the corresponding sequences of Nufs112 proteins differed to an extent. Based on the phylogenies, the selected Nufs112 proteins were most similar to the homologs from the To get a better proteome-based overview of the place of the studied phages within the context of cultured and sequenced phage diversity, 18,553 complete phage genomes  available at INPHARED  were suEscherichia (35 phages), and other bacterial genera were represented by only up to eleven different phages infecting them. Apart from each other, there were no other first neighbors designated as Pantoea phages identified for either Nifs112 or Nufs112. However, three Pantoea agglomerans phages  were among the first neighbors of Nafs113, with the phage LIET2, expectedly from the previously determined BLASTN search, being the most similar one. Notably, VC_437_0, to which Nifs112 was clustered based on its proteome, otherwise comprised exclusively Erwinia-infecting phages, either officially or tentatively representing the genus Eracentumvirus.From the vConTACT2-generated network, only the first neighbors of the studied phages were of interest, excluding the studied phages themselves\u2014six unique neighbors were identified for Nafs113, nine for Nifs112, and 22 for Nufs112, whereas sixty-three of the phages were the first neighbors for both Nifs112 and Nufs112 . The stuAutographiviridae, with sixty-seven being further classified into the Molineuxvirinae subfamily comprising the clusters VC_436_0 (at least six genera representatives) and VC_437_0 (Eracentumvirus), and nineteen into the Colwellvirinae subfamily . Interestingly, while fifty-eight Molineuxvirinae phages were the first neighbors of both Nifs112 and Nufs112 and only nine representatives of this subfamily were uniquely identified as the first neighbors of Nifs112, all nineteen Colwellvirinae representatives were neighbors of Nufs112 exclusively . However, some of the (mostly tentative) designations in the taxonomy associated with the phage genome submissions were not supported at the chosen intergenomic distance criteria\u2014Vectrevirus representatives were split into two VIRIDIC clades; Uliginvirus were split into five VIRIDIC clades at the given intergenomic similarity threshold; the bacteriophage Reminis was tentatively classified as Gajwadongvirus (likely incorrectly); the tentative Tuodvirus Escherichia phage vB_EcolP_P433.1 seemed way more similar to two clades, in which recognized or tentative Vectrevirus representatives are found, than to the officially recognized Tuodvirus phD2B.Most of the genus-level designations in the retrieved complete-genome sequence-associated taxonomy coincided with the VIRIDIC clusterization at a 70% intergenomic similarity threshold, and such clades were monophyletic in the generated intergenomic distance NJ tree  to be so similar to a plethora of Autographiviridae podophages. A look at the ICTV virus metadata resource (VMR_20-190822_MSL37.3) revealed that this stemmed from the misattribution of a taxonomy, as the Vibrio phage Vc1  is a completely different phage to the Vibrio phage VC1 , but the taxonomy associated with the latter was mistakenly expanded to the former in the GenBank.In addition, it seemed very unbelievable to find a single alleged Eracentumvirus, interestingly, so far comprising only Erwinia phages;Nifs112 was a representative of the genus Gajwadongvirus representatives but did not look like a representative of that genus itself;Nufs112 was related to Lietduovirus, but the question of whether it represents a novel species therein remains.Nafs113 could firmly be considered a representative of the genus Nevertheless, combining the results of the performed \u201coverview\u201d analyses for our studied phage place within the so-far-uncovered phage diversity, it was revealed that:Pantoea phage Nifs112 is a linear 46,202 bp long dsDNA molecule  with a GC% content of ~50.2%. The genome of Nifs112 (OK570184.1) was found to contain 59 open reading frames, all of which were located on a direct strand; no tRNA genes were found. ATG was predicted to serve as a start codon for fifty-one of the ORFs; GTG\u2014four, CTG\u2014two, and TTG\u2013four ORFs. The majority of the predicted ORFs had a well-identifiable Shine\u2013Dalgarno motif in the sequence span 20 bp upstream of the selected start codons  of the phages . Moreover, twenty of them were found in all three of the phages even at the 90% identity threshold set for Roary. All three of the phages shared a conserved genome architecture, with the main differences being in the presence or absence of short ORFs encoding different hypothetical proteins without an annotation. Pairwise homologous protein identity percentages clearly indicated that Nifs112 was more closely related to the phage vB_EamP-S2 than to Era103 .Acinetobacter bacteriophages identifiable using CD search, and Acinetobacter podophages are known to have a host-capsular-polysaccharide-degrading ability coupled to their tailspikes [Most of the ORF products of Nifs112 and vB_EamP-S2 were highly similar, showing exceptional conservation within the most functionally annotated products. The difference of interest, however, lay in the tailspike protein that was thought to contain an EPS depolymerase activity and showed a conservation only of about 50% between the two phages, with the sequence encoding the second half of the respective proteins being particularly different . Respectilspikes . Previouilspikes  and was ilspikes . Thus, wErwinia phage vB_EamP-S2 (YP_009797612.1) seemed to be a fusion of two hypothetical proteins encoding ORFs found in both Nifs112 (UJH95776.1 from ORF4 (+2 frame) and UJH95777.1 from ORF5 (+3 frame)) and Era103 (YP_001039634.1 and YP_001039635.1) that were in close proximity of each other in both cases. The corresponding product/products so far seemed to be unique to Eracentumvirus representatives and remained without even a hint at the possible function. The ORF encoding nucleotidyl transferase in the genome of vB_EamP-S2 was also split in two in the genome of Nifs112, and it looked like it might now be translated with a \u22121 frameshift. Although these two ORFs overlapped, no evident slippery signal was found in the region of the overlap of ORF21 (+1 frame) and ORF22 (+3 frame) or ~60 bases upstream until the in-frame stop codon preceding the start codon of the ORF22, and the raw reads supported this sequence unambiguously. Moreover, both ORF21 and ORF22 had Shine\u2013Dalgarno motifs that could reasonably well support the translation of their products . However, given that most BLASTP-found homologs approximately correspond to their concatenated length, the question of whether such products would be functional is open.Additionally, a region encoding the second hypothetical protein in the genome of the Pantoea phage Nifs112 might not show a sufficient amount of similarity to representatives of either of the Eracentumvirus S2  might not be an optimal host for Nifs112 could further be reasoned by the fact that, for example, the closely related Erwinia phage VyarbaL formed plaques of 3\u20133.5 mm in diameter and a well-marked halo on its host strain of E. amylovora  [Erwinia phage S2, representing the same genus Eracentumvirus species as VyarbaL, was able to lyse not only multiple strains of E. amylovora but also at least a single strain of both P. agglomerans (strain Em283) and P. ananatis (strain 351 Lys) [Summing up the analyses, it seemed that the isolated and characterized _EamP-S2  and Vyar_EamP-S2 ) or EracphiEa100 ) speciesublished , which, VyarbaL) . Additio351 Lys) .Pantoea phage Nufs112 is a linear 45,951 bp long dsDNA molecule  with a GC% content of ~47.7%. The genome of Nufs112 (OK570185.1) was predicted to have 67 open reading frames and no tRNA genes; every ORF was detected on a direct strand. ATG was predicted to be a start codon for fifty-eight of the ORFs, TTG for 6, GTG for 3, and no ORFs were presumably starting with a CTG. Only several of the identified ORFs did not have a highly preserved Shine\u2013Dalgarno motif complementary to the 16S rRNA tail of P. agglomerans in the sequence span 20 bp upstream of the selected start codons . Nearly all of the functionally annotated ORF products of Nufs112 were revealed to perform either analogous or similar functions to those identified for Nifs112 products and are typically expected to be present in an ntatives ), and sePantoea phage Nufs112 was most closely related to the Klebsiella phage 6939 and the phage Reminis . From the taxonomically officially recognized phages, Nufs112, indeed, was so far most similar to the representatives of the Gajwadongvirus phage genus (Escherichia phage ECBP5 and Pectobacterium phage PP99).The data presented previously indicated that the Pantoea phage Nufs112 only shared a hypothetical product of ORF67 with Reminis under such a strict threshold. Notably, it was shown that both the recognized Gajwadongvirus species (represented by ECBP5 and PP99) shared up to 32 of the proteins, having a >90% pairwise identity, whereas Reminis had only a single hypothetical protein shared with a recognized representative of Gajwadongvirus at such a threshold  comprised one hundred and thirty-five products, with twenty-nine of them being encoded by the genome of each phage in the comparison. At this threshold, Nufs112 encoded up to twenty-one proteins not found in the proteomes of its closest relatives, nineteen of which remained without any functional annotation , whereas at the 90% threshold there was no protein shared between the five phages, which was not very surprising given their large pairwise intergenomic distances. The hreshold . In linePantoea phage Nufs112 (OK570185) and closely related phages, genomes of the Ralstonia phage Reminis (MN478376) and the Klebsiella phage 6939 (OL362271) were rearranged to ensure collinearity with Nufs112, for which the exact genome termini were identified. In addition, it was revealed that all of the five genomes should be colinear, as seen within the virions of the respective phages. Consistent with the expectations based on the results described previously, nearly all of the proteins found in Nufs112 that were also present in the Klebsiella phage 6939 and the Ralstonia phage Reminis, and the Gajwadongvirus representatives  and theeminis MN8376 and Eracentumvirus representatives, the genomes of Nufs112 and its closest relatives also had a well-defined modular structure . The region from 7.5 kbp up to ~23 kbp contained ORFs encoding proteins responsible for DNA replication, modification, and repair, as well as several additional functions not reliably falling within any of the defined protein functional groups. Functionally annotated proteins encoding ORFs in this part of the genome were interspaced by up to another 16 hypothetical proteins encoding ORFs. The remainder of the genome (~23 kbp to ~46 kbp) was mostly comprised of ORFs encoding virion morphogenesis and putative host lysis proteins that did not form a lysis cassette with sequential ORFs encoding up to four of the lysis proteins  but were interspaced by other morphogenesis proteins coding ORFs.As in the case of the Nifs112 comparison with tructure . The genKlebsiella phage 6939 (URY99237.1) and pairwise similarities. Considering that tail fiber/fibers (their sequence) is one of the factors influencing the host range of phages, it is not uncommon for phages of different hosts to have these analogous proteins be diverged. However, it is worth noting that the Gajwadongviruses ECBP5, PP99, and MR4  were all isolated on different hosts , despite having very similar putative tail fiber proteins (pairwise identity >90% over 306 aa length of the respective proteins).The main difference that stood out when comparing the proteome contents of these phages was, however, a putative tail fiber encoding ORF .These ORF products all had an identifiable T7 tail-fiber-protein conserved domain (pfam03906) at their N termini but differed greatly in lengths (242 aa in Reminis (QGH45085.1); 306 aa in Gajwadongviruses PP99 (YP_009788795.1) and ECBP5 (AID17699.1); 506 aa in Nufs112 (UJH95883.1); 769 aa in The predicted tailspike protein of Nufs112  had an identifiable pectate lyase superfamily protein domain (pfam12708), which was previously also identified in other phages . The actPantoea phage Nufs112 was sufficiently divergent from any of the so-far-uncovered phages, which allows us to propose it to be the founding member of a novel phage genus within the family Autographiviridae.Consolidating the results, the Ralstonia solanacearum was indicated as the host in the metadata of MN478376, the genome of Reminis (MN478376) was not organized taking the SDTRs of 377 bp mentioned by the authors into account, and no SDTRs were present and/or annotated in MN478376, proteome contents and evolutionary analyses using MN478376 indicated that it is a podophage, and not a siphophage, among others).Of note, during the preparation of the genome architecture comparison figure between the phages related to Nufs112, and other analyses performed within this study, several discrepancies between the results presented in the article describing the phage Reminis  and refePantoea phage Nafs113 is a linear 75,899 bp long dsDNA molecule with a GC% content of ~54.1%. No defined termini could be predicted as an implication of the presumed headful packaging strategy that results in the progeny virions each having terminally redundant genomes that are circularly permuted when compared to each other. The scaffold representing the genome of Nafs113 was opened in a way that ensured collinearity with the closest relative, which in this particular case also followed the convention of cutting the circularized scaffold before ORFs encoding terminase subunits. Although, frankly, in the case of both phages, at the time there seemed to be no reason to assume that the ORF preceding TerL-encoding ORF encodes TerS, which is a protein known not to show such a high degree of evolutionary conservation as its larger counterpart, other than empirical phage genomics observations of the common TerS- and TerL-encoding ORF appearance in tandem. However, it is worth noting that with the growth of the number of phage genome sequences in the public biological sequence repositories, more and more exceptions to most \u201cphage genomics rules\u201d begin to appear.The complete genome of the The genome of Nafs113 (OK570186.1) was found to contain 130 open reading frames, 43 of which were located on a direct strand, whereas 87 were located on a reverse strand. No tRNA genes were found in the genome of Nafs113. ATG was predicted to serve as a start codon for one hundred and sixteen of the ORFs, GTG for nine, TTG for four ORFs, and a single ORF was predicted to have CTG as a start codon.P. agglomerans antiSD sequence)) of Nafs113 was only \u22124.57 \u00b1 2.36 kcal/mol (average \u00b1 SD) as calculated by free_align.pl script [Interestingly, despite the fact that most of the predicted ORFs had a well-identifiable Shine\u2013Dalgarno motif in the sequence span 20 bp upstream of the selected start codons , the onel script , whereasPantoea phage Nafs113 was demonstrated to be rather unique among the so-far publicly available sequenced phages (bar the Pantoea phage vB_PagM_LIET2) based on its complete-genome nucleotide sequence, it was not unexpected that only 30% of the predicted Nafs113 ORF products could be assigned a putative function. Nevertheless, a number of the reasonably well-conserved proteins responsible for virion morphogenesis/structural features expected for a tailed dsDNA phage were identified in the genome of Nafs113 despite its genomic nucleotide sequence being very uncommon among the so-far-uncovered phage diversity. Both tail-tape-measure-protein- and tail-sheath-protein-encoding ORFs were identified, which would be sufficient to identify Nafs113 as a myophage had the TEM virion analysis not yet been made. Upon closer inspection, holin, endolysin, and putative inner and outer spanins, representing the lysis proteins expected for a phage of a Gram-negative host bacterium, could be identified. In addition, a number of nucleases and other proteins involved in DNA replication, modification, and repair were annotated. The genome of Nafs113 contained several additional functions performing proteins as well; however, their annotations did not allow us to reliably categorize them into any of the phage product groups we categorized proteins into according to their function , representing a Pantoea stewartii WceF\u2014a glycan biofilm-modifying enzyme with a bacteriophage tailspike-like parallel beta-helix fold. It was recently shown that WceF is a glycosidase active on stewartan, which acts as the main P. stewartii EPS biofilm component, and that WceF is very similar to bacteriophage tailspike proteins [Another marked difference between the two genomes was that one of the two ORFs that were either fused into one (LIET2) or split into two (Nafs113) encoded what was annotated as a 1218 aa long EPS depolymerase-domain-containing protein in LIET2 (YP_009843765.1) or two putative tail-spike (UJH95983.1 and UJH95984.1)-encoding ORFs in Nafs113. While a CD search failed to identify any conserved domains that would imply EPS depolymerase activity for either of these three proteins, the top HHpred hit for each of the three of them was to a PDB structure , whereas the same search using the amino acid sequence of the putative ORF108 product (UJH96005.1) as a query revealed only a match to the hypothetical protein HWC07_gp107 from the Pantoea phage vB_PagM_LIET2.A similar fusion/split was observed for two Nafs113 hypothetical proteins (UJH96005.1 and UJH96006.1) encoded, this time, from frames \u22121 (ORF107) and \u22123 (ORF108) in Nafs113, respectively, whose products appeared to be fused in LIET2 (YP_009843788.1). Again, in this case, too, no apparent +1 frameshift signal, was observed, and the presumed Shine\u2013Dalgarno motif for translation of the ORF107 product appeared \u201cstronger\u201d than that of ORF108. However, in this case, the ORF107 product (UJH96005.1) had a lot of BLASTP hits to hypothetical proteins of similar length (~208 aa) from the bacteria  collected geographically  close and temporally proximal  to each other. Speculatively, it can be proposed that the consequences of differences in horizontal gene exchange events by their ancestral phage populations  might explain most of the differences observed between genome architectures and proteome contents as, we believe, the ecological niches and environmental factors naturally influencing both LIET2 and Nafs113, as well as their respective host species strains, might naturally be very similar.All things considered, there is undeniably a very close evolutionary relationship between the Pantoea phages LIET2 and Nafs113 were higher than the same phage species level threshold of <5% differences over complete genome lengths, and some genomic features were different, there is no sufficient evidence in favor of creating a novel phage species within the genus Lietduovirus for Nafs113 instead of considering it another isolate representing the Lietduovirus LIET2 species. Additional research on both of these phages might, however, shed light on more of their functional differences that may warrant their possible delineation at the species level in the future.Given their great difference from any other phage sequenced so far, at this time, we are rather convinced that, for practical reasons, even though intergenomic distances between Orthoptera) specimen, as described previously for the phage Nocturne116 [Studied phages were isolated alongside their host bacteria from a dead unidentified Latvian grasshopper , and subjected to a double agar overlay assay . Phage-nSeveral rounds of individual plaque purifications were carried out, and three different lines of plaques consistently giving the same negative colony appearances were established and assumed to represent three distinct phages.g, 30 min), and the resulting supernatant was decanted and filtered through a 0.45 \u03bcm pore size syringe filter. For all three of the investigated phages, such propagation yielded filtrate with a titer of up to 1\u20132 \u00d7 1010 PFU/mL.For phage propagation, soft agar layers from 20 double agar overlay plates  showing confluent lysis of the bacterial lawn were collected and homogenized by vortexing with the addition of 5 mL LB liquid medium per plate. Next, the homogenized soft agar layer mixture was subjected to centrifugation . The obtained phage pellets were dissolved in ~4 mL of TE buffer . Acquired phage suspensions were next layered on top of a CsCl solution (CsCl\u20140.6 g per mL of the TE buffer). For each phage, a single Ultra-Clear centrifuge tube  was filled with 11.5 mL of the CsCl solution, and 2 mL of a concentrated phage sample was loaded on top of the CsCl solution. Solutions were then spun at +4 \u00b0C for 20 h  in an SW 40 Ti rotor (Beckman Coulter)-equipped Beckman Optima L-100XP ultracentrifuge. Distinct horizontal phage bands were formed for each of the phages, and these were collected by pipetting. Collected phage-containing bands were desalted using NAP-25/Sephadex G-25 columns  and PBS as an exchange buffer.Phages were then concentrated by centrifugation of the filtrates in a Beckman Optima L-100XP ultracentrifuge with a 70 Ti rotor  for 5 min, rinsed with 1 mM EDTA solution, and negatively stained using 0.5% uranyl acetate before being left to dry for 2 h. After drying, the stained phage-sample-containing grids were examined using a JEM-1230 transmission electron microscope , and virions were pictured with a Morada 11 MegaPixel TEM CCD microscope-mounted camera using iTEM imaging software .Phage particle dimensions were determined with the help of ImageJ software  utilitiTo release the phage genomic DNA, the purified and concentrated phage sample was incubated at +56 \u00b0C for 1 h with the addition of proteinase K and SDS . DNA extraction was performed with the Genomic DNA Clean & Concentrator-10  kit as per the manufacturer\u2019s protocol. Quality and approximate concentration of the obtained phage genomic DNA were evaluated on a NanoDrop ND\u20141000 spectrophotometer  and then diluted accordingly to verify the specific dsDNA amount using a Qubit fluorometer  dsDNA high-sensitivity quantification assay (Invitrogen).The TruSeq DNA Nano Low Throughput Library Prep Kit  protocol, compatible with the TruSeq DNA Single Indices Set A (Illumina), was followed to prepare the Illumina MiSeq-compatible DNA libraries . To prepare the appropriate input for the library preparation step, 200 ng of the dsDNA from each of the studied phages was randomly fragmented with a target fragment length of 550 bp in mind using a Covaris S220 focused ultrasonicator .Resultant library quality control was performed using an Agilent 2100 bioanalyzer  with a high-sensitivity DNA kit (Agilent) and a Qubit fluorometer (Invitrogen) dsDNA high-sensitivity quantification assay (Invitrogen). Libraries were then sequenced on the Illumina MiSeq system (Illumina) using a 500-cycle MiSeq Reagent Kit v2 nano (Illumina) for three of the twelve uniquely indexed libraries comprising the run.Overnight culture of the bacterial host isolate LS5-2 was used for genomic DNA isolation as described for the phage genomic DNA extraction  recommendations , 5 min at +70 \u00b0C, followed by a hold at +4 \u00b0C). The ABI PRISM 3130xl system (Thermo Fisher Scientific) was used as a sequencer. Raw read chromatograms were manually inspected and trimmed in GeneStudio (v. 2.2.0.0.). After, the trimmed reads were joined into a contig and used for consensus sequence calling  in the same software.Sanger-based sequencing of the extracted amplified partial 16S rRNA sequence of the host was carried out in two reactions (27F primer for forward and 1492R primer for reverse read) according to the BigDyePectobacterium carotovorum strain NCPPB 312 (JQHJ01000001) representing an outgroup. Multiple sequence alignment (MSA) was performed using MAFFT  with further manual visual refinement in Inkscape .The resulting partial 16S rRNA gene sequence of the host was queried against the bacterial 16S rRNA gene sequences publicly available at EzBioCloud . All val. 7.453; ), and th. 7.453;  reconstr. 7.453; . The NJ . 7.453; . The resPrior to de novo assembly, demultiplexed read datasets were inspected with the help of FastQC , and coDe novo assembled \u201ccircularized\u201d contigs were next used alongside the corresponding library untrimmed reads for possible genome termini identification using PhageTerm . Next, https://phagesdb.org/DNAMaster/ accessed on 12 April 2021) and were followed by putative ORF product functional annotations using publicly available biological sequence repositories and web services as described previously [P. agglomerans 16S rRNA tail . Longer possible coding sequence overlaps were also evaluated when annotating phages within this study. Additionally, TMHMM [The open reading frame (ORF) and tRNA gene predictions were performed with the corresponding tool implementations in DNA master  were usCUAG-5\u2032;  when evay, TMHMM  and Phoby, TMHMM  web servBacillus phage BJ4 (AOZ61694) and Bacillus phage SBP8a (AOZ62321), as well as the TerL sequences of the studied Pantoea phages Nufs112 (UJH95886.1), Nifs112 (UJH95823.1), and Nafs113 (UJH95900.1)). Multiple TerL sequence alignment was performed using MAFFT .To further verify the packaging strategies/termini types the isolated phages employed, the terminase large subunit (TerL) amino acid sequence ML tree was reconstructed using an extended dataset of Merrill and colleagues . Extensi. 7.453; ), and ML. 7.453; ), select. 7.453;  inferenc. 7.453; ) replicaFirst, a BLASTN search against all the viral sequences (taxid:10239) publicly available at the non-redundant nucleotide collection was performed for complete-genome sequences of studied phages to evaluate their novelty and get a hint at the most appropriate further analyses for the task of elucidating the place of isolated phages within the so-far-uncovered phage diversity.Pantoea phages Nifs112 (OK570184.1), Nufs112 (OK570185.1), and Nafs113 (OK570186.1) were already publicly available in GenBank in late January 2022, we took advantage of the INPHARED [As complete annotated genomes of INPHARED  databaseINPHARED ) proteomINPHARED ). Nodes http://tree.bio.ed.ac.uk/software/figtree/ accessed on 10 May 2021) and Inkscape .The vConTACT2-identified first-neighbor genomes were next subjected to VIRIDIC  analysiIndividual protein phylogenies were reconstructed for the major capsid protein , terminase large subunit , endolysin , and phosphatase  of the studied phages .First, these sequences were subjected to a BLASTP search against the non-redundant protein sequences of viral origin (taxid:10239) under other default settings. For a given protein from the studied phages, the ten highest-scoring hits encoded by other cultured phage genomes were selected and downloaded regardless of the annotations, combining hits into multifastas comprising proteins of presumably analogous functions.Next, respective analogous protein amino-acid-sequence-containing datasets  were aligned using MAFFT  and subhttp://tree.bio.ed.ac.uk/software/figtree/ accessed on 10 May 2021). Inkscape  was then used to combine, as well as annotate, the trees based on the phage complete-genome-associated metadata .The resulting tree was midpoint rooted and visualized in FigTree  and Cliv.2.2.2; ) were sePantoea phage Nifs112 (OK570184) was compared with two representatives of the genus Eracentumvirus\u2014Erwinia phages vB_EamP-S2 (NC_047917) and Era103 (NC_009014).That way, the complete genome of the Pantoea phage Nufs112 (OK570185) was compared with the genomes of the Klebsiella phage 6939  and the Ralstonia phage Reminis , both of which had to be rearranged taking the identified SDTR location in the genome of Nufs112 into account. Additionally, genus Gajwadongvirus representatives Escherichia phage ECBP5 (KJ749827) and Pectobacterium phage PP99 (NC_047802) were also used for Nufs112-related comparisons.The Pantoea phage Nafs113 was compared with the sole representative of the genus Lietduovirus, Pantoea phage vB_PagM_LIET2 (NC_048751).The https://inkscape.org accessed on 10 May 2021).Generated figures were further visually refined in Inkscape  was empPantoea agglomerans LS5-2. The isolated phages were very different from each other and, to our knowledge, were so far the first three Pantoea sp.-infecting phages isolated from an insect source.Three bacteriophages infecting the same bacterial strain were isolated from an unidentified Latvian grasshopper species alongside its host Pantoea podophage Nifs112 (OK570184) was proposed to represent a novel species within the phage family Autographiviridae, subfamily Molineuxvirinae, genus Eracentumvirus (comprising so far exclusively phages infecting Erwinia).The Pantoea podophage Nufs112 (OK570185) was evolutionary moderately related to two of the phages yet without a standing in the official phage taxonomy  and, to a lower extent, to representatives of the phage genus Gajwadongvirus. The sufficient amount of difference Nufs112 showed in comparison to either of the so-far-sequenced phages, however, allowed us to propose it as an exemplar isolate for the creation of a new phage species that would represent a novel phage genus.The Pantoea myophage Nafs113 (OK570186) had quite an unusual capsid with an approximately 3:1 length-to-width ratio. Nafs113 was very distantly related to any of the so-far completely sequenced phages that are publicly available, except for the Pantoea phage vB_PagM_LIET2, with whom Nafs113 shared a very close evolutionary relationship. For practicality reasons, at this time we propose to consider Nafs113 as an isolate of the phage species Lietduovirus LIET2, the so-far single phage species within the genus Lietduovirus, currently represented by the Pantoea phage vB_PagM_LIET2 alone.The Pantoea spp. but also of closely related bacteria .Additional microbiological characterization of these three lytic phages will next be undertaken to determine their possible host ranges, lifecycle characteristics, and virion stability, ultimately to evaluate their potential to be used for biocontrol of not only"}
{"text": "Phages are efficient in diagnosing, treating, and preventing various diseases, and as sensing elements in biosensors. Phage display alone has gained attention over the past decade, especially in pharmaceuticals. Bacteriophages have also found importance in research aiming to fight viruses and in the consequent formulation of antiviral agents and vaccines. All these applications require control over the stability of virions. Phages are considered resistant to various harsh conditions. However, stability-determining parameters are usually the only additional factors in phage-related applications. Phages face instability and activity loss when preserved for extended periods. Sudden environmental changes, including exposure to UV light, temperature, pH, and salt concentration, also lead to a phage titer fall. This review describes various formulations that impart stability to phage stocks, mainly focusing on polymer-based stabilization, encapsulation, lyophilization, and nano-assisted solutions. Bacteriophages are viruses infecting bacteria. The name is derived from \u201cbacteria\u201d and the Greek \u03c6\u03b1\u03b3\u03b5\u1fd6\u03bd (phagein), meaning \u201cto devour\u201d. Bacteriophages are obligate parasites that annex the host\u2019s molecular machinery to complete their life cycle. Hundreds of new virions are folded inside a single bacterial cell. In most cases, bacteria are disrupted, and phages are released (lytic cycle). Chronic phages, e.g., filamentous phages f1, fd, or M13, do not lyse their host cells, but progeny virions are secreted continuously ,3. In thStraboviridae , the Caudoviricetes class , or the family Autographiviridae . Tail fibers are attached to the tail and have a substantial positive charge. The head has a significant negative charge [Caudoviricetes class phages are filamentous  or nearly spherical (isometric) phages .The average size of a virion (single phage) is around 50 nm to 200 nm. However, the largest bacteriophages are more than 800 nm . In oceae charge . Such asCaudoviricetes, initial recognition is based on electrostatic interactions\u2014positively charged tail fibers are attracted to the negatively charged surface of bacteria. The following steps of host recognition utilize specific receptor-binding proteins, which discriminate the appropriate host. This selectivity is the basis for the utilization of phages in sensing.Only recognizing a proper and viable host assures the multiplication of virions and the completion of the phage life cycle. Thus, very often, a multi-step \u201cidentification\u201d process is used. In the case of The phage display method boosts the importance of bacteriophages. The possibility of directly studying the link between genotype and phenotype has allowed for numerous applications. Phage display is quite similar to the immunodetection assay but, instead of relying on antibodies, it relies on phages as recognition elements. Briefly, a library of genetically modified phages expressing different peptides on their capsids is prepared. Then, the phage library is exposed to the molecule (embedded on the surface) to determine specifically binding peptides .Confidence in phage therapy is re-emerging in the pharmaceutical world. The FDA approved the use of phages in critically ill COVID patients with secondary bacterial infections in 2020. Several phage-based products emerged shortly after, for example, GangaGen, ContraFect, Phagelux, and Phagomed, to fight S. aureus in India, the USA, China, and Austria, respectively . In addiApproximately 13% of deaths are related to bacterial diseases . BacteriFoodborne illnesses remain a significant cause of worldwide death despite many advances in pathogen surveillance and food sanitation methods. According to the WHO, 600 million foodborne infections occurred in 2010, resulting in over 400,000 deaths. Besides being a huge social burden, it is also a massive drain on the economy of nations. The average incident is estimated to cost around USD ~1500 per person . MoreoveBeing natural antibacterial agents, bacteriophages are considered an alternative to antibiotics, especially at the dawn of the antibiotic-resistance era. There are several advantages to using phages to fight bacteria.Phages can be produced easily and cheaply in large quantities and easily purified. By only infecting a bacteria solution, one can obtain a large number of progeny phages.E. coli phage PK1A2 in vitro [Bacteriophages are considered non-toxic to eukaryotes because structural elements of the virion cannot bind to eukaryotic cells . There ain vitro . The virThe lysis of bacteria resulting from phage infection supports the inflammatory response against bacteria . TherefoPhages undergo evolution, and thus, they remain effective against bacteria . DespitePhagoburn project) [Bacteriophages have been proposed for medical use since the early 1900s ,30. Due project) . The firproject) .de facto makes phages incapable of infecting bacterial cells [Phage administration without any stabilizing additives triggers an inflammatory response as a result of releasing pathogen-associated molecular patterns (PAMPs) from lysed bacteria  . Howeveral cells .Phages are also tested for biocontrol applications, e.g., in the food industry and agriculture. Phage biocontrol is increasingly accepted as green and natural technology for the specific targeting of pathogens . Phages Among the many bacteriophages, some examples are considered good surrogates for studies on eukaryotic, often dangerous, viruses. The most common examples are MS2 , Phi6 55,56, PhiAs such, methods developed for phage stabilization might be applicable to the stabilization of other viruses. This is still important, as bacteriophages can be used to formulate vaccines. Depending on the particular type of vaccine, phages can either carry the genomes of other viruses, affinity bind eukaryotic virus antigens on the capsid\u2019s surface ,62, or pThe major drawback of using phages as antimicrobials is the stability of virions. The primary criteria are virulence, selectivity, host range, ease of manipulation, and modification. Even though phages are capable of retaining their activity after exposure to stress factors , about 5 log below the required rate of infection (ROI) [The matter of phage suspension stability is not to be underestimated. Improper preparation of such suspension can affect the effectiveness of the phage therapy approach. For instance, in the on (ROI) .With the shift in attention towards phage-based pharmaceutics, the precise nature and uniformity of bacteriophages are gaining increased appreciation. The preliminary testing of phage therapy did not concern the stability of phages; however, commercial circulation would require stable concentrations and known dosages. Phages often lose activity during formulation and storage. The most common factors affecting the viability of phages (and phage proteins) include exposure to organic solvents, high temperatures, pH, and ionic strength .Encyclopedia Britannica). Because of a large number of subunits, removing one of them does not change the properties of the entire molecule. Polymers can be divided into natural  and synthetic  [A polymer is a substance or material of large molecular mass composed of repeating subunits (opylene) . AlthougP. aeruginosa. Such hydrogels were effective in the elimination of bacteria embedded on the surfaces of gel beads. The authors, however, did not provide quantitative values of these antibacterial properties. [Listeria monocytogenes. While pullulan itself was not effective for phage stabilization after drying, the combination of pullulan and trehalose as a stabilizing matrix allowed the maintenance of about 7 log active phages after 60 days . In comparison, in the trehalose matrix, only 3 log phages remained active after the same amount of time [The effect of sugars as stabilizers against protein unfolding is known ,76,77,78perties. . Leung e of time .Polyacrylamide can be used to stabilize phage particles for strictly research purposes, such as protein nuclear magnetic resonance spectroscopy. Trempe et al. reported the stabilization of a filamentous Pf1 phage using 5% polyacrylamide as a polymer-stabilized liquid crystal (PSLC). This approach allowed for measurements of dipolar couplings with a single sample and, therefore, a more accurate analysis of protein structure based on the comparison of theoretical and experimental tensor parameters .PEGylation is the attachment of polyethylene glycol (PEG) groups to the target molecule, commonly used in food and drug formulation to increase molecules\u2019 stability. PEG is biocompatible and reduces the immunogenicity of the molecule, but it is non-degradable . PEG is Polymers might also have an indirect effect on phages. Richter et al. explained the phenomenon of phage titer loss when phages are stored in plastic (mainly polypropylene) labware . The autPolymers are also used as protective matrices in which phages are embedded. Encapsulation allows for better stability and modulates the long-term release of active phage particles . VariousSalmonella was encapsulated within alginate microparticles associated with calcium carbonate to prolong their gut residence time [The applications of encapsulation also include phage cocktails. For example, a cocktail of three phages against nce time ,102. Pacnce time .Malik et al. listed the types of polymers used for phage encapsulation as agarose, alginate, chitosan, pectin, whey protein, gelled milk protein, hyaluronic acid methacrylate, hydroxypropyl methylcellulose poly(N-isopropyl acrylamide), poly(DL-lactide: glycolide), polyesteramide, polyvinyl pyrrolidone, polyethylene oxide/polyvinyl alcohol, cellulose diacetate, and polymethyl methacrylate . AlginatSalmonella [Salmonella phages have been produced to maintain elevated levels of phage particles in the gut [Salmonella phages, protecting them against the acidic conditions of the intestine [Liposomes are biocompatible nanoparticles composed of a lipid bilayer surrounding therapeutic cargo. This approach is promising in achieving directed delivery while surviving the extreme conditions of the stomach and intestine when administered orally . Liposomlmonella . Pharmac the gut . Nanoparntestine . This tentestine . Howeverntestine .While aiming for perfection, modifications in the approach to encapsulate phages are gaining popularity. For example, microfluidic devices are used to produce calcium alginate capsules containing bacteriophages . These cLyophilization (dehydration process), first used for food storage, has become a very commonly used method for phage stabilization and long-term storage. At first, lyophilization involved freezing the phage stock, lowering the pressure, and removing the water  . Now, thFor phage storage, freeze-drying and spray-drying methods are the most important. Freeze-drying is, relatively, the cheapest method for preparing phage powders. One of the first attempts was the freeze-drying of mycobacteriophages; when stored in the dark, phage lyophilizates were suitable for over two years . The aveStaphylococcus aureus ISP phage to prolong the storage time up to 37 months, with a titer loss of about 1 log [6000) in the M13 phage model. After seven days, the phage titer was 2 log higher in the sucrose and trehalose solution compared to PEG [The storage time can be extended by adding some cryoprotectants. Merabishvili et al. proposed adding 0.5 M sucrose and trehalose solutions to the ut 1 log . This obut 1 log . Additiod to PEG . A simild to PEG . Recentld to PEG . Other sd to PEG .Pseudomonas phages\u2014PEV2 and PEV40. They showed that phage powder prepared in the mixture containing 70% trehalose and 30% leucine could be stored for 12 months with a titer decrease of about 0.5 log [Spray-drying allows for a reduction in the decrease in the phage titer during the procedure . Still,  0.5 log . Chang e 0.5 log . In addi 0.5 log . The sen 0.5 log . Moreove 0.5 log . Phage s 0.5 log ) contain 0.5 log .Pseudomonas aeruginosa [Acinetobacter baumannii [Burkholderia cenocepacia [Salmonella enterica [Campylobacter jejuni [Mycobacterium tuberculosis [Freeze-drying and spray-drying were combined into a novel atmospheric spray-freeze-drying (ASFD) ,135. Botruginosa ,143,144,aumannii , Burkholocepacia , Salmoneenterica , Campylor jejuni , or Mycorculosis . Howeverrculosis .Another long-term method of storing and stabilizing bacteriophages is freezing mature virions within bacterial cells. In this approach, bacteriophages at a proper multiplicity of infection (MOI) are mixed with their host bacterium and incubated for a short time. Next, infected cells are frozen and stored at \u221280 \u00b0C. After reviving and washing, phages are released and can actively infect bacterial cells. Golec et al. proved that this method allows for phage storage with minor or no losses in phage titer for about 10 months, depending on a particular phage .The implementation of nanotechnology in medicine has the potential to solve the stability issue. Nanoparticles can be viewed as vectors for drug solubilization while overcoming sequential biological barriers in the body . Phages E. coli cells [B. anthracis can be targeted using phage probes modified and stabilized with gold nanoparticles [Nanoscience plays an essential role in the immobilization of phages; bacteriophages can be chemically or genetically modified to bind strongly to nanomaterials . Gold nali cells . Additioarticles . For morarticles . The onearticles .In some experiments on vaccine formulations, nanolayers of aluminum oxide were used to stabilize the \u03bb bacteriophage, which ensured the controlled release of antigens in vivo . VaccineApart from imparting stability, nanoparticles have been shown to enhance the functioning of phage-based biosensors by exploring rapid and sensitive approaches . Carbon-P. aeruginosa planktonic and biofilm states, with high stability under a broad range of temperatures, pHs, and salt concentrations [Salmonella enterica by causing the deformation of biofilms [The synergistic effect of phages and nanoparticles has been widely used for targeting biofilms and eliminating pathogenic infections ,167. Fortrations . In othetrations . Phage vtrations . Metallibiofilms . Alternabiofilms .Vaccination against infectious diseases saves over three million lives yearly. However, some estimations suggest this number could be doubled if all the problems related to proper storage were solved . AccordiDespite the advantages, research on phages is troublesome due to their instability in phage cocktails and varying phage titers . Table 12), but only for up to 5 min [Phage delivery is also often compromised due to its degradation and clearance by the body\u2019s defense mechanisms . Many teto 5 min . Due to This review presents an update on the state of the art of bacteriophage stabilization, including the research published only last year (since May 2021). The review is mostly focused on polymer-based stabilization, encapsulation, lyophilization, and nano-assisted solutions; the problems and future perspectives of such approaches are also highlighted."}
{"text": "Infections caused by multidrug-resistant (MDR) bacteria have highlighted the importance of the development of new antimicrobial agents. While bacteriophages (phages) are widely studied as alternative agents to antibiotics, combined treatments using phages and antibiotics have exhibited Phage\u2013Antibiotic Synergy (PAS), in which antibiotics promote phage replication and extraordinary antimicrobial efficacy with reduced development of bacterial resistance. This review paper on the current progress of phage\u2013antibiotic therapy includes aspects of the mechanisms of PAS and the therapeutic performance of PAS in combating multidrug-resistant bacterial infections. The choice of phages and antibiotics, the administration time and sequence, and the concentrations of the two agents impact the bacterial inhibitory effects to different extents. Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (the ESKAPE pathogens) have developed resistance to oxazolidinones, lipopeptides, macrolides, fluoroquinolones, tetracyclines, \u03b2-lactams, \u03b2-lactam\u2013\u03b2-lactamase inhibitor combinations, polymyxins, glycopeptides, and carbapenems [A. baumannii, P. aeruginosa, and Enterobacteriaceae are listed as the most critical pathogens that require the development of new antibiotics because they have developed resistance to carbapenem, which is the last-line drug for combating resistant bacteria [Acinetobacter, carbapenem-resistant Enterobacteriaceae, and drug-resistant Neisseria gonorrhoeae are listed as urgent threats [Antibiotics are one of the greatest discoveries in modern medicine and have extensively decreased morbidity and mortality caused by different types of bacterial infections, improving the quality of life and life span of patients . Howeverbapenems . In the bacteria ,3. In An threats . This anBacteriophages (or phages) are viruses that specifically target bacteria . Phage tVirulent phages (or lytic phages), which are unable to lysogenise their hosts, multiply in bacterial hosts, and lead to bacterial lysis after each infection cycle, are only used for therapeutic investigation against bacterial infections. Either wild-type lytic phages ,8 or engAntibiotics are chemical compounds that have a specific mechanism for disrupting either the cell wall or the intracellular pathways. For example, carbapenem inhibits cell wall formation, and ciprofloxacin inhibits DNA replication. Because the mechanisms for phages to inhibit bacteria are different from those of antibiotics, phages can treat MDR bacterial infections. However, bacteria can still evolve an antiphage system in a number of ways: (1) mutating bacterial cell surface receptors, preventing the entry of phage DNA ,14; (2) Phage\u2013antibiotic therapy has been successfully shown to reduce the emergence of phage-resistant and antibiotic-resistant strains. An often-cited example is the fact that the degeneration of cell surface receptors caused by phages restores antibiotic sensitivity, as those receptors are responsible for the efflux of antibiotics . SeveralAs a promising therapeutic strategy for bacterial infection, the combination therapy using phages and antibiotics has now been studied extensively, not only because of its improved performance in reducing phage and antibiotic resistance but also for the synergistic antibacterial effects achieved by phage replication enhancement in the presence of antibiotics  ,23.This review focuses on the PAS response and its mechanisms; the development of resistance in bacteria in response to phage\u2013antibiotic combined treatment; and the application of phage\u2013antibiotic treatment in vitro, in vivo, and in clinical case studies.PAS has been observed between phages and antibiotics. This has raised interest in the therapeutic potential of PAS to improve bacterial killing ,25.The term PAS initially described the phenomenon of the improved antimicrobial effect caused by stimulated phage replication in the presence of sublethal concentrations of antibiotics. Nowadays, the term PAS is used in circumstances where synergistic antimicrobial effects occur. In this review, we use the term PAS to describe the phenomenon of increased phage activity in the presence of sublethal concentrations of antibiotics. The terms synergistic effects or synergism are used herein to describe the significant improvement in bacterial growth inhibition with phage\u2013antibiotic co-treatment.Escherichia coli clinical isolate MFP; on T4-like phages against standard laboratory E. coli strains; on T3 and T7 phages against laboratory E. coli strain AS19; and on T4-type Yersinia phage PST against Yersinia pseudotuberculosis in the presence of sublethal \u03b2-lactams such as aztreonam, cefotaxime, ticarcillin, piperacillin, ampicillin, and quinolones such as nalidixic acid. The plaque size enlargement implies enhanced phage production during plaque formation, resulting in increased bacteria growth inhibition and/or lysis. Other than E. coli phage and Y. pseudotuberculosis phage [P. aeruginosa [S. aureus [Bacillus cereus [Enterococcus faecalis [Burkholderia cepacian [PAS was first studied by Comeau et al. , where sis phage , plaque ruginosa ,27, S. a. aureus ,29, Bacicepacian .Plaque size can be affected by multiple factors, including the intrinsic traits of the phage and the infected host, as well as extrinsic factors such as incubation time, temperature, agar density, and bacterial density . As thesP. aeruginosa PA01 from an average of 1.15 \u00b1 0.18 \u03bcm to 1.8 \u00b1 0.18 \u03bcm  [The PAS response in lytic phages is considered directly related to bacterial filamentation, which increases the bacterial size and cellular surface area ,27,37,38.6-fold) .S. aureus ATCC 12201; PBEF7 and PBEF9 against E. faecalis KCTC 2011; PBBC03 against B. cereus KCTC 1012; T4 against E. coli K-12 strain ATCC 700926; PBEC22, PBEC24, and PBEC82 against E. coli Crooks strain ATCC 8739; and PA26, PA22, and PA25 against P. aeruginosa ATCC 13388. Of the 88 tested phage and antibiotic combinations, 56 exhibited an increase in plaque size along with antibiotic-induced bacterial filamentation in rods or bacterial swelling in cocci [P. aeruginosa phage PA22, in which PAS did not occur , yet the bacteria filamented. However, the reason remains unclear.In another PAS study, 11 phages in combination with 8 antibiotics  were tested against their hosts: phage SA11 against in cocci . The preE. coli strains, PBP1s are involved in bacterial elongation, PBP2s are responsible for cell elongation and rod shape maintenance, and PBP3s are responsible for septal wall formation during cell division. Blocking PBP1s leads to direct cell lysis, blocking PBP2s leads to ovoid bacterial cells, and blocking PBP3s leads to bacterial filamentation [Most of the antibiotic-induced filamentation can be explained by two mechanisms: the \u03b2-lactam-induced and SOS-response-mediated mechanisms. \u03b2-lactams alter the morphology of bacteria by binding to penicillin-binding proteins (PBPs) on the bacterial surface. PBPs are key peptidoglycan-synthesizing enzymes that play an integral role in constructing the bacterial cell wall. For example, in entation .E.coli and P. aeruginosa cells [E. coli and P. aeruginosa can form filaments in 3\u20134 h in the presence of sublethal concentrations of cefuroxime and ceftazidime (0.008 \u00d7 MIC~1 \u00d7 MIC) [E. coli and P. aeruginosa cells, \u03b2-lactams such as imipenem and mecillinam have higher affinity for PBP2s and are reported to cause ovoid cells; ampicillin has similar affinity for PBP2s and PBP3s and is reported to cause localised swelling [\u03b2-lactams vary in their affinity for different PBPs, which induce different alterations in bacterial shapes to different extents or result in direct cell lysis . \u03b2-lactasa cells ,41. In tsa cells ,41,42. E1 \u00d7 MIC) ,41. On tswelling  as well swelling . Other tswelling , PAS resConsequently, the choice of a specific \u03b2-lactam antibiotic affects the degree of filamentation, which in turn may affect the adsorption rate of phages by increasing phage targeting receptors on the bacterial cell wall  was detected in the presence of a sublethal dose of ciprofloxacin, whereas other tested bacteria  exhibited both recA expression and filamentation [recA deletion E. coli mutant strain showed filamentation and PAS in the presence of ciprofloxacin even with no recA expression. These results suggest that the SOS response may not necessarily be responsible for bacterial filamentation induced by DNA synthesis inhibitors or DNA disrupting agents; filamentation could also be triggered through other pathways.It is generally believed that the observed PAS response with quinolones is due to filamentation induced by the SOS response. However, exceptions were reported by Kim et al. . P. aeruentation . MoreoveS. aureus, E. faecalis, B. cereus, E. coli K-12, E. coli Crooks, or P. aeruginosa, or in trimethoprim against E. coli K-12. One possible reason is that SOS-response-mediated filamentation did not happen at the tested antibiotic concentration or within the exposure period (not specified in the article) as filamentation is affected not only by the antibiotic class but also by the antibiotic concentration and duration of exposure [recA expression was only tested in ciprofloxacin, it is unknown whether the SOS response is triggered by trimethoprim and sulfamethoxazole.Moreover, although SOS-response-mediated filamentation has been reported in studies with mono-treatment of trimethoprim and sulfamethoxazole , conflicexposure . As the The SOS response was also found to improve temperate phage activity because it triggers temperate phage assembly and release due to the cleavage of phage repressors by RecA ,48. We hBacterium coli [P. aeruginosa [E. coli [Although PAS and filamentation have rarely been observed with sublethal concentrations of antibiotics other than \u03b2-lactam and DNA-disrupting antibiotics ,27, filaium coli , aminoglruginosa ) or RNA [E. coli ). Althou[E. coli ,24,27.In addition to structural changes in bacterial cells , some alterations in the phage lytic cycle were found in the presence of filamentation and the PAS response. To date, three main alterations in the phage lytic cycle have been reported to induce the PAS response . It is uPAS may be due to bacterial filamentation and resultant changes in the phage lytic cycle , but filFilamentation occurred either when PBPs were inhibited by \u03b2-lactam antibiotics (\u03b2-lactam-induced) or as a survival mechanism when DNA was damaged by antibiotics (SOS-response-mediated). When bacteria remain filamented after antibiotic treatment, genome replication and expression continue without separation into daughter cells. Once antibiotics are depleted or the DNA is repaired, bacterial division and replication resume. If, before the antibiotics are depleted, the phages lyse all bacteria cells with only limited numbers of the lytic cycle, no significant phage replication is observed. This does not provide an advantage to phage multiplication, hence no PAS. However, due to early bacterial clearance, phage\u2013antibiotic co-therapy is still beneficial because the phages promote bacterial lysis.Since phage\u2013antibiotic therapies are mainly used for combating MDR infection, it is expected that MDR bacteria cannot be fully killed/inhibited by antibiotics alone and that they continue to grow in the presence of antibiotics; consequently, the phages undergo replication. In addition, if the antibiotic dose is too low for a subinhibitory effect against the bacteria, or the bacteria are highly resistant to the antibiotics and filamentation cannot be induced, PAS may not occur. Thus, PAS may hinge on a balance between the bacterial replication rate in the presence of a sublethal concentration of antibiotics and the phage replication rate to lyse the host. As a result, the choice of the type and dose of antibiotics for a given phage is a critical determinant of the PAS response.Phage adsorption to bacteria, as the first stage of the lytic cycle, is one of the critical steps for phage multiplication. Phage adsorption includes reversible and irreversible binding. Phage adsorption rate refers to irreversible binding.E. coli, S. aureus, and P. aeruginosa in the presence of sublethal concentrations of antibiotics. By pre-treating E. coli B/r H266 with a low concentration of penicillin, the adsorption rate of T4 phages was significantly increased (4.68 \u00d7 107 phage/mL/min) compared to the control (3.36 \u00d7 107 phage/mL/min) [An improvement in phage adsorption efficiency was found in /mL/min) .50), 75% (T75), and 100% (T100) of phage MR-5 to be adsorbed to S. aureus ATCC 43300 pre-treated with antibiotics for 90 min  and a control bacteria culture. Pre-treatment with linezolid and tetracycline significantly decreased T50 , T75 , and T100 , while clarithromycin and telithromycin significantly decreased T50  and T75 .Kaur et al.  measuredP. aeruginosa PA01 in the presence of 1.06 \u00b5g/mL aztreonam lysine was conducted using transmission electron microscopy (TEM). The cell significantly increased in length  and width . Meanwhile, approximately a two-fold increase in phage attachment was detected in the presence of aztreonam lysine: an average of 2 \u00b1 1 phage E79 adsorbed to each P. aeruginosa PA01 cell in the control, while 5 \u00b1 2 phages per cell were detected in an aztreonam lysine-treated culture [Another assessment of the filamentation and improved attachment of phage E79 to  culture . However culture . MicroscIn filamentation-associated PAS, some have proposed that the increase in adsorption rate proportional to the increased bacterial surface area corresponded to the increased number of receptors on the bacterial surface ,52. EvidE. coli B/r outer membranes regardless of cell sizes. This was surmised from the proportional increase in the irreversible phage adsorption rate and surface area [P. aeruginosa cells was reported [LPS density was reported to be consistent on ace area . A reducreported , but wheChanges in the bacterial cell wall receptors during filamentation are important because they affect PAS. However, limited investigations have been conducted on phage receptor densities during filamentation.E. coli bacteria cells [Cell lysis is the last stage in the infection cycle of lytic phages . The peria cells ,27.E. coli AS19 [E. coli ATCC 11303 [S. aureus ATCC 43300 decreased from 33.3 min to 16.6, 18.3, 23.4, and 23.4 min in the presence of sublethal 1 \u03bcg/mL linezolid, 0.25 \u03bcg/mL tetracycline, 4 \u03bcg/mL clarithromycin, and 4 \u03bcg/mL telithromycin, respectively [P. aeruginosa PA01 from around 9 to 6 min [Accelerated bacterial lysis has been reported to be accompanied by PAS in many studies. The addition of 0.030 \u00b5g/mL cefotaxime reduced the latent period of T4 and two other T4-type phages (RB33 and RB49) from 2 h to 75\u201390 min in oli AS19 . MoreoveCC 11303 . The latectively . Aztreonto 6 min .Bacterial filamentation is an intermediate state where bacterial chromosomes continuously replicate without cell division . Hence, E. coli. A 2.1-fold increase in green fluorescence was detected using a fluorometer [Further confirmation of holin expression was measured using engineered T4 phage with an enhanced green fluorescent gene in filamented orometer . In addiorometer ,26.E. coli K-12 strain with subinhibitory concentrations of cefotaxime and ciprofloxacin, which was due to a decreased holin concentration in the filamented bacterial cell [Although holin is increased in filamented cells, its concentration depends on not only the protein expression rate but also the cell dimensions . Conflicial cell . While Kial cell  detectedial cell .Both accelerated and delayed lysis have been found to be associated with PAS. A shortened latent period or accelerated lysis time increases the rate of repeating the infective cycle, while delayed lysis may allow more time for phage assembly and consequently enlarges the phage burst size .The burst size  refers tE. coli B/r cells induced by pre-treatment with a low concentration of penicillin. Compared with the normal non-treated cells, the filamented cells increased around 4-fold in size along with a 2.5- to 4-fold increase in single burst sizes, suggesting a correlation between bacterial cell size and burst size. Hadas et al. [Hadas et al.  compareds et al.  proposedE. coli K-12 cells induced by sublethal ciprofloxacin treatment. They postulated that the increased burst size was related to the increased availability of viral components. Further quantification of mRNAs, DNA, and proteins was conducted in these filamented E. coli cells, showing a 2-fold increase in the mRNA that encodes T4 phage DNA polymerase, which resulted in also a 2-fold increase in the T4 phage DNA. In addition, a 1.5-fold increase was observed in the mRNA that encodes the phage major capsid protein, but no increase was detected in the capsid protein production. Thus, not all viral components  are necessarily increased in the filamented cells. Overall, the relationship between the increased bacterial cell size and increased viral components or other factors is still not well established.Kim et al.  also obsE. coli culture from 20 min to 28 min. The single burst size was enhanced from 270 to 700 plaque-forming units per cell [Another hypothesis is that delayed cell lysis see  allows aper cell . Kim et The increased burst size is one of the contributors to PAS, but it is not the only factor determining the overall phage production. Other factors, such as the latent period, should also be considered for PAS investigation.Other than filamentation-induced PAS in lytic phages, a recent in vitro study also reported temperate phage\u2013antibiotic synergy, so-called \u201ctPAS\u201d . TemperaE.coli K-12. Eradication (\u22658 log reduction) was achieved by HK97 (MOI \u2265 10) with either MIC or \u00bd \u00d7 MIC ciprofloxacin after overnight incubation, whereas HK97 alone was ineffective against E. coli K-12. The study suggests that the latent period and burst size were not affected by the addition of ciprofloxacin. One of the underlying mechanisms was improved prophage induction triggered by ciprofloxacin via the recA-mediated SOS response. As mentioned in recA with gene recA deletion, which did not exhibit synergy with ciprofloxacin [Al-Anany et al.  examinedfloxacin . Anotherfloxacin .The antimicrobial effects achieved by HK97 with a sublethal concentration of ciprofloxacin are significant. However, \u201ctPAS\u201d is as of yet poorly studied. More investigations with other bacteria species with in vitro and in vivo models are needed for implementing its use therapeutically.P. aeruginosa phages with ciprofloxacin in the absence of bacterial filamentation [P. aeruginosa phage PA22 with tetracycline showed no significant plaque size enlargement even when bacterial filamentation occurred [PAS has been assessed by bacterial filamentation and plaque size enlargement; however, these effects may not always occur. The PAS response was found in entation . Moreoveoccurred .In another study tested with ampicillin, penicillin G, kanamycin, rifampicin, and tetracycline, tetracycline was found to produce the greatest plaque size increase, yet the bacterial cell size increase was the smallest , suggestDue to the large number of available antibiotics and the variations between different phages and bacterial hosts, it is very difficult and time-consuming to systematically assess the PAS response as well as the corresponding characteristics of the phage lytic cycle. This has hindered researchers in further exploring the underlying mechanisms.To date, there are various marketed antibiotics for treating bacterial infections. However, after prolonged exposure to the same or similar antimicrobial agents, bacteria can evolve and develop resistance by reducing drug uptake, increasing drug efflux, modifying drug target sites, and inactivating the drug ,64.Various approaches to using phages and antibiotics, with different mechanisms of inhibiting MDR infections, have been widely investigated. While a single-agent treatment kills its sensitive population and selects resistant bacteria, treatments with two or more agents with different mechanisms of action cover larger host populations. For instance, the combined use of phages and antibiotics leads to the simultaneous selection of phage-sensitive and antibiotic-sensitive populations, resulting in a very small or non-existent population resistant to both agents .In addition, with lethal concentrations of antibiotics and phages, bacteria are unlikely to rapidly evolve and acquire resistance to multiple agents that target different pathways in a short period of time. In particular, when the development of resistance to either agent adversely alters the bacterial components/functions involved in the attacking pathways of the other agent, adaptation trade-offs happen .For example, one of the known adaptation trade-offs between antibiotic resistance and phage resistance is multidrug efflux pump development and alleviated phage attachment. Some bacteria developed a resistance to antibiotics by expressing multidrug efflux pumps that remove the drug from the cells, hence the bacteria become multidrug- or pan-drug-resistant . On the P. aeruginosa phage OMKO1 binds with hosts at the outer membrane porin M (OprM) of the multidrug efflux pump. OprM was knocked out in engineered phage-resistant P. aeruginosa strain to force phage resistance. The engineered strain then regained sensitivity to antibiotics such as ciprofloxacin, tetracycline, ceftazidime, and erythromycin. The minimal inhibitory concentrations (MICs) of these antibiotics decreased by 12-fold compared to non-gene-modified strains [Pseudomonas-targeting phage PEV31, phage-resistant isolates of P. aeruginosa regained sensitivity to ciprofloxacin [Several studies observed gene mutations or reduced protein expression of bacterial drug efflux pumps after combined treatment with phages and bacteria ,21. This strains . The MIC strains ,20. In afloxacin ,67.Phage\u2013antibiotic therapy has promising therapeutic potential for enhancing antimicrobial effects due to PAS and decreasing the likelihood of resistance development. Moreover, it reduces the required antibiotic concentration compared to mono-antibiotic treatment.Pseudomonas-targeting phage PEV20 and ciprofloxacin enhanced P. aeruginosa biofilm eradication, highlighting the potential for reducing the antibiotic concentration required to combat highly recalcitrant infections associated with biofilms [P. aeruginosa biofilms were treated with phage EPA1 and ciprofloxacin, meropenem, and gentamicin, synergism was observed at 1 \u00d7 MIC with a 4.7, 4.1, and 2.6 log reduction in biofilm density, respectively. However, antagonism of antimicrobial activity was observed in ciprofloxacin and meropenem at 8 \u00d7 MIC [Decreased antibiotic MIC when used in combination with phages was observed in several studies ,69,70,71biofilms . Althoug 8 \u00d7 MIC . This isE. coli in combination with phage ELY-1, without compromising the antimicrobial effect [A. baumannii phage Ab105-2jDCI was combined with meropenem and imipenem at 1/8 \u00d7 MIC. On the other hand, synergism occurred with no regrowth of bacteria when the antibiotic concentration was increased to \u00bc \u00d7 MIC [In contrast, increasing the ciprofloxacin dose from a sublethal dose (1/10 \u00d7 MIC and 1/5 \u00d7 MIC) to a lethal dose up to 1 \u00d7 and 2 \u00d7 MIC successfully inhibited the regrowth of l effect . In anot \u00bc \u00d7 MIC . Thus, dSeveral studies ,70,72,73Aminoglycosides, which inhibit protein synthesis, did not show significant synergism with phages in simultaneous administration but showed better bacterial killing and enhanced phage production in sequential administration ,74,75,76P. aeruginosa strains were initially treated with phage LUZ7 (105 pfu/mL). Simultaneous treatment strongly suppressed the growth of P. aeruginosa with no regrowth over 70 h. The most significant synergistic bactericidal effect (p = 0.027) occurred when streptomycin was administered 12 h after phage treatment.Torres-Barcelo et al.  conductep < 0.04). Synergism occurred when the antibiotics were administered 24 h after pre-treatment with the phage cocktail, showing a significant reduction (from 8 to 2 log) in P. aeruginosa biofilm density. In another study, two aminoglycosides (kanamycin and neomycin) were compared with another protein synthesis inhibitor antibiotic, tetracycline, in co-treatment with phage T3 on E. coli biofilm. Interestingly, tetracycline showed no inhibition of phage infectivity even though it has a similar antibacterial mechanism (inhibiting protein synthesis) to kanamycin and neomycin, which decreased the phage burst size, replication, and efficiency of plating [Moreover, co-treatment with an anti-pseudomonal phage cocktail (phage NP1 and phage NP3) combined with gentamicin and tobramycin (8 \u00d7 MIC)  hampered plating . This anAminoglycosides led to a misreading of different cistrons or genes on the 30 s ribosomal subunit, which eventually inhibited the initiation of different proteins. For example, kanamycin and gentamicin inhibited the translation of coat proteins, whereas kasugamycin inhibited maturation proteins . HoweverBased on the above findings, a delay in the administration of antibiotics (about 6\u201312 h) after phage pre-treatment led to stronger antimicrobial activity; this observation was more prevalent with aminoglycosides than other antibiotic classes . To maxiP. aeruginosa, E. coli, and Salmonella typhimurium.DNA synthesis inhibitor ciprofloxacin seemed to yield contradictory results, as a phage\u2013ciprofloxacin combination can exert similar killing effects regardless of the administration time. Both simultaneous and sequential treatment (antibiotics administered after phages) exerted synergism against bacteria P. aeruginosa was observed despite titer reduction (1.8 log) as compared with sequential treatment, although it was not statistically significant (p > 0.05) [E. coli [E. coli. The bacterial killing was maximised when ciprofloxacin (1 \u00d7 MIC) was added 6 h after the phage pre-exposure. Bacterial density was significantly reduced  as compared to other administration times , with no bacterial regrowth observed at the end of the experiment. The phage titer in all delayed ciprofloxacin treatments increased significantly (1.7\u20132.0 log) compared to simultaneous administration. These results agreed with those from another study [S. typhimurium pre-treated with the phage for 6 h and followed by ciprofloxacin showed the highest reduction (3 log) in bacteria count as compared with using antibiotics or phages alone. Contradictory results were observed with tetracycline, where simultaneous treatment with phages showed antagonism in S. aureus [E. coli [When ciprofloxacin (1 \u00d7 MIC) was given simultaneously with phage PA14, a synergistic effect against  > 0.05) . This de[E. coli . However[E. coli . Ciprofler study , in whic. aureus  but not [E. coli , showingRecently, Wang et al.  indicateS. aureus reference strain and five rifampin-resistant S. aureus (RRSA) strains, three of which were methicillin-susceptible and two were methicillin-resistant. Synergistic effects on biofilm reduction were compared between simultaneous and staggered administrations of the antibiotics and phage. Most strains were susceptible to the antibiotics, whereas two strains showed resistance only to doxycycline and levofloxacin. No synergistic effects were observed in the inhibition of anti-biofilm activity in any of the combined treatments with simultaneous application  on all the strains, except the phage\u2013rifampin combination on the reference strain. However, in sequential treatment, synergism was observed in doxycycline (16\u2013128 \u00b5g/mL) and linezolid  after 24 h of phage (106 PFU/mL) pre-exposure. Biofilm density  was strongly reduced in all six strains, including the resistant strains, by the phage\u2013levofloxacin (16\u2013128 \u00b5g/mL) and phage\u2013clindamycin (64\u2013256 \u00b5g/mL) treatments, with five and four strains, respectively, out of the six showing synergistic effects on reducing biofilm density. Rifampin was the only exception; the combination therapy had no bactericidal synergism in any RRSA strain. Rifampin was the only antibiotic that showed minimal antimicrobial activity in all RRSAs, and this was due to the high resistance of the clinical strain, as the binding site of rifampin was altered by the mutation of rpoB on RNA polymerase. The binding affinity of rifampin to bacterial RNA polymerase was reduced [Phage Sb-1 and an antibiotic  were used together on an  reduced .Although staggered administration is an essential factor in exerting synergism, the host strain is also important. Synergism was observed in some RRSA strains but not others, showing that biofilm inhibitory effects were different even when using the same phage\u2013antibiotic (levofloxacin) combinations. The above results demonstrated that host strains can show different inhibition profiles and synergistic effects , but theP. aeruginosa and S. aureus [P. aeruginosa biofilms, whereas S. aureus biofilms only had a 2 log decrease. This was due to the specificity of the phage, as EPA1 is an anti-pseudomonal phage. The reduction in S. aureus biofilms was caused by the administration of a high concentration of gentamicin (8 \u00d7 MIC).These studies only demonstrated the phage\u2013antibiotic treatment on a single bacterial species. When phage EPA1 and gentamicin were used on biofilms of dual bacterial species, . aureus , no syneE. coli infection with phage \u03d5HP3 and ceftazidime in human pooled urine and heat-inactivated serum showed completely different results compared to that in Luria-Bertani (LB) medium [9 PFU/mL of phage, which showed an additive effect. Similar results were found in urine in which only high doses of phage (109 PFU/mL) and ceftazidime (256 \u00b5g/mL) produced synergism. In contrast, synergism was observed in LB medium at low doses of ceftazidime (2 \u00b5g/mL) and phage (104 PFU/mL). The overall reduction in the bacterial level in serum and urine may not be due to the high doses, but the low starting bacterial inoculum, as untreated bacteria gradually decrease in the presence of urine and serum. This phenomenon was also observed in another study with E. coli isolates in urine samples [p < 0.001). It was found that urea was the main factor for reduced E. coli growth. Increased urea concentration corresponds to reduced bacteria count [As the pharmacokinetics of antibiotics in physiological media such as serum and urine can vary, phage\u2013antibiotic efficiency is altered accordingly. Treating ) medium . The ant samples : bacteriia count .Overall, due to the specificity of phages and the diverse pharmacokinetics of antibiotics, phage\u2013antibiotic efficiency depends on the host strains and physiological media.Galleria mellonella were extensively used for testing a wide range of bacterial infections.Although phage\u2013antibiotic treatment is widely studied in vitro, little research focuses on preclinical in vivo trials to evaluate the efficacy of combination treatment. Promising results in treating bacterial infection showed the potential of phage\u2013antibiotic treatment in vivo . Mice anK. pneumoniae in larvae of moth Galleria mellonella. The survival rate of the larvae significantly increased to 50% and 75% when co-treated with phage and mitomycin C or imipenem, respectively, compared to either antibiotic or phage monotherapy, except phage\u2013imipenem co-treatment of the imipenem-resistant strain. This was due to the hydrolysis of imipenem by \u03b2-lactamase in the resistant strain. Nonetheless, the mortality rate of the larvae significantly decreased with all other combined treatments [Galleria mellonella models, achieving a 75% survival rate in treating E. coli infection when phage \u03a6WL-3 was used in combination with fosfomycin [A. baumannii increased the survival rate by approximately 30% (p < 0.05) compared to phage monotherapy [Galleria mellonella model has enabled high numbers of replicates, but their physiological characteristics are much different to those of mammals, so rodent infection models would be more relevant.Mitomycin C and imipenem were combined with lytic phage vB_KpnM-VAC13 to treat imipenem-resistant strain K2534 and persisted strain K3325 of Gram-negative bacteria eatments . Additiosfomycin . In addiotherapy . UtilisaP. aeruginosa lung infection. Co-spray dried phage and ciprofloxacin powder administrated intratracheally significantly reduced the bacterial density compared to single treatment with either agent [P. aeruginosa, a phage cocktail combined with ciprofloxacin was highly synergistic, resulting in negative vegetation in 7 out of 11 rats with more than a 6 log reduction in bacterial concentration, as opposed to zero rats with either phage or ciprofloxacin monotherapy [S. aureus (MRSA) ATTC 43300 strain to induce prosthetic joint and diabetic foot infections [A neutropenic mouse model was used to determine the efficacy of phage PEV20 combined with ciprofloxacin on er agent . In addiotherapy . Bacterifections ,88. The These findings on animal studies of phage\u2013antibiotic combined treatments demonstrated a reduction in bacterial burden caused by both Gram-positive and Gram-negative bacteria, and the ability to recover from the infections increased considerably.Phage therapy was applied to patients in the early years of its discovery, but its use was decreased due to the lack of regulation or standardised methodology, and for safety reasons . In viewK. pneumoniae in a 63-year-old female with Type-2 diabetes and hypertension. Phage monotherapy failed twice, as phage resistance caused re-emergent bacterial isolates. When trimethoprim-sulfamethoxazole was administered orally twice a day with the phage cocktail once a day through bladder irrigation, the resistant strain was completely inhibited, and the urinary tract infection did not recur 6 months after discharge from hospital [A phage cocktail that consisted of six lytic phages combined with trimethoprim-sulfamethoxazole was used to treat a recurrent urinary tract infection with drug-resistant hospital .A. baumannii (CRAB). Phage Ab_SZ3 was administrated with tigecycline and polymyxin E. The phage was given by a vibrating mesh nebuliser once daily, with gradually increasing doses from 5 \u00d7 106 PFU to 5 \u00d7 1010 PFU on Day 13. Tigecycline was given intravenously for the first 5 days of treatment, followed by oral polymyxin E for another 5 days. In a total of 16 days of treatment, no CRAB cultures were detected in the patient\u2019s sputum from the 7th day onwards, with only one exception on the 15th day, when a positive CRAB culture was found. One month after phage treatment, the patient developed sepsis caused by E. faecium and S. haemolyticus. During treatment with vancomycin, colonisation by P. aeruginosa was also found in the patient\u2019s bronchoalveolar lavage fluid (BALF) culture. Even with the occurrence of different types of bacterial infection and the pressure of antibiotic use, CRAB showed no reappearance after the combination treatment [However, not all patients with chronic infections had the same recovery profile after treatment. An 86-year-old female patient with chronic obstructive pulmonary disease was infected by carbapenem-resistant reatment .As an intact immune system plays an important role in suppressing bacterial infections with opportunistic pathogens , chronicAchromobacter species, the combination therapy of phage Ax2CJ45f2 and two antibiotics (cefiderocol and meropenem/vaborbactam) by intravenous administration improved forced expiratory volume in one second  from 33% to 60%. The symptoms were alleviated with decreased sputum production and reduced cough. No adverse effects were observed with the combined treatment [In a pediatric patient with cystic fibrosis female infected with pan-drug-resistant reatment .Above all, phage\u2013antibiotic combinations have produced encouraging outcomes in treating multi-drug-resistant bacterial infections. However, standardisation of the treatment method is difficult, as patients have different medical histories and varying levels of immunity. The administration route, time, and dose of phage and antibiotics differed for each patient. Formulating personalised treatments is time-consuming and expensive, but standardised regimens may not exert the same efficiency in all patients.While most studies focused on the synergistic effects of phages and antibiotics against one bacterial strain or species, there is a demand for investigations of the phage\u2013antibiotic treatment of polymicrobial systems. In clinical settings, bacterial infections are usually associated with multiple strains or species. As a result, the genetic pool is enlarged in these polymicrobial systems, and there are more opportunities for mutations to occur in bacteria as well as phages. These uncertainties may produce varying efficacies in phage\u2013antibiotic therapy.P. aeruginosa phage 14/1 with gentamycin reduced bacteria density in biofilm for the first 12 days, but then regrowth occurred due to resistance development [Another challenge of polymicrobial systems is the typical formation of biofilm. The spatial structure of biofilm may prevent antimicrobials from reaching sensitive populations. Hence, their efficacy against biofilms is reduced compared to planktonic cells. Biofilms also allow more time for co-evolution between phage- and antibiotic-selected populations under prolonged exposure to phage and antibiotics. For instance, an in vitro study reported that co-administration of elopment .P. fluorescens SBW25 was found to be faster than that of bacteriophage SBW25\u03a62 [It is believed that phages have the advantage of co-adapting with bacteria; hence, they can overcome the development of bacteria resistance to phages in recurrent or chronic infections. However, the mutation rate of bacteria  SBW25\u03a62 .P. fluorescens SBW25 was the lowest after 66-days of co-treatment with SBW25\u03a62 (MOI = 0.01) and streptomycin  compared to SBW25\u03a62 with streptomycin at 0.2 \u00b5g/mL or without streptomycin. The phage titer decreased more significantly with a higher concentration of streptomycin, and no phage was detected with 2 \u00b5g/mL (1/10 \u00d7 MIC) streptomycin by Day 14, whereas only a small decrease of around 1 log in phage titer was observed on Day 66 without streptomycin. The phage titer started to decline as bacteria hosts developed resistance to the phage [p < 0.001 for 0.2 and 2 \u00b5g/mL streptomycin compared with no streptomycin. No statistical significance was detected between streptomycin treatments of different concentrations) [Furthermore, Cairns et al. reported that the bacterial density of he phage . Anotherrations) . This obrations) .As there is only limited research that investigated the long-term mutation dynamics of bacteria and phages in the presence of antibiotics, it is unknown whether the presence of antibiotics promotes the mutation rate of bacteria and/or phages, or whether the changes in mutation rates vary depending on the bacteria species, antibiotic class, or phage type.P. aeruginosa lung infection [So far, phage-based clinical therapies are mainly personalised. Unlike antibiotics, the specificity of phage has hindered its wide use as predefined formulations on the market. In response to that, strategies commonly considered include the modification of the antimicrobial spectrum of phages through genetic engineering , the expnfection ,99 and hnfection  have shonfection ,98. Stilnfection ,101.As antibiotic resistance is a life-threatening danger to global health, phage therapy has attracted attention over the past few decades as a potentially efficient alternative solution. The abundance, specificity, and versatility of phages are their advantages. Since antibiotics have been found to enhance phage production and further increase antimicrobial activity, phage\u2013antibiotic combination treatments have been extensively studied. In the presence of antibiotics, instead of dividing, bacteria undergo elongation (filamentation), which facilitates phage adsorption, modifies bacterial cell lysis time, and/or increases phage burst size, which in turn results in enhanced phage replication. In vitro experiments showed synergistic antimicrobial effects with phage\u2013antibiotic co-treatment, resulting in a significant reduction in bacterial growth. Moreover, antibiotic doses and administration sequence are two factors contributing to the variability in synergism. High antibiotic doses do not necessarily produce better bactericidal effects with phages, as inhibition driven by antibiotics may adversely affect phage production. The antibiotic suppression of phage replication in simultaneous administration was demonstrated in several studies, while sequential treatment with phages followed by antibiotics showed better antibacterial effects. The order of administration depends on the specific antibiotic. In vivo efficiency generally agreed with in vitro findings. Promising outcomes with better survival and faster healing have been presented in rodent models and larvae models when combination therapy was given. Further investigations with other animal models or systems need to be developed for a better understanding of phage and antibiotic co-therapy. In clinical case studies, phages combined with antibiotics improved treatment outcomes and reduced bacterial growth. Nevertheless, recurrent bacterial infections in immunocompromised patients are difficult to treat.Successful clinical cases of personalised phage\u2013antibiotic treatment in tackling persistent MDR infections have been reported, but much progress needs to be made before it becomes popular. Firstly, as phages are bacteria-specific, a rapid diagnosis should be developed for precise phage identification. Secondly, world-wide phage banks are needed for the quick selection of suitable phages. Thirdly, dose regimens, including dosage and administration sequence, must be optimised for safe and efficient phage\u2013antibiotic treatment for infections of different severity. Current or completed clinical trials on bacteriophages lack data for their combined use with sublethal dose of antibiotics. Although phage with sublethal doses of antibiotic reported more benefits than those with lethal doses of antibiotics in in vitro models, it may be not ethical for clinicians to prescribe sublethal doses of antibiotics based on existing clinical trials. Hence, further dose optimisation and clinical trials are needed. Fourthly, to prevent a regrowth of uncleared MDR bacteria, researchers should develop a strategy or dose regimen to achieve full pathogen eradication. In addition, phage\u2013antibiotic therapy in association with innate immune system responses is another possible and promising method for eradicating MDR infections. Finally, rapidly formulating methods must be developed for improved delivery to sites of infections.While individualised treatment is the current mainstream of phage-based therapy, it is time-consuming and expensive. Predetermined phage\u2013antibiotic formulations are considered for improved cost-effectiveness, but selecting suitable phage\u2013antibiotic combinations is another challenge because they are not always synergistic against all bacterial strains. Extensive foundational work in synergy screening is needed for selecting phage\u2013antibiotic combinations against different species. Moreover, formulating methods must be developed or optimised for improving stability for convenient storage and transport, and for adjusting the release of phages and antibiotics to achieve either simultaneous or sequential treatments. We believe that with increasing evidence from in vitro and in vivo works, as well as future clinical trials, it is feasible to develop predetermined formulations.Current methods in treating bacterial infections are not necessarily limited to conventional phage\u2013antibiotic combined treatments; genetically engineered phages and phage-coded enzymes have also attracted recent attention. By modifying the tail fibre genes of phages to increase the host range for multiple targeting, the bactericidal effects would be significantly enhanced. Endolysin is an example of a phage-coded enzyme. Its rapid and potent killing of bacteria without apparent resistance development is attractive and may become a potential treatment for MDR infections. By combining new techniques and agents with antibiotics, phage\u2013antibiotic combined treatment is clearly more promising for treating resistant bacterial infections than mono-phage therapy."}
{"text": "Salmonella spp. is a relevant foodborne pathogen with worldwide distribution. To mitigate Salmonella infections, bacteriophages represent an alternative to antimicrobials and chemicals in food animals and food in general. Bacteriophages (phages) are viruses that infect bacteria, which interact constantly with their host. Importantly, the study of these interactions is crucial for the use of phages as a mitigation strategy. In this study, experimental coevolution of Salmonella Enteritidis (S. Enteritidis) and a lytic phage was conducted in tryptic soy broth for 21 days. Transfer to fresh media was conducted daily and every 24 hours, 2 mL of the sample was collected to quantify Salmonella OD600 and phage titter. Additionally, time-shift experiments were conducted on 20 colonies selected on days 1, 12, and 21 to evaluate the evolution of resistance to past (day 1), present (day 12), and future (day 21) phage populations. The behavior of the dynamics was modeled and simulated with mathematical mass-action models. Bacteria and phage from days 1 and 21 were sequenced to determine the emergence of mutations. We found that S. Enteritidis grew for 21 days in the presence and absence of the phage and developed resistance to the phage from day 1. Also, the phage was also able to survive in the media for 21 days, however, the phage titer decreased in approx. 3 logs PFU/mL. The stability of the lytic phage population was consistent with the leaky resistance model. The time-shift experiments showed resistance to phages from day 1 of at least 85% to the past, present, and future phages. Sequencing of S. Enteritidis showed mutations in genes involved in lipopolysaccharide biosynthesis genes rfbP and rfbN at day 21. The phage showed mutations in the tail phage proteins responsible for recognizing the cell surface receptors. These results suggest that interactions between bacteria and phage in a rich resource media generate a rapid resistance to the infective phage but a fraction of the population remains susceptible. Interactions between Salmonella and lytic phages are an important component for the rational use of phages to control this important foodborne pathogen. Salmonella spp. is a relevant zoonotic pathogen transmitted to humans through food and contact with animals   arms race dynamics where new alleles allow bacterial resistance and phage infectivity over time, in which bacteria become more resistant to phages from the past than to present and future phages ; and ii)s SBW25) . To our ublished . TherefoSalmonella Enteritidis and a lytic phage in a high level of nutrients media. To assess the effect of the interactions between S. Enteritidis and the phage; we studied the abundance of S. Enteritidis and the phage, modelled their interactions, characterized the evolutionary model, and identified genomic changes in S. Enteritidis and the phage upon 21 days of coevolution.In the present study, we performed a coevolution assay for Salmonella Enteritidis strain DR016 was isolated from chicken feces in backyard farms of central Chile . Salmonella was grown in TSB for 16 h at 37\u00b0C and stored with 20% of glycerol at -80\u00b0C. Phage vB_Sen_STGO-35-1 (STGO-35-1) was isolated from a backyard chicken flock, using a strain of S. Enteritidis as host, according with the protocol described by al Chile . This isS. Enteritidis DR016 and phage STGO-35-1 at MOI 0.01 .The coevolution experiment one-host-one-phage was conducted using MOI 0.01 . The bacMOI 0.01 . Four reS. Enteritidis was measured with a spectrophotometer using OD600nm . The phage titer was determined using unevolved S. Enteritidis and titer was conducted using the \u201cspot-test\u201d, as previously described  with binomial errors and the logit- link function. Replicates were included as a random effect and both bacterial or phage presence and time sample were included as fixed factors. The analysis was conducted using RStudio statistical software, version 3.5.2. Model was run with the \u2018glmer\u2019 function in the \u2018lme4\u2019 package in R.During the experiment, daily samples of 2 mL were collected in each replicate. The estimation of escribed  , present (day 12), and future (day 21). For the phage, 1 mL of lysate was stored as described above. For S. Enteritidis, cultures were serially diluted to 1\u00b7106 CFU/mL, a total of 100 \u00b5L were streaked on TSA plates and 20 colonies were randomly selected for purification and frozen with 20% glycerol at -80\u00b0C. The proportions of S. Enteritidis resistance to phage and phage infectivity in S. Enteritidis was calculated from the three times by cross streaking the isolated 20 colonies across lines crossed by streaked phages, as previously described  with binomial errors and the logit- link function to test whether phage local adaptation was affected by the time sample in which bacteria was collected using RStudio (version 3.5.2). In this analysis, replicates were included as a random effect and both bacterial and phage time samples were included as fixed factors. As the replicates had a similar behavior, we used a generalized linear mixed model (GLMM) to examine variation in bacterial resistance where replicates were included as a random effect and both bacterial and phage time samples were included as fixed factors.To evaluate bacterial resistance and phage infectivity, generated through the coevolution experiment, phage and escribed   a reference genome represented by S. Enteritidis before the coevolution assays, ii) S. Enteritidis coevolved from day 1 from all 4 replicates, iii) S. Enteritidis coevolved from day 21 from all 4 replicates, and iv) controls  from days 1 and 21 (S. Enteritidis before coevolution (reference genome) were assembled using the \u2018isolate\u2019 mode in SPAdes v3.14.0. Then, scaffolds with length <1kb were removed from the assembly. Blasting resulting scaffolds against an NCBI database of Salmonella enterica, showed that all scaffolds presented >95% of identity. One scaffold with 100% identity to Salmonella enterica plasmid pSJTUF10978 (CP015525) was removed. The final assembly had a consensus length of 4,647,970 bp spanning 24 scaffolds, with an N50 value of 490,728 bp. The average genome coverage was 134X with a GC % of 52.1%. Finally, collinearity with S. enterica (CP050716) was evaluated using D-GENIES (S. enterica genome (98.79%) was covered by the assembly. Gene annotation of this assembly was performed with RASTtk  reference phage represented by STGO-35-1 before the coevolution, ii) coevolved phage from day 1 from replicate 1, and iii) coevolved phage at day 21 from replicate 1. Phenol/chloroform extraction of DNA and precipitation with ethanol were performed for phage STGO-35-1, as previously described . DNA conSalmonella and phage genomes, we used Bowtie2 (version 2.4.1), using default parameters , which splits adjacent SNPs into individual SNPs, left-aligns indels, and regularizes the representation of complex variants. SnpEff (version 4.0e)   was usedS. Enteritidis showed that it survived in the presence of the phage for 21 days . In general, the four replicates behaved homogeneously, with an exponential multiplication on day one and then maintaining an optical density between 1.3 and 1.6 until day 21. In the control, where only S. Enteritidis was inoculated, the optical density was similar among the replicates, with no significant changes. These results suggest that the phage STGO-35-1 in the media did not affect the abundance of S. Enteritidis using a MOI of 0.01. We also observed a homogeneous behavior among the replicates in terms of phage populations. On day one, the phage had a higher titer and then declined steadily over time but did not go extinct. The control with only phage inoculation was indetectable by PFU quantification after day 3 . Our results could indicate that S. Enteritidis of day 1 (past) developed resistance to the phage. Still, a minor population of S. Enteritidis remains susceptible and allows the phage to replicate for 21 days. Our results indicate that a subpopulation of Salmonella is still phage-susceptible in all the replicates tested. We observed that the phage titer decreased over time (104 PFU/mL) . Similar behaviors of host-phage coexistence were reported for Streptococcus thermophilus and a lytic phage (The quantifications of  21 days . In gene 21 days . In geneic phage .R) at 24 hours, which explains why the phage titer decreased . This result is repeated when different rates for the transition of susceptible to resistant bacteria (\u03bcN) are used in the simulations. Interestingly, in all the cases, a minor population of coexisting susceptible bacteria (N) is present in the culture, which could explain why the phage survive for the 21 days, even when the dominant population of Salmonella are resistant. The latter observation could support the hypothesis of the cohabiting population of susceptible bacteria being able to host the phage multiplication and its maintenance in the culture; however, the density of this population (1\u00b7102 CFU/mL) might be too low to satisfy the probability of infection (E. coli (R) but maintaining the resistance generation rate constant over time. The results indicate that dominance of resistance at 24 hours occurs in most cases . In this model, a considerable rate of genetic and/or phenotypic reversion of resistant to susceptible bacteria occurs, and then populations can find stability through low rates of multiplication . The leaky resistance model was described for E. coli and a virulent mutant of phage Lambda; and in Salmonella, a previous study that coevolved S. Enteritidis for 12 days with a lytic phage, did not show population density for susceptible bacteria  the past (day 1), ii) the present (day 12), and iii) the future (day 21); (S. Enteritidis developed a high resistance proportion (0.75 \u2013 1.00) against the phages for all the different times  in all four replicates, without significant differences between the different phage times or replicates  . We also tested phage infectivity of isolated Salmonella obtained from i) the past (day 1), ii) the present (day 12), and iii) the future (day 21). Conversely, phage infectivity was found in low proportions (0.00 \u2013 0.25) in all three times and replicates as well, similarly, without significant differences between Salmonella time or replicates  . Overall, our results show a bacterial resistance mechanism emergence during the first day of coevolution that lasted through the 21 days of the experiments. Over time, high resistance to phage was reported for other bacterial genera such as Vibrio sp. and E. coli with lytic phages . Importantly, the interactions between bacteria and lytic phages depend on the host and the environment with controlled parameters as temperature, resources, and multiplicity of infection  . We alsoc phages . But thiis s25pp . Importanfection .S. Enteritidis could have an advantage over phages in this model of TSB, suggesting, for instance, that it could be easier for Salmonella to mutate the phage receptor than for the phage to find the exact mutation for the new receptor under these experimental conditions   . This inovement) . Then, eruginosa . Thus, fS. Enteritidis populations from days 1 and 21 in all four replicates to determine SNPs in coevolved Salmonella with the lytic phage. We found 31 SNPs distributed in the replicates for days 1 and 21 . SNPs were classified into four categories according to nucleotide changes and putative impact. Specifically, in i) missense presenting a moderate impact (13 SNPs), ii) frameshift presenting a high impact (3 SNPs), iii) stop gained presenting a high impact (1 SNP), and iv) intergenic mutations presenting a lower impact (9 SNPs). At day one, 6 SNPs were identified, most of them being missense (5 SNPs), followed by intergenic (1 SNP). These SNPs, were found in genes encoding membrane proteins as the oxaloacetate decarboxylase and undecaprenyl-phosphate-galactosyltransferase, involved in the synthesis of antigen-O for the LPS , suggesting that the mechanism for resistance is dynamic and can change through, and between, replicates. It would be interesting to evaluate if mutations observed at day 21 are more stable or can still be subjected to further modifications. In a previous study conducted in S. Enteritidis, most of the SNP at coevolved Salmonella with a lytic phage were associated with cell wall proteins and capsule proteins (Pseudomonas after exposure to DSM3 were also on membrane receptor. Additionally, different studies report that mutation in LPS genes induces phage resistance to Salmonella (In general, identified SNPs were present in genes encoding proteins involved in different processes, such as metabolism, virulence factors, LPS biosynthesis, and membrane protein biosynthesis proteins . Other mlmonella . This inS. Enteritidis. However, our results indicate a potential counter resistance mechanism in phage STGO-35-1 to overcome the bacteria resistance developed.To determine SNPs in the lytic phage STGO-35-1, we sequenced phage isolated from day 1 and 21. A comprehensive report of genomic characteristics of this phage was recently reported  and a shSalmonella Enteritidis and a lytic phage vB_Sen_STGO-35-1 in a rich nutrient media for 21 days. Our study highlights the potential of S. Enteritidis to co-evolve with lytic phages in a high nutrient environment by rapidly developing a resistant population but maintaining a low frequency of susceptible bacteria available to the phage for replication. The genomics data showed that Salmonella have different SNPs in response to the phage presence and the phage had less SNPs than the bacteria, but in important proteins described as bacterial receptors While most of the changes occurred during the first 24 hours, initial resistance mechanisms could change throughout the 21 days as observed in our sequencing results. Understanding these interactions contributes to the responsible use of the phages as biocontrol tools.Results presented here contribute to expand our current knowledge on coevolution dynamics using a model of The datasets presented in this study can be found in online repositories. The name of the repository and accession number can be found below: NCBI; PRJNA821546.RB-M, DR, FP\u00c1, and AM-S: conceived the idea, designed and conducted the experiments; MS, RG, JB, FA, SR, and CH-W: analyzed and interpreted data; RB-M, DR, D\u00c1, RG, and AM-S: wrote the manuscript, RB: discussed the results and critically revised the manuscript. All authors contributed to the article and approved the submitted version.We acknowledge the funding sources: 1 [ANID FONDECYT 1181167 to AIMS], 2 [ANID Millennium Science Initiative/ Millennium Initiative for Collaborative Research on Bacterial Resistance NCN17_081 to AM-S], and ANID FONDECYT 3210317 to DA.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
{"text": "Mycobacterium abscessus lung disease previously developed anti-phage neutralizing antibodies while receiving intravenous phage therapy. Subsequent phage nebulization resulted in transient weight gain, decreased C-reactive protein, and reduced Mycobacterium burden. Weak sputum neutralization may have limited the outcomes, but phage resistance was not a contributing factor.An elderly man with refractory  Mycobacterium abscessus is an emerging opportunistic pathogen of increasing clinical significance [M abscessus lung disease is often complicated due to limited numbers of effective antibiotics, prolonged duration of therapy, and frequent treatment-related adverse events [M abscessus infections have prompted exploration of mycobacteriophage therapy for these organisms, although the optimal patient selection, dosing, and route of phage administration for nontuberculous mycobacteria (NTM) remain unclear [ificance . The mede events . Bacterie events . Poor tr unclear , 6.M abscessus. The first patient was a 15-year-old girl with cystic fibrosis (CF) whose post\u2013lung transplant course was complicated by treatment-refractory disseminated M abscessus infection, which was successfully controlled with the use of an IV mycobacteriophage cocktail [M abscessus subspecies massiliense lung disease [M abscessus treatment failure and led to its discontinuation. Here we report the outcomes of this patient after switching the route of phage administration to aerosolized delivery.Two recent case reports , 8 descrcocktail . A secon disease ; howeverM abscessus lung disease was treated for 6 months with an IV cocktail of 3 mycobacteriophages  in addition to a multidrug antibiotic regimen, as previously described [M abscessus burden in sputum at month 1 (C), with subsequent rebound in mycobacterial colony-forming units (CFUs) that temporally correlated with the emergence of a robust anti-phage immunoglobulin M and immunoglobulin G (IgG) neutralizing antibody response, specific to the phages used therapeutically [An 81-year-old man with non-CF bronchiectasis and refractory macrolide-resistant  month 1 C, with sutically . After aH), the phage cocktail was administered twice daily via nebulization using a Philips Respironics InnoSpire Essence Compressor Nebulizer System at a dose of 1\u2009\u00d7\u2009109 plaque-forming units in 3\u2005mL of 0.9% normal saline. Safety and efficacy monitoring during nebulized therapy included serial clinical and microbiological assessments, in-home spirometry with the Microlife PF 100 digital peak flow and forced expiratory volume in 1 second (FEV1) meter, and serial chest computed tomography (CT). Standing weight was recorded regularly by the patient (A) and quantitative assessment of M abscessus counts in expectorated sputum was calculated approximately monthly as the mean CFUs/mL\u22121 (C). Serial M abscessus isolates were assessed for changes in antibiotic susceptibility and for acquisition of phage resistance.To overcome serum neutralization and to increase phage delivery to the site of infection, the patient was started on nebulized administration of the same triple phage cocktail. Regulatory and institutional approval was obtained from the United States Food and Drug Administration and the Johns Hopkins Institutional Review Board and written informed consent was obtained prior to initiation. While continuing multidrug antibiotic therapy for NTM lung disease H, the ph patient A and quaFUs/mL\u22121 C. Serial1 of 1.71\u2005L (A). Following initiation of nebulized phage therapy, FEV1 transiently increased to 2.13\u2005L on day 3, and later remained relatively stable over time, with a 0.12\u2005L decline during the 9-month nebulized treatment period (B). Changes in companion antibiotics during phage therapy were motivated by adverse events suspected to be antibiotic-related. These events included development of eosinophilia, prompting discontinuation of IV imipenem, and nausea and mild liver function test abnormalities, leading to discontinuation of omadacycline and clofazimine . Parenchymal lung abnormalities on chest CT appeared relatively unchanged over time, with only minor interval changes . C-reactive protein (CRP) decreased from 69.3\u2005mg/L prenebulized phage to a nadir of 23.9\u2005mg/L 4 months into nebulized phage (B). While M abscessus burden in sputum fluctuated (C), weight gain during nebulized phage therapy correlated temporally with a relative quantitative reduction in M abscessus in sputum and a decrease in CRP\u2014consistent with an improved systemic response to infection with the use of nebulized phage.With initiation of nebulized phage therapy, there was a reduction in subjective sputum production. By approximately 3.5 months into nebulized phage therapy, the patient\u2019s body weight increased by 8.7% for a total weight gain of 4.3\u2005kg (9.5\u2005lbs) from a prenebulized phage baseline weight of 49.7\u2005kg (109.5\u2005lb) A. C-reaced phage B. While uctuated C, weightA\u20131C). Because of concerns for potential physical destruction and phage loss with the jet compression-type nebulizer [Approximately 4 months after initiation of nebulized phage delivery, these apparent benefits from phage administration dissipated, and the subsequent 7 weeks of treatment were accompanied by weight loss, increase in CRP, and rising sputum bacterial counts A\u20131C. Becebulizer , 11, admM abscessus isolates recovered 7 and 8 months after the start of phage nebulization were tested for phage susceptibility; these specimens had similar profiles to earlier isolates and remained fully sensitive to all 3 phages in the regimen (A). The emergence of bacterial resistance to phage therapy was not observed, nor were there changes in antibiotic susceptibilities , and a weak but notable response to all 3 phages prior to nebulization was observed, which fluctuated after the start of nebulization but did not increase consistently or substantially; sputum IgA titers remained about 100-fold lower than serum IgG levels to the same phages . This sputum IgA reactivity may have occurred, in part, as a response to the 9 months of prior IV phage administration. However, sputum samples showed only weak phage neutralization , which was about 4 logs lower than observed with serum samples 8 months post\u2013phage nebulization .To determine if treatment failure was caused by the emergence of phage resistance,  regimen A. The embilities . Sputum B and 1C). After the start of aerosolized delivery, weight gain, fall in CRP, and decline in mycobacterial load were initially encouraging. These clinical improvements lasted longer (3\u20134 months) than those observed after start of IV therapy (1\u20132 months). However, these gains with nebulized phage were again only transient, and the mechanisms underlying the limited duration of the benefits of phage aerosolization in this patient are unclear. We anticipated that emergence of phage resistance might occur, but after 8 months of nebulization\u2014and 15 months of total phage therapy\u2014the strain remained fully sensitive to phages Muddy and BPs\u039433HTH_HRM10, and with only a slight reduction in sensitivity to ZoeJ\u039445 (A), which was also observed after IV treatment alone [M abscessus clinical isolates is consistent with in vitro studies [Mycobacterium infections. Aerosolized treatment failure did not result from antibody-mediated phage neutralization either, as only relatively weak sputum IgA responses to the phages were observed, and only mild neutralization (E and 1G). However, neutralization did increase at later times (7 and 8 months) after the start of nebulization, and it is plausible that this contributed to limitation of treatment effect.The clinical progression following the switch to aerosolized phage administration mirrors what was observed after the start of IV administration: some benefits initially, followed by subsequent dissipation of those clinical gains. For IV administration, the emergence of strong neutralizing antibody response to the phages was temporally correlated with increases in mycobacterial burden and rises in CRP B and 1C. ZoeJ\u039445 A, which  studies  and is alization E and 1G.Mycobacterium infections. Some patients may see clinical improvement and long-term benefits [This case study highlights the challenges with using aerosolized bacteriophages for controlling benefits  whereas Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.ofac194_Supplementary_DataClick here for additional data file."}
{"text": "Antibiotic resistance is one of the major challenges that humankind shall face in the short term. (Bacterio)phage therapy is a valuable therapeutic alternative to antibiotics and, although the concept is almost as old as the discovery of phages, its wide application was hindered in the West by the discovery and development of antibiotics in the mid-twentieth century. However, research on phage therapy is currently experiencing a renaissance due to the antimicrobial resistance problem. Some countries are already adopting new ad hoc regulations to favor the short-term implantation of phage therapy in clinical practice. In this regard, the Phage Therapy Work Group from FAGOMA (Spanish Network of Bacteriophages and Transducing Elements) recently contacted the Spanish Drugs and Medical Devices Agency (AEMPS) to promote the regulation of phage therapy in Spain. As a result, FAGOMA was asked to provide a general view on key issues regarding phage therapy legislation. This review comes as the culmination of the FAGOMA initiative and aims at appropriately informing the regulatory debate on phage therapy. The emergence of multi-drug-resistant (MDR) bacteria seriously undermines our ability to control bacterial infectious diseases. A recent study shows that MDR pathogens already cause more than one million deaths a year, and the prospects are even more concerning ,2. There(a)Activity against antibiotic-resistant bacteria. Phages can infect and kill bacteria, including MDR strains . This is(b)Specificity. The high specificity of many phages towards their host bacterial strains makes them a highly selective therapy that prevents the dysbiosis of the healthy microbiota. Contrary to antibiotics, only strains of the same genus or species\u2014and often just one or very few strains within a species\u2014are susceptible to infection by a given phage, protecting the normal microbiota and reducing side effects . The spe(c)Multiplication at the site of the infection (auto-dosage). Phages can multiply at the site of the infection. Once the phages reach the targeted bacteria, they will replicate and generate progeny. Therefore, if sufficient phage particles are able to reach the infection site, phage therapy can be considered as auto-dosage treatment. Furthermore, once the infection is successfully controlled, phages would be eliminated in the absence of bacterial hosts. Thus, whenever an auto-dosed, \u201cactive\u201d treatment is achieved, it can elicit the infection eradication by only a single administration .(d)Ubiquity and diversity. Phages can be found in virtually any environment , and the(e)Evolvability. Phages are evolving entities and, therefore, can be optimized using directed evolution techniques. This opens up many possibilities compared to conventional treatments, which are stable chemical compounds. Phage evolvability can be exploited in many ways, such as increasing lytic capability, improving particle stability, expanding the host range, or counteracting bacterial resistance. For instance, the Appelmans\u2019 protocol uses spontaneous mutation and recombination among phages present in a cocktail to produce phage variants capable of infecting initially non-susceptible bacterial strains ,20.(f)Safety. Humans are carriers of many different phages forming the phageome ,22. Thei(a)Phage-resistance. In the same way that resistance to antibiotics emerges, bacteria can become resistant to phage infection. The most common solution to address this involves the use of cocktails of different phages, rather than a single phage, and/or the \u201c\u00e0 la carte\u201d selection of phages for each particular infectious isolate. This makes it much less likely that the host will become resistant to all phages at the same time . The so-(b)Specificity. This feature can be a double-edged sword. Phage specificity requires careful susceptibility testing of each bacterial pathogen before treatment, which may be viewed as an issue for certain acute infections that require urgent action. In addition, this specificity may require either the development of large phage libraries and/or extensive sampling and screening efforts to provide sufficient coverage of bacterial diversity. This can be a daunting task and has posed major regulatory issues, since, according to the current framework, each individual phage should undergo review and approval. In addition, the eventual need to develop a different phage preparation for each bacterial pathogen, as a personalized medicine, reduces business profitability and can be viewed as a serious drawback by pharmaceutical companies. Again, phage cocktails targeting different receptors or different bacterial strains would be a potential solution.(c)In vivo phage activity. There is not necessarily a correlation between the in vitro and in vivo behavior of a phage, particularly regarding its propagation ability. This is due both to the complexity of body fluids and the ecological in vivo interactions ,30. In a(d)Immune response. Since phages are made of biomacromolecules, they are potentially capable of eliciting an immune reaction upon administration . General(e)Gene transfer. Phages potentially have the ability to modify the genome of the host bacteria, which may increase their virulence or dissemination of antibiotic resistance genes . Indeed,The main steps for obtaining phage suspensions suitable for use in clinical settings are summarized in 31 particles [The process of phage therapy begins with the identification and selection of phages and bacterial strains suitable for phage production. Phages are found in any environment, even under extreme conditions, being the most abundant biological entities on our planet, with a current total estimate of 10articles . Phage \u201carticles , since particles .Mycobacterium smegmatis is strongly preferred for the isolation of mycobacteriophages over the pathogenic, slowly growing Mycobacterium tuberculosis [Once samples are collected, phage screening parameters will depend on the target bacterium. In general, phages can be isolated using reference strains or clinical isolates as hosts for propagation . In somerculosis ). If a brculosis . Apart frculosis ,48. In gPhage production is typically carried out by the double-layer agar method, which allows isolation of individual phages, and then liquid culture of single plaques . A pure \u00ae (CIM) columns [The main goal of this process is to avoid the presence of bacterial toxins, lipopolysaccharide, or other cellular debris, in the phage suspensions . In addi columns . These m2+ and Ca2+ ions (around 10 mM in the form of CaCl2 or MgSO4) are the most used supplements since they are added to the culture medium before infection to facilitate adsorption and are then present in the recovered lysate [Depending on the phage, phages can be stored at different temperatures, commonly at 4 \u00b0C, \u221280 \u00b0C, or in liquid nitrogen (\u2212196 \u00b0C), or can be freeze-dried ,55. Protd lysate ,56. Othed lysate ,58. Adsod lysate .Phages are basically protein structures and, therefore, they are susceptible to proteases, certain chemical compounds, high temperatures, pH, and ionic strength. Thus, it is important to use an appropriate formulation to ensure that the phage titer remains stable both during formulation and storage and in the in vivo environment where they are applied. Again, the optimal formulation conditions may vary depending on the specific phage, so this deserves careful consideration in the case of phage cocktails, as each type of phage may require individually tailored conditions . However(a)Oral administration is appropriate for gastrointestinal infectious diseases. In some cases, oral phages given without additional protection, as water-based liquid suspensions, reportedly survived gastric passage and were recovered in the feces ,61,62. T(b)Topical administration of phages is chosen for skin infections, wounds, burns, ulcers, and osteoarticular infections . Phages (c)The local phage treatment of respiratory infections requires preparing phages either as stable liquid formulations for intranasal instillation or nebulization, or as a solid powder in an inhalable form . The mos(d)Intravenous administration is recommended in the treatment of systemic infections . In this(e)Intravesical instillation of liquid phage preparations has also been used to treat genitourinary tract infections ,78.(a)Phage identity. The identity of each phage is defined by its specific genomic sequence ,80. Meta(b)Phage Titer. The titer of each individual phage is classically assessed by the double-layer agar method. An alternative is lethality curves, in which the kinetics of phage-induced lysis are assessed by measuring the optical density of phage-infected bacterial cultures . Other m(c)General Purity. For biopharmaceuticals, the purity and correct composition is classically assessed by high-performance liquid chromatography, combined with mass spectrometry if necessary. These methods can be used to identify phage capsid proteins, toxins, or other bacterial proteins. Because of the potential risk posed by the necessary production with pathogenic bacterial hosts, quality criteria should specify maximum levels for contaminants such as toxins or bacterial DNA, which normally must be tested with specific and appropriate molecular biology methods as specified below.(d)Toxins. Several in vitro methods have been developed for endotoxins quantification: gel-clot, turbidimetric, and chromogenic tests. Among the latter, the limulus amebocyte lysate assay is the most widely used . When th(e)Contaminating Nucleic Acids. Quality controls may also be required to determine the concentration of contaminating nucleic acids . The presence and concentration of residual nucleic acids are typically checked by qPCR.(f)Other Quality Controls. Current regulations on sterility or general quality parameters in pharmaceutical products should also apply to phage-based pharmaceuticals . Some paAs products intended for human therapy, phage preparations must comply with certain quality criteria that ensure their safety for clinical use, as well as exhaustive traceability documentation. The production and delivery parameters must be set up by the phage preparation supplier in accordance with the applicable regulations see  and to mPerhaps the greatest hurdle to the implementation of phage therapy in Western medicine is the lack of appropriate regulation. If phage preparations are considered \u201cclassical\u201d medicinal products, they must comply with the corresponding legislation concerning medicinal products production and quality. This essentially means that they should follow GMP (good manufacturing practices) requirements, which has imposed an important problem for the development of medicinal phage preparations in terms of increasing costs or complicating management for developing large-scale production .Although therapeutic phages shall be considered indeed a medicinal product, the nature itself of the phage makes it essentially different from common antimicrobial chemotherapy. Indeed, phage specificity, virus\u2013host co-evolution, or a complex in vivo pharmacological behavior have negatively influenced the outcome of many of the phage therapy clinical trials conducted in contemporary times ,89,90,91A recently adopted strategy in Belgium has allowed a more systematic and practical approach to personalized phage therapy, which also provides production and handling flexibility . The key to this approach is to consider phage products as magistral preparations, since, unlike medicinal products, these are subject to less strict regulation in terms of their production and marketing ,94. In tRegardless of the sub-optimal regulatory status of phage therapy in most countries, including Spain, phage preparations for therapy are available either commercially  or through the request to academic or clinical institutions devoted to phage therapy research and promotion.Pseudomonas aeruginosa) and testing the identity and quantity of bacteria present in every patient [Although potentially controversial , an adap patient . Further(a)In the case of a severe infection produced by an MDR bacterium.(b)When the infection occurs in an area reluctant to the use of antibiotics, such as in prosthetics.(c)Or, in general, whenever there is no standard of care option available, such as patients suffering from hypersensitivity to the antibiotic treatment.In our opinion, the use of phage therapy would be advisable:In these situations, i.e., in the absence of any other satisfactory therapy and when there is a real risk to the patient\u2019s life or significant deterioration of their quality of life, the use of phages, possibly in combination with standard-of-care antibiotics, would be advisable. Conversely, whenever the patient\u2019s infection can be satisfactorily treated with antibiotics, those will always be the therapy of choice. Additionally, a proper diagnostic of the infection-causing bacteria must be conducted see , as wellHaemophilus influenzae, P. aeruginosa, Stenotrophomonas maltophilia, S. aureus, Achromobacter spp., Burkholderia cepacia, and Mycobacterium abscessus, usually resistant to many antimicrobials. Among them, the main pathogens in Spain are S. aureus and P. aeruginosa , with the latter being significantly associated with worse pulmonary function. According to a Spanish multicenter study, 55% of P. aeruginosa isolates from CF patients were MRD, while 16% were extensively drug-resistant [P. aeruginosa, is expected to have a remarkable impact on the life quality and expectancy of these patients.Cystic fibrosis (CF) is a rare disease . Althouesistant . In addiesistant ,105. CF P. aeruginosa and S. aureus resistant to methicillin, fluoroquinolones, and rifampicin. In these cases, treatment options are often limited to antibiotics of last resort, such as polymyxins which have been shown to have higher relapse rates and higher frequency of complications.These are a particular form of deep, localized infections with poor response to antibiotic therapy. The diffusion of antibiotics into bone tissue is often low and is negatively affected by the presence of bacterial biofilms at the contact between bone and prosthetic material. Repeated surgeries to eradicate the biofilm mechanically, i.e., removing the implant and/or resecting the infected bone, and sometimes amputation, is the only infection control option . Most ofThere are some recent examples of clinical cases (CF and osteoarticular infections) treated with phages that support the feasibility of this therapy .In addition to phages, their lytic enzymes have also been explored as antimicrobials . Phage lSince peptidoglycan is present exclusively in bacteria and not in mammalian cells, the risk of cytotoxic effects to humans and animals is minimized. However, since they are proteins, they may induce an immune response. Indeed, in vitro and in vivo studies have shown that neutralizing antibodies can be generated upon repeated exposure to lysins. However, although these antibodies reduce the antibacterial activity of the enzybiotics, they do not completely neutralize them ,123, whiSystemic administration of enzybiotics may also release cellular debris from lysed bacteria, which may induce a proinflammatory response. These bacterial cell debris include lipopolysaccharides, (lipo)teichoic acids, and peptidoglycan through membrane fragmentation and can potentially lead to severe complications such as septic shock. However, allergic or severe inflammatory reactions have not been described so far upon administration of enzybiotics, even in clinical trials with human subjects ,131. The\u00ae SAL200 (Tonabacase) [S. aureus bacteremia without observing any adverse effects. Tonabacase is currently in phase II [S. aureus [In recent years, the way has been paved for recombinant lysins to enter different phases in preclinical and clinical trials. Accelerated clinical advances and their high technical feasibility make them a good alternative therapy to replace or to be used in combination with conventional antibiotics in the short term, even with some authors pointing out that lysins may be able to enter the clinic in a shorter term than phages . Some coabacase)  and Exebabacase) , againstphase II , and Exe. aureus .As discussed in this review, the application of phage therapy is experiencing a considerable boom in recent years. Since 2017, the global phage therapy market reached USD 567.9 million and growth expectations at compound annual growth rate (CAGR) are of 3.9% for the period from 2018 to 2026 . In 2016However, some potential hurdles may still be found in the way towards a successful market entrance, such as the already mentioned regulatory issues , the quaIn this context and given that large pharmaceutical companies have practically stopped investing in new antimicrobial compounds, many scientific startups are focusing on studying and characterizing phages and their products for their therapeutic application. These new companies are located mainly in the USA, India, Korea, Canada, and some European countries. In Spain, the first company focused on the development of endolysins (Telum Therapeutics) has recently been created . An optiBacterial resistance is a major global threat and a leading cause of mortality worldwide. The lack of effective antibiotics against MDR strains urgently requires therapeutic alternatives, and phages or their derivatives can be a promising approach with sufficient scientific and technological know-how in place. However, the lack of both specific regulation for their clinical use and public awareness stands out, in our opinion, as the major hurdles to be presently faced. With this review, we would like to emphasize the advantages of phages as therapeutic tools and how they can be used to combat MDR bacteria. Moreover, despite some foreseeable drawbacks also covered here, phages are nowadays the only short-term solution for many patients. Taking this into account, a suitable regulatory initiative from the competent authorities would be welcomed in Spain in the short term. Both scientific literature and the lead examples of other countries prove that phage therapy is already mature to be translated into a regulation that must ease, protect, and help developing the phage therapy market and infrastructure."}
{"text": "Acinetobacter baumannii (MDR A. baumannii) is an emerging pathogen in the ESKAPE group. The global burden of antimicrobial resistance has led to renewed interest in alternative antimicrobial treatment strategies, including phage therapy. This study isolated and characterized a phage vB_AbaM_ ABPW7 (vABPW7) specific to MDR A. baumannii. Morphological analysis showed that phage vABPW7 belongs to the Myoviridae family. Genome analysis showed that the phage DNA genome consists of 148,647 bp and that the phage is a member of the Phapecoctavirus genus of the order Caudovirales. A short latent period and a large burst size indicated that phage vABPW7 was a lytic phage that could potentially be used in phage therapy. Phage vABPW7 is a high-stability phage that has high lytic activity. Phage vABPW7 could effectively reduce biofilm formation and remove preformed biofilm. The utility of phage vABPW7 was investigated in a human A549 alveolar epithelial cell culture model. Phage vABPW7 was not cytotoxic to A549 cells, and the phage could significantly reduce planktonic MDR A. baumannii and MDR A. baumannii adhesion on A549 cells without cytotoxicity. This study suggests that phage vABPW7 has the potential to be developed further as a new antimicrobial agent against MDR A. baumannii.Multidrug-resistant  MDR baumanniiA.  has become resistant to many antibiotics, including colistin, the last-resort antibiotic [baumannii in patients is associated with a variety of severe conditions such as bacteremia, pneumonia, ventilator-associated pneumonia, central nervous system infections, central line-associated bloodstream infections, catheter-associated urinary tract infections, wound infections, and surgical site infections, leading to increased morbidity and mortality, severe clinical outcomes, and longer lengths of stay at hospitals. In addition, one of the main concerns with MDR baumanniiA.  is its ability to form biofilms, the community of multiple bacterial cells associated with either biotic or abiotic surfaces. During infection by MDR baumanniiA.  in humans, MDR A. baumannii interacts and then adheres to epithelial cells. During the adherence, biofilm formation is induced, and bacterial adherence to the host cells is an essential step in MDR baumanniiA.  colonization resulting in bacterial infection and subsequent pathogenesis [A. baumannii has developed resistance to almost all of the available antibiotics [baumannii control is urgently required.Multidrug-resistant tibiotic . Infectiogenesis ,3,4. Nowibiotics . TherefoAcinetobacter phage AB02 was reported, and the phage showed therapeutic efficacy against MDR baumanniiA.  [baumanniiA.  cells and biofilm formation [Galleria mellonella [Clostridium phage phiCDHS1 reduced the bacterial attachment and cell number in human colon tumorigenic HT-29 cells [Escherichia coli (E. coli) K1 in human epithelial cells was decreased by phage K1F [Escherichia phage P1 resulted in a significant reduction in E. coli in human colon HT29 cells [baumanniiA.  in mammalian cells.Bacteriophages or phages are bacterial viruses that enter and infect bacterial cells. The replication of the phage in host cells causes bacterial lysis and death, making them an alternative antibacterial treatment ,7. Moreoaumannii . Treatmeormation ,12. Furtormation ,14, a coormation . Even thormation , Gallerillonella ,18, mousllonella , and clillonella , only a 29 cells . The inthage K1F . The tre29 cells . Howeverbaumannii.A.  The biology of the phage was characterized, and the efficacy of the phage in vitro was determined, including antibiofilm activity. To the best of our knowledge, this is the first study on the antibacterial activity of the phage against MDR baumanniiA.  in an A549 cell culture model.This study aimed to isolate a phage infecting MDR v/v) glycerol solution and were then stored at \u221280 \u00b0C.MDR A. baumannii , E. coli , K. pneumoniae , methicillin-resistant Staphylococcus aureus (MRSA) , and P. aeruginosa  were kindly provided by the Natural Product Research Center of Excellence, Faculty of Science, Prince of Songkla University, Thailand. A. baumannii ATCC 17978 was included. After receiving the bacterial isolates, Gram-staining and biochemical tests  were performed to confirm they were A. baumannii species. Routinely, bacterial isolates were cultured on tryptic soy agar plates . After overnight incubation at 37 \u00b0C, bacterial colonies were transferred to 3 mL of sterile tryptic soy broth . The bacterial suspension was incubated overnight at 37 \u00b0C with continuous shaking at 150 rpm. For the long-term storage of bacteria, bacterial isolates were mixed with sterile 40% (A. baumannii strains to amikacin (30 \u00b5g), ceftazidime (30 \u00b5g), gentamicin (10 \u00b5g), imipenem (10 \u00b5g), colistin (10 \u00b5g), and tigecycline (15 \u00b5g)  were tested by the disc diffusion technique. The susceptibilities of 2 in a humidified incubator.The human alveolar basal epithelial cell line A549 (ATCC Cat No. CCL-185) was provided by Professor Dr. Duncan Richard Smith, Institute of Molecular Biosciences, Mahidol University, Thailand. The cells were grown and maintained in Dulbecco\u2019s modified Eagle\u2019s medium  supplemented with 10% heat-inactivated fetal bovine serum  at 37 \u00b0C with 5% COA. baumannii strain ABPW063 was employed as a host for phage isolation. The wastewater samples were centrifuged to remove the debris at 6000\u00d7 g, 4 \u00b0C for 10 min. The supernatant was transferred to a new tube and then filtered. Ten milliliters of the filtrate were added to 10 mL of tryptic soy broth , followed by the addition of MDR A. baumannii. The mixture was incubated at 37 \u00b0C for 24 h in a shaking incubator (150 rpm). The mixture was centrifuged to remove bacterial cells at 6000\u00d7 g, 4 \u00b0C for 10 min. The supernatant was filtered through a syringe filter. A spot test was performed to detect phages. Briefly, MDR A. baumannii was mixed with top agar, and 10 \u00b5L of the filtrate was spotted onto the plate. The plates were incubated overnight at 37 \u00b0C and the lytic zone in the bacterial lawn, which indicates the presence of phages, was detected.The phage was enriched and isolated from wastewater samples collected from Thasala hospital, Nakhon Si Thammarat, Thailand. The MDR 4. 7H2O, 50 mM Tris-HCl pH 7.5). After incubation at 4 \u00b0C overnight, the samples were centrifuged at 6000\u00d7 g for 10 min at 4 \u00b0C and filtered through a sterile 0.22 \u03bcm filter. The filtrate was diluted with SM buffer in a series of ten-fold dilutions followed by the double-layer agar technique. The phage was purified five times to obtain purified phage.A single plaque observed in the overlays was picked with a sterile pipette tip which was then soaked in an Eppendorf tube containing 500 \u03bcL SM buffer  and then quickly poured onto a TSA plate. Plaques were counted after incubation overnight at 37 \u00b0C.The supernatant was diluted with SM buffer by a series of ten-fold dilutions. Two hundred microliters of the phage-diluted solution was mixed with 200 \u00b5L of log-phase MDR g for 20 min at 4 \u00b0C. The supernatant was filtered through a sterile 0.22 \u03bcm filter and then concentrated using an Amicon\u00ae Ultra-15 centrifugal filter units, Ultracel 100 kDa membrane  [To amplify a phage stock, the phage supernatant was diluted with SM buffer by a series of ten-fold dilutions. The dilution (200 \u03bcL) was mixed with exponential-phase bacterial culture (200 \u03bcL) in top agar. The mixture was poured on a TSA plate and incubated at 37 \u00b0C overnight. The phage was eluted by adding 5 mL SM buffer onto semi-confluent plates. The supernatant was pooled and centrifuged to remove the bacterial cells at 6000\u00d7 MA, USA) . The supv/v) uranyl acetate (pH 6.7). The phage was visualized using a JEM 2010, JEOL electron microscope  at an operating voltage of 160 kV. The phage classification based on the morphological characteristics was determined according to the standard phage classification method [A morphological observation of the phage was performed by transmission electron microscopy (TEM) as described previously . The phan method .A. baumannii, A. baumannii ATCC 17978, E. coli, K. pneumoniae, MRSA, and P. aeruginosa strains. Two hundred microliters of exponential-phase clinical bacterial isolates was mixed with top agar and poured onto a TSA plate. Ten microliters of the diluted phage stock containing 104 PFU/mL was dropped on the surface of the top agar inoculated with 200 \u03bcL of each bacterial culture. The plates were left to dry and then incubated overnight at 37 \u00b0C. The presence of a clear zone in the bacterial lawn was regarded as the lytic activity of the phage, while the absence of the clear zone indicated no lytic activity. Experiments were undertaken independently in duplicate.The standard spot test was performed to evaluate the lytic activity of the phage as previously described . The hosA. baumannii isolate. The mixtures were incubated at room temperature for 20 min, followed by the double-layer agar technique. After incubation overnight at 37 \u00b0C, the plaques were counted. The EOP value was calculated as the ratio of the titer of the phage on a test isolate to the titer of the phage on the host bacterium. Experiments were undertaken independently in duplicate.The phage lytic activity was confirmed by calculating the EOP as previously described . The spoA. baumannii was measured following a previously reported methodology [A. baumannii at an MOI of 0.1, followed by incubation at 37 \u00b0C without shaking. Samples were taken every minute for the first 10 min, after which samples were taken every 5 min from 10 to 30 min post-infection. The samples were immediately added to pre-chilled TSB supplemented with chloroform. The phage titer was then determined by double-layer agar assay. Experiments were undertaken independently in duplicate with the duplicate plaque assay. The adsorption rate constant, k, in mL/min, was calculated by following the equation as previously described [k is the adsorption rate constant (mL/min); B is the bacterial cell concentration; t is the time; Po is the stating titer; P is the final titer.The attachment rate of the phage to MDR hodology . The phaescribed .k = 2.3A. baumannii was centrifuged at 6000\u00d7 g for 20 min at 4 \u00b0C. The supernatant was removed, and the bacterial pellet was re-suspended in TSB. The bacteria were infected with the phage at an MOI of 0.1. To allow for the absorption of the phage into bacterial cells, the mixture of bacteria and phage was incubated for 20 min at 37 \u00b0C. After centrifugation at 6000\u00d7 g for 20 min at 4 \u00b0C to remove any unabsorbed phage, the pellet was re-suspended in TSB and then incubated at 37 \u00b0C with shaking at 150 rpm. Samples were taken every 10 min for 120 min, and the phage was titered by the soft-agar overlay method. The time between phage adsorption and cell lysis was the latent period. The burst size and the average number of phages released per bacterium was calculated from the ratio of the total number of the released phage progeny at the end of one growth cycle to the initial count of infected bacterial cells. Experiments were undertaken independently in duplicate with the duplicate assay [The latent phage period and burst size were inferred from a one-step growth curve as previously described ,27. Briete assay .A. baumannii growth was assessed following the previously established methodology [A. baumannii was cultured at 37 \u00b0C overnight, and the concentration of bacterial cells was adjusted to 1 \u00d7 108 colony-forming units per mL (CFU/mL). MDR A. baumannii was infected with phage at MOIs of 0.1, 1, and 10. The samples were incubated at 37 \u00b0C with constant shaking. The supernatant was taken every hour for 8 h to measure the bacterial growth at 600 nm. Experiments were undertaken independently in duplicate with the duplicate assay [The effect of the phage against MDR hodology . MDR A. te assay .A. baumannii cells were visualized under SEM. MDR A. baumannii cells were infected with the phage at an MOI of 1 and then incubated at 37 \u00b0C for 4 h. The samples were centrifuged at 6000\u00d7 g for 10 min. The supernatant was removed, and the bacterial pellets were washed twice with 1 mL of PBS. The bacterial cells were re-suspended in 200 \u00b5L of PBS, and the bacterial suspension was dropped onto glass slides. After cell fixation with 2.5% glutaraldehyde in 0.1 M PBS, the cells were washed twice with PBS. The cells were dehydrated in a series of ethanol solutions  after which the bacterial cells were dried in a critical point dyer and then coated with gold. The ultrastructure of bacterial cells was observed under a field emission SEM (FE-SEM) .The effects of the phage on MDR 8 PFU/mL. The bacteriophage in the SM buffer was incubated at different temperatures  for 2 h. After incubation at specific temperatures, the samples were cooled in an ice-water bath, and the phage viability was determined by the double-layer agar method. The phage incubated at 4 \u00b0C was used as a control. For the ability of the phage to tolerate varying pH values, the phage was incubated in SM buffers ranging from pH 1 to pH 14 for 2 h, after which the samples at varying pH levels were neutralized to pH 7, and the phage viability was measured by the double-layer agar method. The phage incubated in the SM buffer at pH 7 was used as a control. The phage stability in glycerol stock was evaluated. The phage was mixed with glycerol in the SM buffer. The final concentrations were 25% (v/v) and 50% (v/v) glycerol [The effects of different temperatures, pH values, and UV radiation on the phage stability were measured according to the previously described methods . The phaglycerol . After iThe phage whole-genome de novo sequencing was undertaken commercially  on the Illumina sequencing platform. The phage DNA was extracted, and the quality was verified by the Picogreen method using Victor 3 fluorometry . The library was prepared using the TruSeq Nano DNA library preparation kit . After checking the library quality, the library was sequenced, and the quality of raw sequence data from high throughput sequencing pipelines was evaluated by FastQC (version 0.11.5), a quality control tool. The results from FastQC were assembled de novo into a contig by SPAdes de novo (version 3.13.0) with the k-mer parameter. Prokka (version 1.12) was used to predict the locations of protein-coding sequences, tRNA genes, tmRNA genes, and rRNA genes. BLAST and EggNOG were used to annotate the functions of genes. Blastx was performed against standard databases with an E-value cutoff of 0, a query coverage cutoff of 80%, and an identity cutoff of 80%. The genome map was constructed on the CGview server.Staphylococcus phage JD419 (QOI66744). All pairwise comparisons of the nucleotide sequences were analyzed by the Genome-BLAST Distance Phylogeny approach under settings recommended for prokaryotic viruses [The relationship of the phage to other phages in the NCBI database was determined by a Blastn search with an E-value cutoff of 0, and an identity cutoff of 70%. The outgroup was  viruses ,32,33. T viruses . Branch  viruses , and the viruses , using t viruses . The gen viruses . EMBOSS Staphylococcus phage JD419 (QOI66744) was used as an outgroup.The tail fiber gene was selected to investigate the genetic relationships using the maximum-likelihood phylogenetic tree and the Jones\u2013Taylor\u2013Thornton matrix-based model based on the amino acid sequence alignment. The gene was compared with other gene sequences in the NCBI database by Blastx . The mulA. baumannii was cultured and diluted with TSB. One hundred microliters of MDR A. baumannii was added into a flat-bottomed 96-well microtiter plate (107 CFU/well). Subsequently, 100 \u03bcL of diluted phage (101\u2013108 PFU/well) was added to the wells, and the plates were incubated without agitation at 37 \u00b0C for 24 h. For the disruption of preformed biofilm, bacteria were added into a flat-bottomed 96-well microtiter plate and incubated at 37 \u00b0C for 24 h without shaking to allow for biofilm formation. At the indicated time point, the supernatant was removed. The biofilm was washed twice with PBS, and 100 \u00b5L of the phage (101\u2013108 PFU/well) was added to the wells. The plates were incubated at 37 \u00b0C for 24 h. At the indicated time points, the plates were washed twice with PBS and then air-dried. Two hundred microliters of 0.1% crystal violet was added to stain the biofilm biomass. After incubation at room temperature for 30 min, the excess stain was washed away with PBS, and 200 \u03bcL of absolute ethanol was added to solubilize the biofilm. The biofilm biomass was measured at 595 nm using a standard microplate absorbance reader. To determine biofilm viable cell numbers, the supernatant was removed, and the biofilm was washed twice to remove planktonic bacteria with PBS. The biofilm was re-suspended in 100 \u00b5L of PBS by vigorous pipetting. The supernatant was serially diluted in PBS. Ten microliters of the supernatant were dropped onto TSA plates and air-dried. The colonies were counted after incubation at 37 \u00b0C overnight. Experiments were undertaken independently in triplicate with the duplicate assay.The ability of the phage to prevent biofilm formation and to remove biofilm was assessed according to the methods described previously . The bioA. baumannii towards A549 lung epithelial cells was examined using the MTT -2,5-diphenyltetrazolium bromide) cell proliferation assay. The cells were grown in 96-well tissue culture plates at 37 \u00b0C with 5% CO2 until the cells reached approximately 90% confluence. The medium was discarded, and cells were then washed with PBS. The cells were incubated with 200 \u00b5L of the phage (MOIs of 0.01 to 100) or MDR A. baumannii (MOIs of 0.01 to 100) diluted in cell culture medium. After incubation under standard conditions for 24 h, the medium was removed, and the cells were washed twice with PBS. One hundred microliters of complete fresh medium were added to the wells followed by the MTT solution. After incubation under standard conditions for 4 h, DMSO was added into the well to solubilize the water-insoluble formazan. Absorbance was measured at a wavelength of 570 nm using a standard microplate absorbance reader. Cells incubated with only a combination of cell culture media and SM buffer (or only cell culture media) and cells incubated with 5% DMSO were included as negative and positive controls, respectively. The experiments were undertaken independently in triplicate with the duplicate assay.The cytotoxicity of the phage and MDR The adsorption of phage to A549 cells was undertaken as described by others . BrieflyA. baumannii infection under the cell culture model was determined following the methodology in a previous report [A. baumannii at an MOI of 1. After incubation for 2 h, the cells were washed twice with PBS and then incubated with the phage at MOIs of 0.01 to 100 under standard conditions for 2 h. The medium was removed, the cells were washed twice with PBS, and complete medium was added; the cells were then cultured under standard conditions. At 24 h post-incubation, the supernatant was removed and replaced with 100 \u03bcL serum-free media containing 0.2% resazurin. The plates were incubated under standard conditions for 3 h and the fluorescence was taken at 530/590 nm to measure the reduction in bacteria. On the parallel plates, the MTT assay was undertaken as previously described. The experiments were undertaken independently in duplicate.The phage efficacy in reducing an MDR s report . The celA. baumannii were centrifuged at 6000\u00d7 g for 10 min. The supernatant was removed, and the bacterial pellet was washed three times with PBS. The bacterial cells were resuspended in DMEM and then adjusted to a final OD600 of 1.0 before proceeding with the bacterial adhesion assays. After removing the cell culture medium, the A549 cells were washed with PBS and incubated with MDR A. baumannii at an MOI of 1 and phage at MOIs of 5 and 10. The cells were incubated under standard condition, and the supernatant was collected at 0, 1, 2, 4, and 6 h post-incubation. The production of planktonic bacterial cells was measured by counting the colony-forming units after plating cultures. The supernatants were serially diluted ten-fold and then spread onto TSA plates. The production of free phage was determined by counting plaques using a double-layer agar method. At 6 h post-incubation, the viability of A549 cells was assessed by a trypan blue exclusion test. The kinetics of the antibacterial activity of the phage against planktonic bacteria under the model without cells were evaluated in parallel. The experiments were undertaken independently in duplicate with duplicate plaque assay.The anti-planktonic property of phage vABWU7 under the cell culture model was assessed, and the number of planktonic bacteria and free phage production were evaluated at time intervals for 6 h. A549 cells were cultured in 6 well tissue culture plates under standard conditions and then grown until the cells reached approximately 90% confluence. Overnight cultures of MDR A. baumannii to A549 cells was evaluated as previously described [A. baumannii infection for 2 h. For the therapeutic treatment, the cells were infected with MDR A. baumannii for 2 h and then treated with the phage for 2 h. The cells were incubated under standard conditions, and the bacterial number was assessed at 0, 1, 2, 4, and 6 h post-incubation. The supernatant was discarded at the indicated time points to remove non-adherent bacteria. The cells were washed three times with PBS to remove non-adherent extracellular bacteria and phage. Subsequently, the cells were treated with 1 mL of PBS containing 0.1 mM EDTA and incubated at 37 \u00b0C for 10 min, followed by vigorous pipetting. The supernatant was serially diluted ten-fold and then spread onto TSA plates. The viable bacterial cells were determined by counting colonies formed after overnight incubation at 37 \u00b0C. In addition, the cell viability was also determined in parallel by the trypan blue exclusion test. The experiments were undertaken independently in duplicate with the duplicate assay.The effect of the phage on the adhesion of MDR escribed ,41,42,43A. baumannii that attached to A549 cells were determined following the protocol as described above, and the ultrastructure of the bacteria on the cell surface was visualized. Briefly, the phage at an MOI of 5 were added to the cells 2 h before MDR A. baumannii infection (at an MOI of 1) in prophylactic phage administration, while the cells were infected with MDR A. baumannii cells (an MOI of 1) before the phage treatment (at an MOI of 5) in the therapeutic treatment. The cells were incubated under standard condition for 4 h and then washed twice with PBS. The cells were fixed for SEM analysis following the protocol as described above. The ultrastructure of bacterial cells attached to A549 cells was observed under an FE-SEM .The effects of the phage on MDR https://www.graphpad.com/scientific-software/prism/ (accessed on 1 September 2022)) was used to analyze all data. Statistical significance analysis was undertaken by unpaired T-test using the GraphPad Prism, p < 0.05 for significance.The GraphPad Prism program, version 5 , moderate (0.5 > EOP \u2265 0.1), low (0.1 > EOP > 0.001), and no activity (EOP \u2264 0.001). Three, four, and two isolates were found to exhibit a high EOP value (EOP = 0.61\u20131), moderate EOP value (EOP = 0.1\u20130.4), and low EOP value (EOP = 0.01\u20130.04), respectively ) ). The phage induced blisters and pores on the bacterial cell membrane. MDR A. baumannii cells had burst, with cell lysis, leading to cell death ) c. MDR A.ll death d.v/v) and 50% (v/v) glycerol after storage at \u221220 and \u221280 \u00b0C for 30 days when compared with the stability of the phage in SM buffer at 4 \u00b0C, as a control  (Escherichia phage vB_EcoM_ESCO37 (OM386659.1), Escherichia phage phAPEC8_ev052 (LR597654.1), Escherichia phage vB_EcoM_ESCO25 (OM386656.1), Escherichia phage ESCO13 (NC_047770.1), Dompiswa phage TSP7_1 (NC_062742.1) and Klebsiella phage ZCKP1 (NC_047994.1), respectively. Staphylococcus phage JD419 (QOI66744) was used as the outgroup. The sequencing results showed that phage vABPW7 was a member of the genus Phapecoctavirus, subfamily Stephanstirmvirinae, class Caudoviricetes, and order Caudovirales.To evaluate the relationship of phage vABPW7 and other phages on a nucleotide level, the whole-genome sequence of phage vABPW7 was searched against the Blastn database and the data were further analyzed by VICTOR, a genome-based phylogeny and classification of prokaryotic viruses program. Phage vABPW7 was the most closely related to 05078.1) B. Other Escherichia phage BI-EHEC, was analyzed by ViPtree. A high identity of both genomes was observed across the majority of the genomes, although regions of lower identity were seen  and Escherichia phage vB B_EcoM_ESCO25  , a putative structural protein of Klebsiella phage ZCKP1 , a putative tail fiber protein of Enterobacteria phage ECGD1 , and a putative tail protein of Salmonella phage GEC vB MG , respectively. The tail fiber of Staphylococcus phage JD419 (QOI66744) was used as an outgroup.To analyze the relationship of phage vABPW7, a phylogenetic tree analysis was carried out with the tail fiber protein encoded by ORF159. The nucleotide sequence of the gene was searched with Blastx against the NCBI database with an E-value cutoff of 0, a query coverage cutoff of 80%, and an identity cutoff of 70%. The phylogenetic tree results showed that phage vABPW7 shared a common clade ancestor with the tail fiber protein of dentity) b. The taA. baumannii was incubated with phage vABPW7 at various concentrations. Phage vABPW7 at 101 to 108 PFU/wells were significantly effective in preventing biofilm formation by reducing approximately 9.25 to 67.27% biofilm biomass, and 91.5 to 99.4% (1.2 to 2.05 log) of viable cells compared to a control, not treated with phage ) ) ) d. Phage (n = 3)) e,f. Some(n = 3)) . The phaA. baumannii is an emerging pathogen associated with increased morbidity and mortality rates worldwide. MDR A. baumannii is frequently involved in hospital-acquired and ventilator-associated pneumonia. However, MDR A. baumannii has become a common pathogen, which can infect the respiratory tract, blood, soft tissues, and urinary tract, resulting in septicemia, meningitis, endocarditis, pneumonia, wound, and urinary tract infections. MDR A. baumannii rapidly develops resistance to all antibiotics, including colistin and tigecycline\u2014the only effective antibiotics in clinical treatment [A. baumannii strains including the development of new antibiotics, nanoparticles, peptides, and phages. To date, various phages infecting A. baumannii have been studied and reported, and Acinetobacter phages have been investigated in vitro and in vivo, including their use in clinical trials in the US [Acinetobacter phage in a cell culture model. To further shed some light on phage therapy, this study focused on the isolation and characterization of a phage infecting MDR A. baumannii, determined the effects of this phage on biofilm, and evaluated the efficacy of phage in vitro, including in an A549 cell culture model.MDR reatment . Given tn the US ,48. HoweA. baumannii lawn, and the plaques have a halo around them indicating that the phage might encode depolymerases with polysaccharide-degrading activity. A phage host range and EOP analysis showed that phage vABPW7 was able to infect 45% of 20 clinical strains. These results indicated that phage vABPW7 could possibly be used in phage therapy, especially as part of a phage cocktail. An increase in the number of bacterial strains for host range testing might provide useful additional information. Many common factors affecting phage viability, such as temperature, pH, UV radiation, and phage infectivity over long periods, have been reported [A. baumannii completely, highlighting the high lytic activity of phage vABPW7.Phage vABPW7, which has a long and contractile tail, was enriched and isolated from a wastewater sample. The phage produces small plaques on an MDR reported . Phage vPhapecoctavirus, subfamily Stephanstirmvirinae, class Caudoviricetes, and order Caudovirales. The comparative whole-genome sequencing results showed no Acinetobacter phage-sharing whole-genome similarity with phage vABPW7. Interestingly, phage vABPW7 shares the highest whole-genome identity with Escherichia phage BI-EHEC. The phage also shares high sequence similarity with Klebsiella phage ZCKP1, Dompiswa phage TSP7_1, and Myoviridae sp. Even though most of the studies on phages have reported that phages are highly host-specific, some phages can infect many species and different genera of bacteria [Escherichia phage, could infect A. baumannii. The identity of phage vABPW7 and Escherichia phage BI-EHEC was 95.1%, indicating that phage vABPW7 might be a typical phage in the genus Phapecoctavirus, subfamily Stephanstirmvirinae, class Caudoviricetes, and order Caudovirales. Furthermore, there was no virulence factor or resistance gene in the genome of phage vABPW7, suggesting the potential as a reliable therapeutic agent for phage therapy against MDR A. baumannii. Due to the high similarity of phage vABPW7 and Escherichia phage-specific avian pathogenic E. coli, the lytic activity of phage vABPW7 towards Avian pathogenic E. coli should be investigated in the future.The genome study of phage vABPW7 showed that the phage is classified as a member of the genus bacteria . Thus, iEscherichia phage vB B_EcoM_ESCO25 and Escherichia phage ESCO13, demonstrating that phage vABPW7 was grouped into the genus Phapecoctavirus, corresponding to the whole-genome analysis. In addition, previous studies have shown that the tail fiber proteins of phage, such as Acinetobacter phage IME200 [Acinetobacter phage phiAB6 [Klebsiella phage SH-KP152226 [The process of a phage entering bacterial cells is mediated by a specific interaction between the bacterial cell membrane and the tail fiber protein, which is a phage receptor-binding protein functioning in phage adsorption and penetration into bacterial cells . The bine IME200 , Acinetoe phiAB6 , and KleKP152226 , possessKP152226 . CapsulaKP152226 . It is wKP152226 , and as KP152226 . MoreoveA. baumannii can cause various diseases, including pneumonia, an infection that inflames the alveoli in the lung. Cases of nosocomial pneumonia, severe community-acquired pneumonia, and ventilator-associated pneumonia have been reported to continuously increase [A. baumannii can adhere, colonize, and invade human epithelial cells, followed by intracellular multiplication, dissemination to other tissues, persistence, and activation of cell death pathways [A. baumannii infections in mammalian cells. A. baumannii interacts with A549 cells through fibronectin and then enters the cells, leading to pathogenesis [MDR increase . Previoupathways . Human logenesis ,63. Receogenesis . In addiogenesis . To ensuogenesis . Moreoveogenesis . In an aogenesis . Howeverogenesis , and theAcinetobacter phage vABPW7 has the potential to be used as an alternative antibacterial approach to control MDR A. baumannii infections. Phage vABPW7 is suitable for further development towards its application in phage therapy, including as part of a phage cocktail."}
{"text": "Bacteriophage (phage) therapy is re\u2010emerging a century after it began.Activity against antibiotic\u2010resistant pathogens and a lack of serious side effects make phage therapy an attractive treatment option in refractory bacterial infections.Phages are highly specific for their bacterial targets, but the relationship between in vitro activity and in vivo efficacy remains to be rigorously evaluated.Pharmacokinetic and pharmacodynamic principles of phage therapy are generally based on the classic predator\u2013prey relationship, but numerous other factors contribute to phage clearance and optimal dosing strategies remain unclear.Combinations of fully characterised, exclusively lytic phages prepared under good manufacturing practice are limited in their availability.Safety has been demonstrated but randomised controlled trials are needed to evaluate efficacy. Shigella enteritis (bacillary dysentery) in 1919, in the first recorded description of phage therapy. In the years that followed, phages were used with varying success by d'Herelle and others for a variety of serious infections, including staphylococcal bacteraemia, typhoid fever and osteomyelitis,Caudovirales)  . Phages Box 1A) and electron micrograph of bacteriophage (Myoviridae) with 1% uranyl acetate negative staining, with size marker (B). Bacteriophage preparations were dialysed against 0.1\u00a0M ammonium acetate in dialysis cassettes with a 10\u00a0000 membrane molecular weight cut\u2010off (Pierce Biotechnology), negatively stained with 2% uranyl acetate, and visualised using transmission electron microscopy (TEM). TEM was conducted at the Westmead Scientific Platforms  on a Philips CM120 BioTWIN (Thermo Fisher Scientific) transmission electron microscope at 100\u00a0kV. Images were recorded with a SIS Morada digital camera using iTEM software (Olympus Soft Imaging Solutions).Schematic  in the 1970s of high dose phage for the treatment of cholera found tetracyclines to be more effective.Escherichia coli infection, and only half of these were phage\u2010susceptible in vitro. More recently, a phase 1 double\u2010blind RCT of a topical bacteriophage cocktail in chronic venous leg ulcers found no safety concerns,Pseudomonas with standard care with silver sulfadiazine, found that significant reductions in bacterial counts took longer in the phage group and that the silver sulfadiazine group had higher treatment success than the phage group. The PhagoBurn trial suffered from instability of the phage preparation, but showed that phage\u2010susceptibility of Pseudomonas isolates in vitro is crucial to eventual clinical success, with susceptibility rates of 89% in successful cases compared with 24% in clinical failures in the phage therapy arm  experience in humans is less impressive . The larrapy arm .Box 2Phages usually exhibit a high degree of host specificity and are most easily sourced from the habitat of their usual bacterial hosts. Phages specific for clinically relevant bacteria may be readily sourced from human and animal sources, hospital wastewater, and environmental soil and water. Their reproduction is dependent on the bacterial host, either integrated into the bacterial genome as a prophage or acting in a purely parasitic manner , the latBox 3A), the phage DNA (purple) is classically either reproduced and packaged as new virions at the expense of the cell  or reproduces with the host DNA .After infection  susceptible isolate \u2014 productive infection and formation of individual plaques detectable at serially diluted phage lysate \u2014 and (B) resistant isolate \u2014 single plaques absent and early bacterial lysis present only at high concertation of phages. Liquid culture\u2010based system: (C) across a gradient of multiplicity of infections (MOIs) against eight bacteria on a single 96\u2010well plate. There are seven MOIs tested from highest to lowest (left to right). Column eight and nine are phage\u2010only and bacteria\u2010only controls. (D) Bacterial growth kinetics in presence of phage at differing MOIs.Phage\u2010susceptibility testing of bacterial isolates with the standard double\u2010layer method:The plaque assay is time\u2010consuming and operator\u2010dependent, with automated methods still in their infancy. Liquid culture\u2010based systems that continuously monitor bacterial growth in the presence of bacteriophage in a standard 96\u2010well plate format , C may bAcinetobacter infection when antibiotics were failing.The development of in vitro bacterial resistance was a prominent feature of phage therapy for a high burden multidrug\u2010resistant input)A good understanding of the non\u2010linear kinetics of bacteriophage distribution in blood and tissues is necessary to maximise efficacy of future therapeutic protocols.1\u2013105  colony\u2010forming units per mL of blood in severe sepsis,9 plaque\u2010forming units (PFU) into the human blood volume (~\u00a05\u00a0L) is expected to yield an MOIinput over 200.Numerous methods of bacteriophage delivery have been explored, including topical, inhalational, oral and injectable . When administering phages orally, concern has been raised regarding recombination between modular phage genomes in the gut, although there has been little evidence from trials to date to resolve this one way or the other.The paradoxical persistence of a narrow host range for phages is not fully understood, given the presumed ecological advantagesE. coli have added additional barriers (curli fibres) as a collective protection.Urine concentration of bacteriophage may be dose\u2010dependant,Optimised preparation of phage combinations (cocktails) for use in humans requires well characterised bacteriophages, a good understanding of the bacterial target population, and highly effective purification protocols to avoid inflammatory responses to contaminating residual bacterial endotoxin and protein.9\u20131010\u00a0PFU/mL) with unique but overlapping host ranges to guarantee lysis of the bacterial target and clinically relevant variants.General recommendations regarding the standardisation of key components of phage therapy have been published,Phage therapy may be presented as a discrete quality\u2010assured formulation either as several phages admixed to provide a broad spectrum of activity in a single medicine for a specific infection  clinical trials in humans means that phage therapy still has the status of an old technology based on anecdotal evidence, not much different to traditional medicines. Nevertheless, there are some successful phase 1 and 2 studies, including in Australia, which suggest the potential of phage therapy as an alternative or adjunct to antibiotics.S. aureus sepsis and infective endocarditis were treated twice daily for 2 weeks with intravenous AB\u2010SA01  \u2014 a GMP\u2010quality cocktail product with three bacteriophages \u2014 as adjunct to standard care.Recent studies in Australian centres of both intranasal instillationNatural bacteriophages are defined as investigational drugs by the Therapeutic Goods Administration in Australia. The regulatory side of bacteriophage therapy is beyond the scope of this review, but most medical communities are both sceptical and curious.To progress, we need at least to guarantee the availability of efficient phage susceptibility testing and of preparations that are safe for intravenous administration, define optimal dosing, and effectively monitor phage\u2013bacteria\u2013human host interactions and any important collateral effects on other members of the microflora.Realistically, application of therapeutic phages alone may never completely replace chemical antimicrobials as the standard of care. For now, it is reasonable to define phage therapy as a promising rescue therapy that has been associated with some spectacular resultsNo relevant disclosures.Not commissioned; externally peer reviewed."}
{"text": "Escherichia phage, YF01, from a municipal wastewater treatment plant in Yokohama, Japan. We demonstrate that the YF01 phage shares a high similarity to a collection of thirty-five Escherichia and Shigella phages found in public databases, six of which have been previously classified into the Kuravirus genus by the International Committee on Taxonomy of Viruses (ICTV). Using modern phylogenetic approaches, we demonstrate that an expansion and reshaping of the current six-membered Kuravirus genus is required to accommodate all thirty-six member phages. Ultimately, we propose the creation of three additional genera, Vellorevirus, Jinjuvirus, and Yesanvirus, which will allow a more organized approach to the addition of future Kuravirus-like phages.Bacteriophages, viruses that infect bacteria, are currently receiving significant attention amid an ever-growing global antibiotic resistance crisis. In tandem, a surge in the availability and affordability of next-generation and third-generation sequencing technologies has driven the deposition of a wealth of phage sequence data. Here, we have isolated a novel  Escherichia coli is a versatile and genetically diverse species commonly inhabiting the intestinal tracts of humans and animals as both a commensal and pathogenic microbe [E. coli infecting humans are routinely released into the environment via sewage systems, which lead to wastewater treatment plants (WWTPs) [E. coli, alternative methods to control these pathogens are highly sought after. Bacteriophages (or phages) have received high attention as a potential therapeutic alternative to antibiotics [ microbe . Pathoge (WWTPs) . Given tibiotics ,4,5. 8\u2013109 particles per milliliter [E. coli in biological WWTPs [Phages are viruses that multiply by infecting bacteria, often very specifically at the species or strain level. They play a key role both in microbial ecology and microbial evolution, having the ability to alter the population dynamics within microbial communities and modify bacterial genomes through horizontal gene transfer ,6,7. In lliliter , which ial WWTPs . The useal WWTPs . The advent of next-generation sequencing technologies, such as those offered by Illumina, has led to a preponderance of genomic sequencing. Newer \u201cthird-generation sequencing\u201d technologies, such as those offered by Oxford Nanopore, facilitate the acquisition of long-sequence reads (up to Mbs), allowing for the generation of complete bacterial and other higher organism genomes. While commonly used in combination with highly accurate short-reads, more recent revisions of the technology, known as Q20, include qualitative improvements that facilitate the generation of near-perfect bacterial assemblies, and perfect viral assemblies, without the need for short-read polishing ,15.Escherichia phage assemblies listed on NCBI Genbank. The International Committee on Taxonomy of Viruses (ICTV) currently recognizes Escherichia phages belonging to at least 11 different families and 100 different genera [Phieco32-like virus genus was created in 2009, in response to the isolation and characterization of the Escherichia phage phiEco32, a novel 77 kb phage with a rare C3 podovirus morphotype [Phieco32likevirus and then Phieco32virus), while also adding five new phage members to its ranks  [Kuravirus in 2019.As of December 2022, there are currently ~1600 rphotype . The gen kv1721) ,19,20. TE. coli phage, YF01, from a WWTP in Yokohama, Japan. The YF01 phage was sequenced and assembled using long-read Q20 Oxford Nanopore technology and shown to be related to phages from the ICTV-classified Kuravirus genus. Modern reticulate network analyses revealed that YF01 and the current six ICTV-classified Kuravirus phages shared high similarity with 29 other phages present in public databases. Using a combination of approaches, we demonstrate that these 36 phages represent four different genera under current ICTV guidelines. We further analyze the genomic features of the YF01 phage in comparison to other Kuravirus-like group phages and elucidate the core proteome of the group to demonstrate that the large terminase subunit and portal protein, among three other proteins, show high conservation and highly correlated phylogenetic reconstruction to whole genome-based methods. Ultimately, we recommend the expansion and reshaping of the Kuravirus genus to four separate genera to accommodate a total of 36 phages isolated worldwide.In this study, we isolated a novel Escherichia coli strain K12 (JCM20135), isolated from feces from a diphtheria convalescent sample, was used in this study. Bacterial cultures were grown in Nutrient Broth with NaCl  and NB with agar (with added 12 g L\u22121 agar) at 25 \u00b0C. Cultures were grown under aerobic conditions.g for 5 min. The supernatant was filtered through a cellulose acetate (CA) filter with a 0.20 \u03bcm pore size . A total of 1 mL of filtrate was added to NB and 100 \u00b5L of log phase E. coli K12. The suspension was incubated overnight at 25 \u00b0C. The overnight culture was centrifuged at 2000\u00d7 g for 10 min and the supernatant filtered through a CA 0.20 \u03bcm pore filter . E. coli colonies from a growth plate were taken with sterile cotton swab and spread uniformly on NB agar medium. A total of 40 \u03bcL of filtrate was added plate dropwise, air-dried, and incubated at 25 \u00b0C overnight. A visible plaque was removed with the wide end of a tip and resuspended in SM buffer , a process repeated twice to ensure a pure phage isolate. Activated sludge mixed liquor was collected from a municipal WWTP in Yokohama in November 2021 where urban wastewater was received. The activated sludge sample was transferred to a 15 mL tube and centrifuged at 2000\u00d7 11 PFU mL\u22121) was incubated on a grid for 10 min. Grids were washed twice in MilliQ water and stained with 2% (w/v) uranyl acetate prior to a final wash in MilliQ water. Grids were dried prior to examination under a JEOL JEM02010HC electron microscope. TEM was performed as previously described . Briefly11 PFU mL\u22121) were mixed in a 9:1 ratio and incubated at 25 \u00b0C for 1 h. Phage PFU mL\u22121 was then determined by spot test (20 \u00b5L) using a dilution series down to 10\u22128 and counting the dilution that resulted in 10\u2013100 plaques. For temperature testing, the NB and YF01 phages (~1 \u00d7 1011 PFU mL\u22121) were mixed in a 9:1 ratio and incubated for 1 h at different temperatures . Phage PFU mL\u22121 was determined as above. Each experiment was repeated two times.To test phage stability, the pH of NB was adjusted to 3, 5, 7, 9, and 11 using NaOH and HCl. The pH-adjusted NB and YF01 phages (~1 \u00d7 1010 PFU mL\u22121) was extracted using a zinc chloride phenol:chloroform extraction method [2 to remove host contaminants before the precipitation of phage virions by the addition of 40 mM ZnCl2. Virions were resuspended in phage extraction buffer  SDS and 50 \u00b5g mL\u22121 proteinase K) and incubated for 1 h at 55 \u00b0C. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added and the top layer was removed and DNA precipitated with isopropanol. The pellet was washed in 70% (v/v) ethanol and resuspended in 10 mM Tris-HCl (pH 8.5). DNA concentration and purity were measured using a Quantus Fluorometer  and NanoDrop One , respectively. DNA integrity was assessed via agarose gel electrophoresis.Genomic DNA from 1 mL phage filtrate (>1 \u00d7 10n method . Brieflyhttps://github.com/rrwick/Filtlong). Filtered mean read size was 17.8 kb with a mean read quality of Q20.3, as assessed by NanoPlot v1.40.0 [https://github.com/nanoporetech/medaka) made no corrections to the assembly. Residual Nanopore adapter sequences were truncated from the termini of the final assembly by screening against Y-adapter top (5\u2032-GGCGTCTGCTTGGGTGTTTAACCTTTTTTTTTTAATGTACTTCGTTCAGTTACGTATTGCT-3\u2032) and Y-adapter bottom (5\u2032- GCAATACGTAACTGAACGAAGT -3\u2032) sequences.DNA libraries were prepared using 1 ug of DNA using the Ligation Sequencing Kit 14 (SQK-LSK114), loaded on a R10.4.1 flow cell (FLO-MIN114) and sequenced using a MinION Mk1B . Simplex and duplex basecalling was performed using Guppy v6.4.2 in super accurate (SUP) mode. Read filtering was performed with Filtlong v0.2.1 . DTR sequences in other eviously . To annoeviously  and manueviously , a profieviously , and theeviously . tRNAs weviously  and Arageviously . Figureseviously . Amino aeviously  in GeneiE. coli K12 MG1655 (NC_000913). Codon usage was determined using the statistics function in Geneious Prime v2022.2.1. Codon usage that differed by \u22652-fold compared to the host were plotted using Prism v7.0.Coding sequences (nucleotide) for YF01, phiEco32, ES17, KBNP1711, and ECBP2 phages were determined using Prokka and directly accessed from Genbank for Kuravirus-like group phages was performed using Roary [The core analysis of the ng Roary . Brieflyng Roary  was usedng Roary  and the Escherichia phage YF01 was isolated from activated sludge collected from a municipal WWTP in Yokohama, Japan. The phage formed small, transparent, circular plaques approximately 1 mm in diameter (\u22121) noted from 60 \u00b0C and no observable phage activity above 70 \u00b0C (diameter a. Phage diameter b. We nexve 70 \u00b0C c. The YFve 70 \u00b0C d. The pHE. coli (~50.8%), a common phenomenon among lytic phages [The YF01 phage was sequenced using long-read Q20 Oxford Nanopore technology, leading to the assembly of a 78,626 bp genome with 704-fold coverage . The GC c phages .Escherichia (and 1 Shigella) phages  . As vCongrouping ) of 24 aKuravirus phages, and performed matrix nucleotide identity analysis to understand the nucleotide-level similarity more precisely. Using nucleotide similarity thresholds of 95% and 70%, for species and genus demarcation, respectively, as recommended by the ICTV, VIRIDIC grouped these phages into a total of thirty-three species and four genera G1-G4  grouped into the largest genus (G1), while KBNP1711 and ECBP2 phages grouped into two separate genera, G3 and G4, respectively. The apparent discrepancy between our genera groupings and the Kuravirus classifications by ICTV occurred due to the nucleotide similarity between the KBNP1711 and ECBP2 phages and the other ICTV-classified phages (such as the phiEco32 phage) falling below the currently accepted threshold of 70% .We took these 36 grouped phages, including the YF01 phage and the six ICTV-classified ra G1-G4 . In contAeromonas, Cronobacter, Escherichia, Proteus, Salmonella, and Vibrio species. VICTOR phylogeny demonstrated that the 36 clustered phages formed a monophyletic group , using whole phage genome sequence inputs . Here weic group , with clic group , G1\u20134. OKuravirus phages occurred between 2009 (the creation of the genus with phiEco32 phage) and 2015\u20132016 . Given the substantial increase in the amount of Kuravirus-like phage genomes deposited in public databases since then (30 new Kuravirus-like phages deposited in the past 9 years) and based upon the current criteria for the genome-based classification of viruses [Kuravirus genus into four separate genera based on the currently accepted similarity thresholds . We will recommend to the ICTV the identification of these four genera as Kuravirus (G1), Vellorevirus (G2), Jinjuvirus (G3), and Yesanvirus (G4), with the names of the new genera (G2\u2013G4) representing either the geographical location of isolation or locality of the research group, of the founding phage member of each respective genus . At the present time we will refer to this grouping of 36 phages as the Kuravirus-like phage group or simply \u201cthe group\u201d  and genes involved in DNA replication and nucleotide metabolism on the right  direct terminal repeats (DTRs) indicative of a T7 phage-like replication strategy where the substrate for capsid packaging are large DNA concatemers. The presence of DTRs in the group was first determined in phiEco32 phage (193-bp DTRs) using a combination of restriction profiling, primer walking, and sub-cloning strategies [rategies . In morerategies . With thrategies ,61. Givegp3-4), which form the molecular motor responsible for packaging of the nascent phage DNA, are the first two genes in this module. While gp3 displayed no known domains via the NCBI conserved domains database (CDD), it did show similarity to the small terminase subunit in the Pseudomonas phage PaP3 using a more sensitive comparison of profile hidden Markov models (HMM) [gp25 in the YF01 phage) annotated as a terminase small subunit. This was likely due to the presence of a HNH endonuclease domain, which are often associated with phage DNA packaging [gp3 as the putative small terminase subunit in the YF01 phage. The virion morphogenesis and lysis module in the YF01 phage shares high homology with other group phages, comprising a total of 20 genes transcribed in the forward direction . The terls (HMM) ,64. We dackaging . Howevergp5-9) encode proteins that assemble to form the rare C3 morphotype (an elongated phage capsid) seen in the YF01 phage (gp5), scaffolding protein (gp7), major capsid protein (gp8), and adapter protein  and a larger product that occurs due to a ribosomal slippage event at a heptanucleotide slippery sequence  near the natural stop codon of gp8, leading to an extended isoform using the -1 frame . This is a well-known phenomenon in the tail assembly chaperone in long-tailed phages (~3.5% slippage rate in the Escherichia phage Lambda [Escherichia phages T3 and T7 (~10% slippage rate) [The next genes in this module  but alsge rate) ,68. Kuravirus-like group phages remains unclear. In the YF01 phage, the extended MCP isoform is substantially larger (892 a.a) than those seen in many other group members (~500\u2013550 a.a) and most similar in size to that seen in the Paul phage (894 a.a.) . As obseInterestingly, the ultrastructural analysis of the group member, SU10 phage, revealed a capsid structure with apparent uniform MCP formation  . Consistgp12) and an endolysin (gp13), which work in concert to induce host cell lysis at the conclusion of the phage lifecycle  and distal (gp11) tail fibers likely assemble into hexameric long tail fibers, which are attached to the adapter complex [gp15), also attached to the adapter complex, is bound by short tail fibers (gp14) [gp16, extends from the channel formed by the nozzle.The next structural genes in this module encode numerous tail proteins ( complex . The nozs (gp14) . The thrs (gp14) , and maygp18-22) and may be packaged inside the phage capsid along with the genomic DNA and play roles during the infection process gp49) and separately encoded 5\u2032-3\u2032 exonuclease (gp27) and 3\u2032-5\u2032 exonuclease (gp70) subunits. The separation of the DNA polymerase and its exonuclease domains is common to all group members and is similarly observed in phages closely related to the group  and NAD-dependent DNA ligase (gp57). The YF01 phage, like many phages, encodes certain enzymes involved in nucleotide metabolism and modification. These include a deoxynucleoside monophosphate kinase (gp28), a thymidylate synthase (gp60), and a deoxycytidine triphosphate deaminase (gp68).The genome of the YF01 phage contains an assortment of genes involved in DNA replication, repair, and nucleotide metabolism. These include a DNA polymerase (gp30) and a small transcriptional regular (gp76), which are highly conserved across the phage , middle genes located in the middle of the phage genome corresponding to DNA replication genes (gp23-81 in the YF01 phage), and late genes located at the left of the phage genome corresponding to the virion morphogenesis and lysis genes (gp1-22 in the YF01 phage). In the phiEco32 phage, RNA polymerase holoenzymes assembled with the phage-encoded sigma factor (gp30 in the YF01 phage) were shown to drive expression of some middle genes and all late genes  [gp76 in the YF01 phage) appeared to have a dual function in the phiEco32 phage by (1) promoting the expression of the phage sigma factor and (2) shutting off early gene expression via physical interaction with host \u03c370-factor, essentially promoting the progression of the phage lifecycle to late stage genes [Within the YF01 phage genome, we also identified genes encoding an RNA polymerase sigma factor (n cycle) . The trage genes .E. coli strain (K12), AGA was the second least used of five possible Arg codons, with a usage share of approximately 3.6%. This represents a ~5.6 fold increase in AGA codon usage in the YF01 phage over the host  revealed a core proteome of 63 proteins, which were shared amongst all member phages (53% of the total proteome in the case of YF01 phage; Finally, we wanted to determine the core protein-encoding genome of the Kuravirus genus from six to thirty-six member phages using a combination of modern bioinformatics approaches\u2014here grouped into four genera, namely Kuravirus and the three newly proposed genera, Vellorevirus, Jinjuvirus, and Yesanvirus. We also described a new member of this group, the Escherichia phage YF01, isolated from activated sludge in Yokohama, Japan. Genomic analyses described the organization of YF01 phage and characterized the function of encoded proteins involved in phage DNA replication and metabolism, virion morphogenesis, host cell lysis, and transcriptional regulation. Lastly, we determined that sixty-three proteins form the core proteome of Kuravirus-like group phages, with five proteins, including the large terminase subunit and DNA primase/helicase, as some of the strongest phylogenetic markers of Kuravirus-like phages.This study describes the expansion and reshaping of the members from the current ICTV-classified"}
{"text": "Bacteriophages (phages) are selective viral predators of bacteria. Abundant and ubiquitous in nature, phages can be used to treat bacterial infections (phage therapy), including refractory infections and those resistant to antibiotics. However, despite an abundance of anecdotal evidence of efficacy, significant hurdles remain before routine implementation of phage therapy into medical practice, including a dearth of robust clinical trial data. Phage\u2013bacterium interactions are complex and diverse, characterized by co\u2010evolution trajectories that are significantly influenced by the environments in which they occur . An understanding of the molecular mechanisms underpinning these dynamics is essential for successful clinical translation. This review aims to cover key aspects of bacterium\u2013phage interactions that affect bacterial killing by describing the most relevant published literature and detailing the current knowledge gaps most likely to influence therapeutic success. This comprehensive review discusses bacterium\u2013phage interactions, antimicrobial potential of phages and research challenges that impact realization of the successful bacteriophage therapy. BacteriophagesBacteriophages, or phages, are viruses that specifically and selectively infect bacteriaBiofilmSurface\u2010attached, structured community of microorganisms embedded in a self\u2010produced extracellular matrix EnzybioticsPhage\u2010derived antibacterial enzymes with therapeutic potential. Depolymerases catalyse the hydrolysis of the capsule polysaccharide of Gram\u2010negative bacteria, while lysins  are hydrolytic enzymes capable of cleaving the cell wall (peptidoglycan) of both Gram\u2010negative and Gram\u2010positive speciesL\u2010formsCell wall\u2010deficient bacteria resistant to supra\u2010therapeutic concentrations of cell wall targeting antibioticsLysogenic conversionPhage\u2013bacterium interaction in which a prophage encodes proteins that enhance bacterial fitness or virulenceLysogeny or lysogenic cyclePhage life cycle in which the viral genome stably integrates in the bacterial chromosome, replicating with itLytic infection and productive lysisInfecting phages replicate their genome and assemble new viral particles (virions) by hijacking host resources. Phage\u2010directed cell lysis then releases this viral progeny ready to infect new cells, in an exponential growth cycle (productive lysis) limited only by availability of bacterial prey and their response/s to phage attackObligate lytic phagesPhages that cannot undergo lysogeny. Preferred for therapeutic applicationsPhage adsorptionMolecular interactions between phage proteins and specific bacterial receptors that bind the phage to the bacterial cell surface allowing for infection (phage genome release into the cytosol) to occurPhage cocktailCombination of multiple phages for therapeutic application. Phages in a cocktail ideally act synergistically against a bacterial target and limit the development of phage\u2010resistant variants. Cocktails combining phages with different host specificity allow for broader therapeutic targetingPhage therapyMedical use of phages as antimicrobials for treatment of bacterial infectionsPseudolysogenyPhage\u2013bacterium interaction in which the phage genome resides within the host cell without chromosomal integration, in an unstable, inactive stateTemperate phagesPhages capable of undergoing lysogeny. These may lie \u201cdormant\u201d within a living bacterial cell while integrated into the host chromosome as \u201cprophages\u201d, but have the potential to enter a lytic infection cycle under certain conditions (e.g. host cell stress). Temperate phages are less preferred for therapyTransductionPhage\u2010mediated horizontal transmission of genetic information from one bacterial cell to another, as opposed to genetic inheritance through reproduction . Mainly associated with the lysogenic life cycleth century saw an unprecedented steady decline in mortality attributable to bacterial infections ], and immediately recognized their potential as antimicrobial agents  of antibiotics has since led to the emergence of globally disseminated bacterial pathogens that are resistant to last\u2010line treatments, and antibiotic resistance now poses a significant global health and economic burden , phages have been in routine medical practice for over 70\u2009years and this experience provides a rich source of empirical data  constitute the largest group described to date  within minutes of infection  is pragmatically addressed by the use of combinations of multiple phages (cocktails) with differing adaptive strategies . StableInoviridae    Fig\u2009  and the absence of classic lysogeny genes , but there is no established genetic marker of pseudolysogenic capacity, as it is not usually a feature of exponentially growing bacteria. Replication of obligate lytic T4\u2010like phages is completely inhibited in nutrient\u2010stressed E.\u2009coli, but it has been reported that under the same conditions, a T4rI mutant (defective in the function of the holin inhibitor) keeps producing viable virions , fully resuming in the rest of the population only upon\u2009nutrient addition with restoration of logarithmic growth. P.\u2009aeruginosa and Yersinia enterocolitica can support pseudolysogenic infection with apparently obligate lytic myoviruses or podoviruses that provide bacteria with immunity from further phage\u2009infection (superinfection exclusion)  physical protection from harsh environmental conditions outside the host   antibiotics with important clinical implications  and host control of the administered therapeutic virus by innate and acquired immune responses  is the term used to indicate the ratio of phages to bacteria in esponses . The phas et\u00a0al, . Carefuls et\u00a0al, .et\u00a0al, et\u00a0al, Phage adsorption to the bacterial cell is a first and crucial step in the\u2009infection process  also play a unique role in controlling phages as they can effectively trap them, preventing host infection   actually able to succeed in nature , resulting in host range expansion , phage type (even very closely related phages can give different outcomes), and timing of administration  , the testing conditions , l, et\u00a0al  presenteet\u00a0al, et\u00a0al, in\u2009vivo can be problematic . Given the ubiquity of phages in nature and the aeons of co\u2010evolution with bacteria, an array of finely tuned and well\u2010established defences against phage attack are also to be expected. The physiological state of the bacterial host population is an important determinant of phage replication , and theet\u00a0al, et\u00a0al, et\u00a0al, et\u00a0al, et\u00a0al, The impact of bacterial stress on the lytic/pseudolysogenic pathways may be therapeutically important. Phages that ordinarily pseudolysogenize stressed bacteria , both Gram\u2010positive and Gram\u2010negative bacteria live in complex sessile biofilm communities  phage preparations may induce the expression of key anti\u2010inflammatory genes, including IL\u20101RA and IL\u201010 family cytokines  in T4\u2010like phages , which can impact phage bioavailability Phages that have evolved to protect their prey populations by down\u2010regulating the host immune response may prove to be difficult choices in therapy. Conversely, phage\u2010mediated immunomodulation may be a good therapeutic trade\u2010off in severe sepsis where attenuation of a lethal cytokine\u2010mediated inflammatory response may be the most important therapeutic goal.In this review, we sought to highlight the main areas of phage and bacterial biology that may directly relate to therapeutic outcome and in need of further investigation Table\u2009.et\u00a0al, However, bringing phages into the pharmacopoeia requires attention to several other areas that we have not fully discussed. The limited host range of most therapeutic phages means that this precision therapy needs well\u2010curated and accessible phage sources, which is a biobanking and information management challenge  \u2014https://phage.directory/Phage Directory \u2014P.H.A.G.E. vzw \u2010 Home (p\u2010h\u2010a\u2010g\u2010e.org)Phages for Human Applications Group Europe \u2014https://phageaustralia.org/Phage Australia \u2014http://www.yalephagecenter.com/Center for Phage Biology and Therapy at Yale \u2014Center for Innovative Phage Applications and Therapeutics (ucsd.edu)Centre on Innovative Phage Applications and Therapeutics (first dedicated phage therapy centre in North America)\u2014Online links to relevant sources"}
{"text": "Validated methods for phage selection, host range expansion, and lytic activity determination are indispensable for maximizing phage therapy outcomes. In this review, we describe some relevant methods, highlighting their advantages and disadvantages, and categorize them as preliminary or confirmatory methods where appropriate. Experimental conditions, such as the composition and consistency of culture media, have an impact on bacterial growth and, consequently, phage propagation and the selection of phage-resistant mutants. The phages require different experimental conditions to be tested to fully reveal their characteristics and phage therapy potential in view of their future use in therapy. Phage lytic activity or virulence should be considered as a result of the phage, its host, and intracellular/environmental factors, including the ability of a phage to recognize receptors on the bacterial cell surface. In vitro quantitative and qualitative measurements of phage characteristics, further validated by in vivo experiments, could be incorporated into one system or mathematical model/formula, which could predict a potential successful outcome of clinical applications. A century ago, bacteriophages (phages) were defined as \u201cdevourers of bacteria\u201d  or \u201cobliPhages are the most abundant and diverse life-like entities on Earth, where they are found in almost all ecospheres, such as seas, rivers, and soil, and within other organisms, including humans. They control the abundance of their bacterial hosts and, as such, also impact global energy and nutrient cycles . Phages virulentus, meaning \u201cfull of poison\u201d. They are used to indicate the relative capacity of a \u201cmicrobe\u201d  to cause disease [The words \u201cvirulence\u201d and \u201cvirulent\u201d come from the Latin word  disease , or in c disease ,13. Lyti disease . Gill et disease . Phage v disease , and may disease . Sometim disease .In fact, virulence is not a distinct phage characteristic, but a complex, dynamic, and variable phenomenon that includes both phage and bacterial factors . Indeed,Pseudomonas aeruginosa than either one acting alone [Nowadays, experts increasingly agree that phages will not replace antibiotics , and coung alone . Phages ng alone  one of tng alone , and preng alone , are of ng alone ,26,27 toMycobacterium smegmatis \u201csurrogate\u201d strain for the propagation of M. tuberculosis phages [Today\u2019s laboratory facilities and materials are more developed than those in F\u00e9lix d\u2019H\u00e9relle\u2019s time. Glass tubes and Pasteur pipettes, for instance, are replaced with Eppendorf tubes or 96-well microtiter plates and multichannel micropipettes. Notwithstanding the modernization of laboratory equipment, there are no significant differences in the techniques used for phage isolation and propagation, the development of phage cocktails, nor the  production of therapeutic phage preparations. In 1930, d\u2019H\u00e9relle recognized that the most effective therapeutic phages could be isolated from patients that had recovered from infection. He also claimed that more than 50 bacterial strains should be used in phage isolation and enrichment methods . Interess phages  to impros phages . Notwiths phages ,32 to des phages  or perfos phages , for inss phages .This review brings together relevant methods for phage isolation, detection, characterization, and selection, including phage activity determination, host range evaluation and expansion, and the translation of in vitro results to clinical practice. We will mostly focus on the practical side of these methods , including some inputs and interpretations based on our personal experiences, as well as the advantages and disadvantages of the methods with regard to developing more standardized approaches.In this section, we will discuss a number of methods that are commonly used for in vitro phage lytic activity determination, including phage detection and enumeration testing and the in vivo translation of results (Diagram 1).Bacteria hooks with hosts covering the wide range of receptors needed to hook the largest variety of potential phages. This requires having a readily available panel of strains with known genetic profiles. Every newly isolated phage can be further studied, e.g., to determine its biology;Bacteria hooks of particular interest can be included. In this case, bacterial strains are selected based on specific features such as antibiotic resistance, and it is not necessary to have an exhaustive list of characteristics or to know their genomic profile. The strains could be objects of further scientific study.Bacterialstrains used for the \u201cfishing\u201d or detection of new phages are referred to here as \u201cbacteria hooks\u201d. For the isolation of potentially new phages, the well-known \u201cphage enrichment\u201d (PE) method is used. It was first developed by Winogradsky and Beijerinck  and lateTwo times  or ten tIt is preferable not to centrifuge/filter the sampling source, unless it contains large contaminants and/or components that will interfere with the incubation process. It is assumed that conditions close to those in the natural source environment will facilitate phage/bacteria interactions and the isolation of phages;Using lower temperatures (25\u201328 \u00b0C) than those routinely used in clinical microbiology (30\u201337 \u00b0C) ,36 and lUsing 96- or 384-well microtiter plates for the incubation of a large number of inoculums of bacteria hooks is more convenient. The bacterial suspensions are collected from each well using a multichannel pipette ;v/v) of chloroform to PL at +4 \u00b0C (temperature shock) kills the remaining intact bacterial cells, including lyrically phage-infected bacteria, and could thus result in substantially increased phage titers [After incubation, the potential phage lysate (PL) is centrifuged and filtered. There is no necessity for the use of chloroform, as this could reduce the viruses\u2019 infectivity  or inacte titers . Chlorofe titers . In addiThe obtained PL could be used further as the second source for another enrichment BP with different bacteria hooks.Bacteria hooks consisting of working host strains, i.e., strains that have already been adapted/approved for phage propagation/production, speed up downstream phage adaptation/training procedures. Newly isolated phages could, of course, also be propagated and trained in other bacterial strains than the ones used for isolation. A scaled-up version of the PE approach is described by Olsen et al. as part of a high-throughput screening (HiTS) method for phages. They propose using 96-deep-well plates, which allows for the simultaneous handling of a large range of environmental samples (water). One single host is used in each well containing 1.5 mL of water sample, and the method is oriented towards predominantly lytic and easily cultivable phages . An outlIt is considered a disadvantage of the PE method that faster-growing phages will outcompete phages with slower-growing populations , maskingGenerally, the PE lysate is first tested against the bacteria hooks used in the PE method, but it could also be carried out using any other relevant BP, for instance, containing strains from available bacterial culture collections . Differe(i)The \u201cdirect spot test\u201d : in which only one dilution of the phage lysate is spotted on bacteria grown directly on solid agar. It is described below;(ii)The \u201cspot test\u201d  (we will(iii)The \u201clysis profile assay\u201d  or, as wSeveral parallel streaks  of bBacterial suspensions are simply spotted  in a griBacterial suspensions are directly streaked on streaks of phages made on solid agar  we call.In the \u201cdirect spot test\u201d, bacteria can be grown either as a series of distinct areas (streaks or spots) or as complete lawns on solid agar (without soft agar overlay). Phage lysates are applied in the areas of expected bacterial growth. Bacteria are commonly applied in three ways:The first two preliminary phage detection approaches allow for the screening of large numbers of BPs and phages. The choice between either of them is a matter of practicality.It is considered that the \u201cspot-on-streak\u201d assay (a variation of the \u201cdirect spot test\u201d) does not allow for the evaluation of a possible emergence of bacterial phage resistance . In factAll the previously mentioned methods should be considered as preliminary detection techniques, as they are merely revealing bacterial lysis on agar or in liquid and do not confirm that these are the result of phage activity.4 colony-forming units (cfu)/mL, which will result in sufficient growth to reveal the activity of phages that are present at a low concentration. The \u201cspot-on-streak\u201d assay allows for the application of multiple phage lysates on multiple bacterial strains, at different dilutions, on one plate. Note that bacteria grow slower on a solid agar surface than in broth, which will help phages that are present at lower titers, or with slower reproduction rates, to pace the bacterial growth and reveal themselves. Moreover, when large-size phage virions cannot diffuse [As PE lysates potentially consist of different phage variants at different concentrations, possibly including rare and interesting variants at low titers, it is reasonable to continue evaluating the PE lysates without diluting them. Bacterial suspensions used in the above-mentioned methods should have a minimum concentration of 10 diffuse  in soft  diffuse , thus in diffuse .Pipetting robots could be used for the \u201cspot-testing\u201d-based methods. A rectangular-shaped tray-plate, from SPL life science, for instance, is perfect to perform the spotting and could be fixed on the pipetting robot workstation. The advantage of that plate is that it has nearly the same dimensions (127.94 \u00d7 85.50 mm) as a 96-well microtiter plate (127.71 \u00d7 85.43 mm), which can be used as a reservoir for the phages that will be spotted. The spotting height should be adjusted correctly to avoid piercing the agar surface or splashing the drop while spotting, and thus generating aerosols and subsequent cross-contamination. In case of the \u201cspot-on-streak\u201d assay, bacterial streaks are pre-prepared, while the \u201cspot-on-spot\u201d method could be performed entirely by the pipetting robot.After visual examination of the lysis zones and interpretation of the preliminary results, several phage/host bacteria combinations are selected to be further submitted to confirmatory methods that are able to reveal true phage plaque formation.Plaque formation is the result of multiple rounds of infection, lysis, and release of progeny , and it While a variety, or the technical modification, of methods are used for plaque formation and enumeration, double agar layer (DAL) methods are the most commonly used.Confirmation of plaque formation;Study of plaque morphology;Enumeration (determination of pfu/mL) of phages.The main reasons for using plaque formation assays are:Phage differentiation/selection;Plaque purification;Phage virulence/lysogeny evaluation procedures.The morphological appearance of the individual plaques is the first parameter that needs to be determined, as it is of great importance for:Plaque diameter;Level of transparency/turbidity of the plaques;Halo formation and size;Motility.The DAL method was independently developed by the Belgian microbiologist Andr\u00e9 Gratia in 1936 (\u201cDes relations num\u00e9riques entre bact\u00e9ries lysog\u00e8nes et particules de bact\u00e9riophage\u201d), and by Hershey, Kalmanson, and Bronfenbrenner in 1943 , to be fAnother phage enumeration method described by Kutter et al. , Kropinski, and Mazzocco et al. (\u201cDrop Plaque Assay\u201d) ,52,53 isThe disadvantage of the MD/SP DAL method is that it is not precise enough. For more accurate counting and a perfect comparison of the plaque sizes and morphologies on each strain , the SD/In addition, some studies have shown that particular phages only reveal clear lysis in the first two dilution spots, with no sign of lytic activity in further dilution spots. The reason for this could be an abortive infection, or \u201clysis from without\u201d , or someFor plaque differentiation and purification, the most commonly used and described method uses phage streaks  on a bacThe distance between the plaques (well isolated discrete plaques);Different dilutions of phage lysate are applied;A certain number of passaging rounds are performed ;Several bacterial host bacterial strains are used;Several growth media are used.To validate the plaque purification method as a confirmatory method, the following criteria need to be considered:For practical convenience, mini petri dishes of 35 mm diameter can be used for plaque formation/passaging assays. It is highly recommended to perform a valid plaque purification procedure before moving on to further characterization and activity evaluation.Bacterial strains for phage host range studies are referred to here as \u201cbacteria kits\u201d. MD/SP DAL is mostly used for the evaluation of EOP . TherefoThe concept of host range or breadth  can be dhttps://www.atcc.org/microbe-products#t=productTab&numberOfResults=24 (accessed on 30 March 2022)) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen  [FDA guidance on antibiotic testing requires the testing of at least 100 bacterial strains, and for some species more than 300 strains, with recent clinical isolates accounting for at least 75% of the strains . Followich 2022)  and inclAt the same time, according to the FDA and some other regulatory bodies for diagnostics , preliminary and confirmatory tests are the main components of systematic qualitative analysis, and this kind of approach needs to be tailored to phage identification, enumeration, and activity evaluation.The phage liquid culturing (PLC) method is considered an alternative approach for phage host range and lytic activity measurement . In addiThe PLC method, or \u201cAppelmans\u2019 method\u201d, was developed in the 1920s by the Belgian surgeon Ren\u00e9 Appelmans ,62. InitPhage enumeration with phage titer expressed as a dilution factor;Estimation of the multiplicity of infection (MOI) , i.e., tEvaluation of host range and lytic activity ;Expansion of host range after multiple passaging.The Appelmans\u2019 technique can be used for different purposes:Nowadays, the PLC, or Appelmans\u2019 method, is mostly used and described for the study of phage host range and lytic activity in view of translation to the in vivo context. Different interfering/misleading factors may arise when using this method, such as the growth of phage-mutants , the \u201crePhage-exposed bacterial growth curves have been extensively studied ,70,71,72P. aeruginosa cystic fibrosis bacterial isolates lead to increased pathogen clearance and a lowered resistance evolution as well [Today, the experimental evolution of individual phages or phage mixtures through serial interactions with one or a mixture of host bacteria is the most used approach to extend the phage host range. Several studies performed in the period 1963\u20131991 describe the benefit of serial passage experiments (SPEs) that allow for molecular and phenotypic evolution in real time . The cha as well .K. pneumoniae [Eastern European researchers, particularly in the Republic of Georgia, used the noted Appelmans\u2019 dilutions method  for passeumoniae .Burrowes et al. designed a 96-well plate formatted for Appelmans\u2019 protocol to analyze the individual phages after every 10 rounds of evolution. They showed that starting with a phage cocktail resulted in a larger host-range expansion than when using individual phages, and based on genomic analysis, they observed a recombinatorial origin for output phages with a broadened host-range .The crucial factors for ensuring a rapid host range extension of phages are (1) the use of a phage mixture from the start, which allows recombination to generate sufficient diversity, and (2) the use of both the original bacterial hosts that had been used for phage propagation and an updated collection of clinical bacterial isolates that are resistant to the given phages, as it is important to produce therapeutically useful phages .While Burrowes et al. state that the Appelmans protocol works predominantly via recombination between phages ,85, MapeSerial passaging for HRE can be performed on agar as well, whenever liquid media are not adequate for demonstration of phage lytic activity. The agar method is more time consuming than the liquid method, but it has the advantage that the obtained phage mixture no longer needs to be processed further for plaque formation .Many studies refer to existing gaps in standardization and validation of assays/methods documenting phage activity and in the translation of their results to in vivo applications ,32,34,87Methods determining phage lytic activity  are baseAs we mentioned earlier, the \u201cdirect spot test\u201d and \u201cspot test\u201d methods should only be considered as preliminary  phage detection (sensitivity) tests, as they are not demonstrating plaque formation, while the MD/SP DAL method can be considered as a confirmation test for phage detection as it demonstrates plaque formation. Since the SD/MP DAL method allows for the most precise phage enumeration and plaque morphology characterization, it could be considered as a confirmatory method for phage enumeration and plaque morphology characterization. Note that the MD/SP DAL method is less time-, material-, and labor-consuming as it allows for the analysis of several phages or phage dilutions on one plate. It would be relevant for host range determination and EOP evaluation. We will not provide a detailed discussion of phage culture purification here, as it is beyond the scope of the present review and would deserve a dedicated paper. However, to ensure that a particular phage lysate (newly isolated or evolved) is a single phage particle product and authentic, it first must go through plaque and then culture purification steps. For adequate phage purification, five or more passages should be performed using the \u201cphage T-streaking\u201d method as a preliminary approach, followed by five or more passaging steps using the SD/MP DAL method, as this method allows for full morphological selection and characterization of phage plaques. Once a particular phage is purified (plaque and culture) using an established and validated procedure, the candidate phage can be submitted to further characterization.The PLC method has also been put forward as an alternative approach for phage host range measurement . It is fTM system provides a high-throughput capability (4800 phage assays) [To analyze phage/bacterium population interaction dynamics in a comprehensive manner, it is advised not to use OD measurement, but to measure the conversion of water-soluble tetrazolium salts, which yields a higher sensitivity and dynamic range. For this, the OmniLog assays)  for the The main reason for attempting to standardize phage lytic activity measurements and make them as effective as possible is to be able to correlate phage in vitro traits  with theThe phage liquid culturing (PLC) method is put forward as the best assay to evaluate phage lytic activity ,17,78, iSome relevant phage infection parameters, such as adsorption rate, latent period, and burst size, can be deduced from monitoring phage growth in liquid media . Phage iEvery phage candidate with the potential to be used in the therapy\u2014be it naturally isolated, with or without expanded activity or host range, genetically engineered or not, or used within a \u2018one-size fits all\u2019 or broad-spectrum approach \u2014should iTherefore, the question remains as to what should be considered and tested to determine a phage\u2019s potential to reduce the bacterial population at different infection loci.S. aureus rarely expresses its capsular polysaccharides, which are typical for clinical isolates, when they are grown in the laboratory [P. aeruginosa possesses an arsenal of virulence factors enabling it to invade host cells and circumvent host defenses [Certain bacterial determinants are critical for the outcome of phage/bacteria interactions. In vitro and in vivo phage/mixture testing is most often performed using homogeneous bacterial populations grown either on agar, as planktonic cells in liquid culture, or in biofilms. However, the bacterial composition of the infection loci to be treated with phages  usually consists of an assembly of different strains belonging the same or different bacterial species  and exhiboratory . P. aerudefenses , which adefenses , body maThe right phage-bacteria ratio or so-called MOI to achieve complete bacterial lysis over a given period of time in a liquid culture should be determined ,51. The The optimal phage-bacteria ratio is correlated with phage = bacteria growth rates, and the balanced combination of phage-bacteria is the main determinant for the successful reduction/delay of the emergence of phage-resistant bacterial mutants. This optimal phage/bacteria ratio can be used in phage-virulence assays or in vitro and animal models.An appropriate phage mixture or cocktail  is belieFinally, we can conclude that, to date, no validation procedure/format has been developed nor approved by the relevant regulatory authorities for the evaluation, categorization/ranking (preliminary or confirmatory), or documentation of the methods used to assess in vitro and in vivo phage activity in a standardized manner. All of the methods commonly shared and used so far are copied, developed, or modified from manuals and scientific papers, mostly dating from d\u2019H\u00e9relle\u2019s time.The level of phage virulence as a whole (phage therapy capacity)\u2014host detection, host range, phage-bacteria growth rate, phage-bacterial interaction , phage survival/sustainability, adaption to the host, and invading ability\u2014is associated with conditional factors such as patient age and physiology , concentration of bacteria, temperature, and pH at the infection site. Thus, as phage-bacterial interactions are continuously evolving, so is phage virulence. Phage virulence capacity could be enhanced in vitro by implementing a good understanding of phage-bacterial interactions under certain specific conditions (resembling those at the infection loci). In vitro evaluation of phage activity, using standardized and integrated criteria, is bound to provide a valuable support for in vivo applications. Every selected method should be rational, reliable and appropriate in a particular situation, feasible, and cost effective, considering timelines, labor, and material consumption."}
{"text": "Anemia is a major health problem in Saudi Arabia and has multiple etiologies. Many studies have been conducted in Saudi Arabia in specific population groups like school children, adolescents, university students, and females in the reproductive age group, and most have reported high prevalence of anemia. This study was conducted in a specialist hospital in Makkah city and includes all outpatients aged 15\u2009years and above.  To study the burden of anemia among hospital attendees, its stratification based on gender and age, and its severity along with the morphological types of anemia.  This is a study conducted at a specialist hospital in Makkah city and one-month data were collected retrospectively from the laboratory database and include demographic and routine hematological results of complete blood count (CBC).  A total of 21,524 patients were included, out of which 9444 (43.9%) were males and 12020 (56.1%) were females. The overall prevalence of anemia was 38.7% (8339). Prevalence was very high in females, accounting for 68.2% (5689), whereas it was 31.8% (2650) in males. There were 39.6% (3301), 43.9% (3657), and 16.6% (1381) cases of mild, moderate, and severe anemia, respectively. In females, anemia was more prevalent in the age group of 15 to 49, which is considered as the reproductive age group. Microcytic anemia was the most prevalent type observed in this age group, accounting for 40.7% of all anemia cases. Normocytic anemia was more prevalent in the males, accounting for 52%.  Our study showed high prevalence of anemia among the patients attending outpatient departments in a specialist hospital. Females have high prevalence of anemia when compared to male population. Microcytic anemia was the most common anemia type among females and was seen in the 15\u201349 age group. There is an increase in prevalence of anemia with age for males, whereas, in females, increased prevalence is observed in the reproductive age groups and the anemia prevalence maintained a steady decrease towards the 5th to the 9th decades. Normocytic anemia was more prevalent in the 5th to the 9th decades, indicating that there are more etiologies other than iron deficiency in the causation of anemia. Macrocytic anemia was the least reported anemia type. Anemia of mild and moderate severity was predominant in both genders, although severe anemia showed higher prevalence in females as compared to males. Conclusion. Anemia is highly prevalent in adolescents, adults, and the elderly in Makkah region. The most common cause is thought to be iron deficiency, although other causes are not uncommon. The authorities need to address the problem of prevention and reduction in anemia prevalence by taking effective measures and interventions. Our study showed high prevalence of anemia among the patients attending outpatient departments in a specialist hospital. Females have high prevalence of anemia when compared to male population. Microcytic anemia was the most common anemia type among females and was seen in the 15\u201349 age group. There is an increase in prevalence of anemia with age for males, whereas, in females, increased prevalence is observed in the reproductive age groups and the anemia prevalence maintained a steady decrease towards the 5th to the 9th decades. Normocytic anemia was more prevalent in the 5th to the 9th decades, indicating that there are more etiologies other than iron deficiency in the causation of anemia. Macrocytic anemia was the least reported anemia type. Anemia of mild and moderate severity was predominant in both genders, although severe anemia showed higher prevalence in females as compared to males.  Anemia is a global health issue affecting a quarter of the global population . It affeWHO has reported global prevalence of 30.2% among nonpregnant women (15\u201349.99 years) and about 33% in Asia and 44.4% in Africa. Among men (aged 15\u201359.99 years) and the elderly (\u226560 years), the global anemia prevalence is 12.7% and 23.9%, respectively [Anemia is a common problem in low- and middle-income countries, particularly among adolescent females, women of reproductive age, pregnant women, and children. Anemia is expected to be reduced in women of reproductive age (15\u201349 years) by 50 percent by 2025, according to the second of the world's six global nutrition objectives. Given the fact that anemia affects half a billion women of reproductive age around the world, eliminating anemia is essential for the health as well as their economic production. According to the Global Health Observatory, anemia prevalence among women of reproductive age ranged from 9.1 percent in Australia to 69.6 percent in Yemen in 2016 .According to reports, anemia is a significant health burden in the Gulf countries, with a high frequency of anemia in females between the ages of 17 and 24 years, as well as males, being reported [According to the findings of a study conducted on Saudi women aged 15\u201349 years, anemia was found in 40% of the participants . Many stThis study was done using the patient's data collected retrospectively for one month from a specialist hospital in Makkah city. The data were collected from the laboratory database and include demographic and routine hematological results of complete blood count (CBC) performed on a fully automated hematology analyzer. Hematological data from all patients aged 15 years and above and attending the routine outpatient departments over a period of one month from January 1, 2019, to January 31, 2019, were included for analysis.The hemoglobin cut-off (g/L) for the diagnosis of anemia and its categorization based on severity into mild, moderate, and severe anemia was done as per WHO recommendation as shown in P value <0.05 was considered statistically significant. Continuous variables were expressed as mean\u2009\u00b1\u2009standard deviation and categorical data were presented as median and interquartile range (IQR). Odds ratios (ORs) and 95% confidence intervals were obtained by logistic regression to determine the impact of age on anemia.Statistical Analysis was done using IBM SPSS Statistic . The specialist hospital lab receives between 400 and 500 blood samples for routine hematological tests every day from patients attending the outpatient clinics. A total of 21,524 patients were included, out of which 9444 43.87%) were males and 12020 (56.2%) were females . The mea.87% wereP < 0.001), 1.011 , and 1.013 , respectively. The OR for microcytic anemia was 0.984  and for macrocytic anemia it was 1.014 .All the anemia cases were categorized into microcytic, normocytic, and macrocytic anemia based on the reference cut-off for mean corpuscular volume (MCV). Microcytic anemia was the most prevalent type among females, whereas normocytic anemia was more prevalent in the males, accounting for 52% of the anemia cases in males Tables  and 5. TThis study was done using the patient's data collected retrospectively for one month from a specialist hospital in Makkah city. In this study, we attempted to analyze the anemia prevalence among the outpatient attendees in various speciality departments. Anemia is a complex disease with nutritional and nonnutritional variables and mechanisms at play. Saudi Arabia, along with the Gulf Arab countries and others, is located in the eastern Mediterranean. In the eastern Mediterranean region, anemia prevalence ranges from 22.6 percent to 63 percent among pregnant women and is 69.6 percent among women of reproductive age . Anemia Owaidah et al.  describeNumerous risk factors for the development of IDA in Saudi women of reproductive age have been identified . DietaryThe cause of anemia in the senior population must be determined, and fresh studies must be conducted to investigate it. Consumption of tea, which contains a high concentration of polyphenols, has been linked to anemia in the elderly. Polyphenols block nonheme iron absorption. Tea drinking is a widespread ritual in Saudi Arabia and is typically drunk before and after meals , 22. AccThe World Health Assembly (WHA) has designated anemia reduction as a global dietary priority for 2025 . There iOur study has some limitations. We included patients who visited a hospital, which may have inflated the numbers, but we excluded hospitalized patients. However, our findings are consistent with those of other research conducted in Saudi Arabia.Our study showed high prevalence of anemia among the patients attending outpatient departments in a specialist hospital. Females have high prevalence of anemia when compared to male population. Microcytic anemia was the most common anemia type among females and was prevalent in the 15\u201349 age group considered as the reproductive age group, whereas normocytic anemia was more common among the male gender. Anemia of mild and moderate severity was predominant in both genders, although severe anemia showed higher prevalence in females as compared to males. The authorities need to address the problem of prevention and reduction in anemia prevalence in Saudi Arabia by taking effective measures and interventions."}
{"text": "Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification.Nontuberculous Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid.No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these.Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections.Phage treatment of  Mycobacterium infections. We observed no adverse reactions, favorable outcomes in at least 50% of patients, no evidence of phage resistance, and neutralizing immune reactions that do not correlate with treatment success.We describe 20 consecutive cases of bacteriophage treatment of  The therapeutic use of phages for treating drug-resistant bacterial infections has received recent attention, but the types of infections and pathogens deemed suitable; routes, dosage, and frequency of administration; interactions with antibiotics; and pharmacokinetics remain unclear , 2. UnliMycobacterium pathogens, alternative therapies are needed [Mycobacterium abscessus\u2014are particularly challenging, as many are refractory to antibiotics and extended drug therapies are poorly tolerated [M. abscessus infections are particularly challenging and typically preclude lung transplantation. Phages have been proposed for managing CF [Mycobacterium infections, including NTM and tuberculosis [Because of increasing and widespread antibiotic resistance among e needed . Nontubeolerated . NTM infolerated , 13. Peoaging CF , but therculosis , 16, remM. abscessus clinical isolates [M. abscessus isolates have a smooth colony morphotype [Mycobacterium smegmatis; few phages have been isolated directly on any strain of M. abscessus [There is great variation in phage susceptibilities among isolates . Approxirphotype , 18, andrphotype . In contrphotype . Nonethebscessus .M. abscessus infection following bilateral lung transplantation and drug-induced immunosuppression [M. abscessus pulmonary infection [9 plaque-forming units (PFUs) per dose for at least 6 months. Both were also treated with concomitant multidrug antibiotic regimens. The first patient had clinical improvement in lung function, radiographic imaging, and clinical signs and symptoms, although without complete clearance of the infection [M. abscessus colony counts in sputum that was abrogated by emergence of a potent neutralizing antibody response to the phages [Two case reports have described compassionate use of phages for NTM infections , 19. Onepression . The secnfection . The secWe report here therapeutic interventions for a pilot cohort of 20 patients with antibiotic-refractory mycobacterial infections. Phage administration was safe, no resistance was observed, and favorable microbiological or clinical outcomes were observed in a majority of cases.M. abscessus infections, but some had other underlying diseases complicated by NTM  infections. The following criteria determined eligibility for compassionate use phage therapy: age >5 years, microbiologic documentation of mycobacterial infection based on at least 2 positive cultures of relevant tissue or body fluids; drug susceptibility testing (DST) documenting resistance to multiple antimycobacterial drugs; clinical signs, symptoms and radiographic findings involving at least 1 organ system , European Respiratory Society (ERS), European Society of Clinical Microbiology and Infectious Diseases (ESCMID), and Infectious Diseases Society of America (IDSA) diagnostic criteria [Since May 2019, we received approximately 200 requests for adjunctive phage treatment for patients with NTM infections that were either refractory to antibiotic therapies or in which extended drug treatments were not tolerated; antibiotic susceptibilities are shown in 9 PFU intravenously twice daily; some patients also received the same dose by inhalational nebulization  rough colony strains were infected and killed efficiently by at least 1 phage. If an isolate was efficiently infected and killed by 1 or more phage, and the patient\u2019s clinical status indicated eligibility for compassionate use intervention as previously described, regulatory permissions were obtained and purified phages were dispatched to a local dispensing pharmacy. Of the 20 patients offered treatment, 17 had M. abscessus infections; 14 of these had underlying CF, 1 had bronchiectasis, 1 had scleroderma, and 1 had hypersensitivity pneumonitis. One patient had a Mycobacterium chelonae disseminated skin infection, 1 had CF with pulmonary Mycobacterium avium, and 1 had a disseminated BCG infection , 1 had disseminated skin lesions caused by M. chelonae, 1 had scleroderma with M. abscessus infection, and 1 had disseminated BCG infection , 1 with pulmonary M. avium infection (patient 17), and another with M. chelonae skin infection (patient 16); details of patients 15 and 16 were recently reported [M. abscessus cultures and successfully underwent a bilateral lung transplant. Two patients (1 and 13) had substantial clinical improvement, but without clear evidence of culture conversion. One of these (patient 1) was the case reported previously [M. abscessus infection and clinical signs and symptoms greatly improved, but some skin nodules persisted after >1 year of phage treatment. These were only intermittently culture positive, but the patient subsequently died from CF-related health challenges and organ failure 44 months after the start of phage treatment. In the other (patient 13), there was substantial improvement of symptoms and forced expiratory volume in 1 second by spirometry, but the patient remained culture positive.Favorable clinical and microbiological responses were observed in 5 patients  and partial clinical or microbiological responses were noted in 6 patients  ; all wernfection . In 5 pareported , 23. ThrMycobacterium infections. One patient (patient 2) had a severe chest infection requiring sternum resection. Phage treatment resulted in AFB smear-negative chest swabs, but the patient died after failing therapy for multiple bacterial and fungal coinfections. For patient 4, phage treatment resulted in conversion to culture negative tracheal aspirates, but systemic adenovirus infection resulted in death. Patient 7 had M. abscessus infection with both rough and smooth colony morphotypes; the rough strain derived from the smooth strain by a mutation in glycopeptidolipid synthesis [For 4 patients , response to therapy was partial and more difficult to assess largely due to complications from other infections, although there was evidence of improved control of the ynthesis . The phaM. abscessus, and active phage was identified only for the rough strain  had inconclusive responses to therapy or had modest short-lived improvements. Patient 8 developed phage neutralizing antibodies and had little clinical improvement with either aerosolized or IV administered phage, likely due to the phage neutralizing immune response. Like patient 7, patient 11 had a mixed infection with both rough and smooth colony morphotypes of h strain . While clization . PatientM. abscessus infections, showed no overall clinical or microbiologic response , all with pulmonary response . All hadM. smegmatis) for phage growth. Eleven patients were treated with just a single phage (33HTH_HRMGD03), and in the 6 patients from whom rough colony M. abscessus isolates were recovered after the start of phage treatment, all remained fully phage sensitive. Indeed, changes in phage sensitivity were only observed in 1 phage in a cocktail of 3 (Phage administration by either IV or aerosolized routes was well-tolerated with no serious adverse reactions related to the phage in any patient. Phage preparations were highly purified, certified to be sterile, and had undetectable endotoxin levels, an advantage of using a lipopolysaccharide-free bacterial host (Sera from 15 patients were tested for immune reactions, and robust neutralization of at least 1 phage was observed in 8 of the patients following IV treatment . AlthougThis series of 20 patients treated with phages on a compassionate use basis provides support for further evaluation of phages for treatment of mycobacterial infections. Phage administration was well-tolerated, and phage resistance was not observed even when using a single phage. Favorable responses were observed in more than half of the patients, including complete resolution of some infections, and successful lung transplantation in 1 patient. However, some patients saw little clinical benefit, and the basis for this variability in response is unclear. Although phage treatment of mycobacterial infections shows promise, this cohort illustrates some key limitations and lessons.M. abscessus infection was fully resolved, the phages were also deposited bronchoscopically, which may have contributed to more effective phage delivery to the infection. Nebulization may also avoid systemic neutralization. The immune status of the patient is also important; immunocompromised patients may tolerate extended phage administration without antibody-mediated neutralization. However, little is known about intracellular penetration or uptake of phages, particularly by macrophages, where most replicating mycobacteria are found. Third, dosage and regimens warrant optimization. As the treatments are well-tolerated, higher doses could be contemplated, using longer interdose intervals. Further exploration of pharmacodynamics and tissue penetration of phage is critical.First, the repertoire of therapeutically useful phages is small, and expansion requires further phage isolation, developing phages induced from lysogenic strains or using synthetic phages , 24. HowM. abscessus strains, the unpredictable specificity for rough strains, and the limited phage repertoire represent current impediments to broad implementation of phage treatments. However, these limitations are not insurmountable, and these case studies suggest that phage treatments may be valuable tools for clinical control of NTM infections. Successful outcomes for M. chelonae, M. avium, and BCGosis, as well as M. abscessus, suggest a large spectrum of target Mycobacterium diseases.The lack of therapeutically useful phages for smooth Mycobacterium infections and the potential for infection control when none other is effective.Although compassionate use case studies such as these lack the rigor and consistency of treatment and patient monitoring possible in carefully controlled blinded clinical trials, they provide a wealth of insights for designing such trials. Variations in antibiotic regimens, surgical interventions, and management of coinfections can all potentially influence patient status, and direct linkage of phage treatments with outcomes in individual patients is perilous. Nonetheless, this series of case studies strengthens the likelihood of direct benefits from phage treatments of ciac453_Supplementary_DataClick here for additional data file."}
{"text": "Pseudomonas aeruginosa, Acinetobacter sp, and Staphylococci. With investment to develop new antibiotics waning, finding and developing alternative therapeutic strategies to tackle this issue is imperative. One option remerging in popularity is bacteriophage (phage) therapy. This review focuses on Staphylococcus\u00a0aureus and how it has developed resistance to antibiotics. It also discusses the potential of phage therapy in this setting and its appropriateness in high-risk people, such as those with cystic fibrosis, where it typically forms a biofilm.The production and use of antibiotics increased significantly after the Second World War due to their effectiveness against bacterial infections. However, bacterial resistance also emerged and has now become an important global issue. Those most in need are typically high-risk and include individuals who experience burns and other wounds, as well as those with pulmonary infections caused by antibiotic-resistant bacteria, such as  Staphylococcus aureus (S. aureus), has developed drug resistance mechanisms to the currently available antibiotics, including cloxacillin, vancomycin, daptomycin, and others, and is resultantly responsible for over two million infections and over 23,000 deaths in the United States alone each year . Ba. Ba87]. d others . Severalbiofilms ,94,95. Is et al. . Howevers et al. . Moreoveproteins .Collectively, as described in this review, staphylococci readily form antibiotic resistance, whether in free planktonic forms or biofilms. It can adapt over very short periods when a new antibiotic is introduced to develop resistance, which ultimately increases these infections\u2019 economic and healthcare burden. Thus, an alternative treatment regimen that permanently eliminates infection is desperately required. One postulated solution is bacteriophage therapy.The term \u201cphage\u201d is defined as a type of virus that can enter and destroy bacterial cells whose potential was appreciated early in history ,100. TheTheir bactericidal activity begins with the binding of the virion to specific bacterial cell-surface receptors via phage receptor binding proteins (RBP), followed by the adsorption of phage DNA into a bacterium. This then initiates various molecular mechanisms which promote phage propagation and resultant bactericidal activity . BacteriAs natural predators of bacteria, bacteriophages have several innate mechanisms to destroy biofilms . For exaP. aeruginosa, due to its significant clinical impact as an opportunistic pathogen, in cystic fibrosis (CF), burns, pneumonia, and urinary tract infections [5 PFU direct in the ear as a treatment strategy for chronic otitis media caused by P. aeruginosa, patients were observed for up to 42 days\u2019 post treatment, and encouragingly, there was a reduction in infection by over 50% [Klebsiella pneumoniae. Results showed that a single intraperitoneal dose provided 1 h post-bacterial inoculation resulted in 100% recovery [P. aeruginosa with two phages together or in combination with three antibiotics . Significantly, only moderate activity was observed when each phage/antibiotic treatment was conducted singularly. However, synergistic activity was observed when phages and antibiotics were applied concurrently [P. aeruginosa  for activity against P. aeruginosa strains isolated from children with CF. Excitingly, the authors identified E79 as a possible new therapeutic phage candidate for P. aeruginosa lung infections in CF due to its broad antibacterial activity (91% of the tested strains were sensitive) [Most phage/antibiotic studies have focused on fections . In one over 50% . Similarrecovery . Anotherurrently . These furrently . Trend ensitive) .S. aureus are well known for their resistance to high concentrations of antibiotics [Although much attention has focused on treating Gram-negative bacteria with phage, Estrella and colleagues demonstrated that many strains of staphylococci carried bacteriophages. These phages were able to lyse some but not all strains. However, by exposing staphylococci to several phages, a susceptibility pattern could be recognized, and similarities or differences between strains could be determined . Since tibiotics ,127. S. aureus phage (AB-SA01) in detail. S. aureus phage (AB-SA01) is a bacteriophage cocktail produced by AmpliPhi Biosciences Corporation that consists of three naturally occurring, obligately lytic myoviruses related to Staphylococcus phage K belonging to the Herelleviridae family. Importantly, it did not contain any bacterial virulence or antibiotic resistance genes when sequenced. In addition, they confirmed that this phage\u2019s inherent characteristics met the human use criteria and was predicted to remain active against circulating multidrug-resistant S. aureus strains for long enough to be useful under good manufacturing practices. Overall, results offered great promise, with AB-SA01 killing ~95% of S. aureus isolates [S. aureus strains have been shown [S. aureus ventilator-associated pneumonia (VAP), Prazack and colleagues demonstrated reduced mortality in mice treated with and without teicoplanin compared to the placebo [S. aureus strains [S. aureus biofilms [Lehman et al. were one of the first to characterize an isolates . Furtheren shown . Anotheren shown . Importaen shown . Using a placebo . Rahman  strains . Specifibiofilms . Althougbiofilms , they cabiofilms . Neverthbiofilms .S. aureus using the SATA-8505 phage with vancomycin, cefazolin, linezolid, dicloxacillin, and tetracycline, simultaneously or sequentially. Results demonstrated minimal bactericidal activity when both phage and antibiotic were concurrently added, which was significantly increased when phage treatment preceded antibiotics, particularly with vancomycin and cefazolin [Staphylococcal epidermis, a normal skin microflora with phage saGU1, inhibited the growth of phage-resistant S. aureus in vitro [Kumaran et al. also studied the synergistic activity of phage/antibiotic treatment against biofilm-forming efazolin . Subsequefazolin ,94. Anotefazolin . Identifin vitro . HoweverS. aureus anti-biofilm agents for the treatment of staphylococcal infections over the current time period, as shown in clinicaltrials.gov website about bacteriophage therapy since April 2022. Phage therapy (PT) is used in the majority of the cases that are related to biofilms in S. aureus infections. These infections are mainly attributed to medical devices [S. aureus on these devices. Phage therapy is also included in the management of patients with deep-seated infections such as osteomyelitis [S. aureus infections [S. aureus [S. aureus biofilm-related infections resolved with PT except in only two patients [However, with all the foregoing data on the diversity of phage therapy, both in vitro and in vivo, which greatly outweigh any specific concerns, we summarize some important clinical studies that used phages or phage-related  devices ,138, imp devices ,146,147, devices ,148, whemyelitis ,150,151 myelitis , chronicmyelitis , and bramyelitis ) and chrmyelitis . Howevermyelitis . Exebacafections . Both si. aureus ,154,156 . aureus  detected. aureus ,146,156 . aureus ,153,154 . aureus  The use . aureus ,153,154 . aureus ,151,156.. aureus ,155, witpatients ,145, indS. aureus infections, as well as to address knowledge gaps or issues that need to be addressed. As highlighted, phage offers an extremely promising therapeutic alternative to combat staphylococcal infection found in various human diseases, including septicemia, pneumonia-like infection, venous leg ulcers, etc. [S. aureus infections, it is important to highlight that relatively few phages would be needed and combined with currently available antibiotics to have an effective outcome. Furthermore, different phages can be combined into unique cocktails and administered via different routes, i.e., topically, orally, or both, to facilitate effective penetration into the system/tissue [The knowledge gained from work performed in this research space will identify opportunities to translate phage therapy into practice for rs, etc. . Phages m/tissue .S. aureus isolates has been shown to be significantly associated with the type of the S. aureus clonal complex (CC). Phage V1SA20 is only active against CC80 strains, while all phages exhibit poor activity against CC7, CC59, CC239, and CC398 strains [However, several challenges have also been identified. For example, lysogenic phages are commonly considered unsuitable for treatment due to their high potential for horizontal gene transfer; however, some lytic phages show no positive value as antimicrobial agents . Perform strains . However strains .S. aureus PT failed to completely cure chronic polymicrobial biofilm infection of a bone allograft in a sarcoma patient. The authors explained the possibility of incomplete coverage of anti-S. aureus PT against non-staphylococci bacteria [Phage therapy (PT) should also be carefully assessed in patients with chronic infections caused by polymicrobial agents. For example, anti-bacteria . In addibacteria . Howeverbacteria . Althougbacteria  demonstrbacteria . The uptbacteria . This fibacteria . Tolerabbacteria . On a sibacteria .Work currently being performed is also identifying phage-associated treatment options. For example, phages can produce specific enzymes , which are involved in the rapid degradation and destruction of the bacterial cell wall. The promising findings on phage-encoded endolysins from in vitro studies have been extensively reviewed, and their synergy action with antibiotics could give insight into new therapeutic options . This enS. aureus that must be addressed and understood in order to facilitate the development of phage therapy into standard clinical practice. Overall, several points emerge from this review. Firstly, positive interactions that are observed between phages and antibiotics provide a strong rationale that phage/antibiotic combinations can be successfully used against many multidrug-resistant bacteria [This review summarises the essential aspects of antibiotic resistance observed in bacteria ,176. Secbacteria . Finallybacteria ,133. Nev"}
{"text": "Vibrio alginolyticus is an important pathogen of marine animals and has been the target of phage therapy applications in marine aquaculture for many years. Here, we report the isolation and partial characterization of a novel species of the Siphoviridae family, the Vibrio phage Artemius. The novel phage was species-specific and could only infect strains of V. alginolyticus. It could efficiently reduce the growth of the host bacterium at various multiplicities of infection as assessed by an in vitro lysis assay. It had a genome length of 43,349 base pairs. The complete genome has double-stranded DNA with a G + C content of 43.61%. In total, 57 ORFs were identified, of which 19 were assigned a predicted function. A genomic analysis indicated that Vibrio phage Artemius is lytic and does not harbor genes encoding toxins and antibiotic resistance determinants. Vibrio alginolyticus is one of the most important pathogenic bacteria of marine organisms and has become a serious threat to the aquaculture industry [industry . It has industry ,3, crustindustry ,5 and moindustry  all overindustry . In Euroindustry . The manindustry . Therefoindustry . In thisindustry ,12,13. Vibrio alginolyticus isolated from the live feeds of a fish hatchery. This work is part of a broader effort of our research team to isolate and characterize lytic bacteriophages against pathogenic vibrios relevant to marine aquaculture [Here, we describe the isolation and characterization of a novel phage infecting aculture ,14,15,16Artemia salina culture tank of the Institute of Marine Biology, Biotechnology and Aquaculture of the Hellenic Centre for Marine Research in Heraklion, Crete (IMBBC-HCMR). HCMR-2 Art1 was identified as Vibrio alginolyticus using molecular methods  [Table 1). All bacterial strains were maintained in microbeads (MicroBank) at \u221280 \u00b0C; when working, they were grown in a lysogeny broth (LB)  at 25 \u00b0C. All strains had been previously identified to a species level using sequencing.The bacterial strain used as a host (HCMR-2 Art1) was isolated from the water of a live feed B, toxR) ,18,19 anArtemia salina culture tank of IMBBC-HCMR following a standard enrichment procedure [Vibrio alginolyticus strain HCMR-2 Art1) and incubated at 25 \u00b0C for 24 h. Following filtration through 0.22 \u00b5m filters, 10 \u00b5L aliquots were plated by a standard double-layer agar method and incubated overnight at 25 \u00b0C to detect and enumerate the plaque forming units (PFU). The clearest plaque formed was picked and further purified by re-plating five times to ensure clonal phage stocks. The purified phage was named Vibrio phage Artemius and propagated to reach a titer of 1010 PFU mL\u22121 and stored at 4 \u00b0C.The phage was isolated from the water of a live feed rocedure . Briefly10 PFU mL\u22121 was negatively stained with 4% w/v uranyl acetate. Vibrio phage Artemius was observed using a JOEL transmission electron microscope operated at 60 kV at the Electron Microscopy Lab of the University of Crete.The phage morphology was observed by transmission electron microscopy (TEM). An aliquot of the phage suspension with a titer of ~107 CFU mL\u22121) was mixed with 3 mL of warm top agar (culture medium with 0.6% agar) and poured onto bottom LB 1/2 agar plates. When the top agar hardened, spots of 10 \u00b5L phage were made and examined after 24 h incubation for lysis. The host range of Vibrio phage Artemius was evaluated by a spot test using the bacterial stains shown in 7 CFU mL\u22121), it was infected with 20 \u03bcL of the phage preparation at 4 different MOIs . Three wells were not infected and were used as the control. The growth curve of the cultures was monitored in real-time over a minimum of 18 h and OD600 measurements were recorded every 10 min.The efficacy of Vibrio phage Artemius on the host strain was assessed by an in vitro lysis assay. Sterile 96-well plates were used and loaded with 180 \u03bcL of a freshly prepared culture of the host bacterium. The plate was placed in a TECAN microplate reader (Infinite PRO 200) equipped with a temperature control and incubated at 25 \u00b0C with orbital shaking. When the bacterial culture was at the exponential phase (~107 PFU mL\u22121) to different temperatures . The aliquots were incubated at each temperature for 1 h and then rested at room temperature (RT) for 10 min. Each aliquot was then serially diluted and spotted (10 \u03bcL/spot) on a host bacterial lawn (HCMR-2 Art1). After a 24 h incubation of the agar plates, the phage titer was determined for each temperature and 4 \u00b0C was used as the control. The thermal stability of the novel phage was examined by exposing 500 \u03bcL aliquots of the phage  was infected with the phage at a multiplicity of infection (MOI) of 0.01. The same amount of phage was added to a tube containing only LB and served as the control. Immediately after the infection, 500 \u03bcL of the infected culture was placed in tubes containing chloroform and stored in ice (4 \u00b0C). The same procedure was repeated for 20 min with a 2 min interval. When all time points were collected, the aliquots were centrifuged at 13,000 rpm for 3 min. They were then serially diluted and spotted onto the host bacterial lawn on LB \u00bd agar plates. The phage titer was determined after 24 h of incubation of the agar plates at 25 \u00b0C.For the adsorption time and one-step growth, we followed the protocol described in Misol et al.  accordin7 CFU mL\u22121) was centrifuged at 13,000 rpm for 3 min. The supernatant was discarded and the pellet was washed with saline. After two repetitions of this step, the culture was finally resuspended in LB, infected with the phage at a MOI of 0.01 and rested for 15 min at RT. Following centrifugation at 13,000 rpm for 3 min, the supernatant was discarded and the pellet was dissolved in 1 mL saline. The infected culture was then transferred to a new tube containing 25 mL LB and the aliquots were immediately removed and placed in empty Eppendorf vials. The aliquots were then centrifuged at 13,000 rpm for 3 min and the supernatant was transferred to a new 2 mL Eppendorf vial, serially diluted and spotted onto the host bacterial lawn on LB \u00bd agar plates. This procedure was repeated every 10 min for 120 min. The phage titer was determined after 24 h of incubation of the agar plates at 25 \u00b0C.For the one-step growth, 1 mL of freshly grown culture at the exponential phase  was used as a negative control. At least 5 \u03bcg of high-purity bacterial DNA, the quality of which was tested using a BioAnalyzer , was used to generate a paired-end 300 PE genomic library with an optimized size selection using magnetic bead purifications based on the standard Illumina protocol and by using a Nextera XT Library Construction Kit . To calculate the average size of the library, we used a Tapestastion 4200 system . The insert size was estimated as the average size of the library minus the Illumina adapter size and was found to be 834 bp. The sequencing was performed using an Illumina NovaSeq 6000 sequencing platform  according to the manufacturer\u2019s protocol at Life Sequencing , which allowed us to obtain at least 1 million paired reads sequences. Possibly contaminated, primer, N-terminus and 3\u2032-, 5\u2032-low quality reads were trimmed off (threshold: 0.05). The raw reads were quality inspected and were assembled in Geneious Prime using the Geneious assembler. RASTk and Glimmer were used as the gene predictors for the structural annotation through the PATRIC webserver [https://phage.ai/, accessed on 1 May 2022). The genome of Vibrio phage Artemius with annotated predicted ORFs was then visualized in a circular representation with Geneious software  and CGview.The DNA extraction of Vibrio phage Artemius was performed using the phenol\u2013chloroform method according to Higuera et al. . The extebserver  and Galaebserver . The preebserver  adjustedebserver , InterPrebserver  and T\u039cHMebserver ,29 databebserver  and Viruebserver  as well ebserver . The phaebserver . The phaTable 2) [A ViPTree was used to build a viral proteomic tree by comparing the proteome of Vibrio phage Artemius with 4982 dsDNA phage proteomes . The phyTable 2) . A totalTable 2) . The gapTable 2)  with a bTable 2)  was usedphage morphology of the virions as observed with TEM classified the novel phage to the Siphoviridae family. The head was 48.7 \u00b1 0.9 nm wide and the tail was 107.0 \u00b1 2.9 nm long  .The Figure 2), with the highest MOI yielding a more rapid decrease in the bacterial titer. The bacterial titer remained low (close to the detection limit) over a period of approximately 10 h.Vibrio phage Artemius was able to lyse the host bacterial population in vitro using approximate MOIs ranging between 0.1 and 100 . According to the host range assay, Vibrio phage Artemius was able to only lyse strains that belonged to p < 0.05) was observed when the phage was exposed to 70 \u00b0C and 80 \u00b0C. The phage was not inactivated at high temperatures. We then exposed the phage to acidic and alkaline pH values ranging from pH = 1 to pH = 10 and compared the titer of the phage with the titer at pH = 7, which served as the control (p < 0.001), but remained stable from pH = 3 to pH = 10 with no statistically significant difference compared with the control. We exposed Artemius to different temperatures ranging from 25 \u00b0C to 80 \u00b0C, with T = 4 \u00b0C serving as the control a, to inv control b. The ph\u22121.The adsorption time assay showed that 90% of the Vibrio phage Artemius virions required 10 min to bind to the bacterial host a. Artemi\u22123) with an average similarity of 52.67%. Overall, 19 ORFs (10.83%) were assigned a function based on the protein homology. No genes associated with integration, virulence or antibiotic resistance were detected. The genome of Vibrio phage Artemius was not modularly organized . However, several gene encoding proteins required for phage assembly  were arranged in subclusters as well as a few genes encoding for DNA replication and nucleotide metabolism proteins . The genes that were functionally annotated are shown in Table 4.The genome size of Vibrio phage Artemius was 43,349 bp with a GC content of 43.61%. The genome arrangement was dense, as suggested by the 1.31 genes per kbp. The Rapid Annotation using Subsystem Technology (RASTk) server and Glimmer.hmm 3.0 revealed that 57 ORFs were present in the genome. Each individual ORF was manually inspected in order to validate the presence of start codons (ATG/GTG or TTG). There was no presence of tRNA in the genome, as shown by ARAGORN. A total of 50 ORFs used a start codon of ATG, 6 ORFs used GTG and 1 used TTG. A search of the NCBI nr database showed that 45 ORFs (78.95%) had significant hits . The proteins required for phage morphogenesis included major capsid protein (ORF 8), tail-length tape measure protein (ORF 17), tail tube protein (ORF 13), tail tubular protein (ORF 22) and neck protein (ORF 16). In addition, the small terminase subunit (ORF 2), large terminase subunit (ORF 5), stopper protein (ORF 10) and phage portal protein (ORF 6), which are involved in DNA packaging for tailed phages, were identified. Proteins for DNA replication, recombination and repair were also detected: these were DNA polymerase (ORF 23), HNH endonuclease (ORF 51), DNA polymerases, DNA helicase  and other regulatory elements (ORF 54). Finally, several transmembrane proteins were detected  . In addition, it was predicted to infect hosts from the Gammaproteobacteria class, which includes the Vibrionaceae family. A closer view of the tree focusing on the nearest relatives  showed that Vibrio phage Artemius clustered together with Shewanella phage 1/44 (Siphoviridae) and Escherichia phage PTXU04 (Podoviridae).A wide genome proteomic tree analysis validated that Vibrio phage Artemius belonged to the Vibrio alginolyticus phages  showed that Vibrio phage Artemius shared a common ancestor with Vibrio phage phi-St2 and Vibrio phage phi-Grn1. However, the phages were rather distant, resulting in low bootstrap values (<75%); thus, the node was not well-supported. A large terminase subunit gene was used for the phylogeny because it is considered to be a signature, well-conserved gene among the phages and is a strong molecular motor associated with phage packaging [Phylogeny using the large terminase subunits of the ackaging .Vibrio alginolyticus has been used frequently as a target for novel phage isolation. Until today, more than 40 different phages have been isolated against V. alginolyticus, which is suggestive of the importance of this opportunistic pathogen for the aquaculture industry [industry . Severalindustry ,42, but industry .V. alginolyticus), Vibrio phage Artemius was closer to Vibrio phage phi-St2 and Vibrio phage phi-Grn1, which belong to the genus of Schizotequatrovirus [Shewanella sp. Interestingly, this phage was isolated from iced water in the Baltic Sea [Vibrio phage Artemius is a novel phage, as indicated by the BLAST search in the NCBI nr database where the closest phage at the nucleotide level was Vibrio phage 2.044.O._10N.261.51.B8 (MG592661.1) with a 79.6% similarity over a 2% query cover. A phylogenetic analysis of Artemius verified the novelty of this phage. According to the phylogeny obtained with the terminase large subunit using phages infecting the same host (trovirus . Accordiltic Sea ,46. Two of the most crucial characteristics of a phage to be considered for therapeutic purposes are the inability to integrate in the host genome and the lack of unwanted genes such as toxins and antibiotic resistance determinants that could be transferred to its host through lysogenic conversion or recombination . A genomSiphoviridae family [High temperatures usually have a devastating effect on phage integrity, causing tail aggregation, detachment of the phage head and denaturation of the nucleic acid . Interese family .Vibrio alginolyticus as it was not able to infect the other congeneric species; thus, it was a species-specific phage. Broad host-range phages are considered to be ideal for phage therapy, especially in aquaculture where the diversity of the pathogenic strains and species is wide. Phages with a broad host range have been reported against Vibrio alginolyticus [The host range of Vibrio phage Artemius was limited to strains of olyticus ,41. In tSiphoviridae family that infects V. alginolyticus and, based on its biological and genomic characteristics, could be considered for efficient and safe phage therapy applications.In conclusion, Vibrio phage Artemius is a potential new species of the"}
{"text": "This cross-sectional study uses Centers for Medicare & Medicaid Services payment data to examine use of short-course radiotherapy from 2015 to 2019 among Medicare beneficiaries with indolent lymphoma. We evaluated SC-RT use with US episode-based payment data from the Centers for Medicare & Medicaid Services.6With increasing medical costs and concerns about COVID-19 exposure, reducing financial and time burdens of cancer treatment is critical.STROBE reporting guideline. Radiotherapy episodes from 2015 to 2019 were analyzed for Medicare beneficiaries 65 years or older who did not receive systemic therapy and lived more than 90 days after RT for lymphoma . Most patients (71%) received LC-RT. Receipt of SC-RT was associated with older age , hospital-affiliated vs free-standing site of care , and conventional RT vs IMRT   used conventional RT; however, IMRT use increased from 17% in 2015 to 25% in 2019 (45-6.02) . Increas45-6.02) .P\u2009<\u2009.001), conventional RT, younger patients, and hospital-affiliated sites (P\u2009<\u2009.001), younger patients, conventional RT, and freestanding sites  vs $8484  for LC-RT. For SC-RT, median total spending was $8048  with IMRT vs $4121  with conventional RT. Median total LC-RT spending was $13\u2009085  with IMRT vs $7657  with conventional RT. Professional services\u2013related spending was reduced with SC-RT (adjusted \u03b2, $572; 95% CI, $550-$594; ed sites . Technicng sites . In lineMost Medicare beneficiaries treated with radiation monotherapy for lymphoma between 2015 and 2019 received LC-RT. Use of LC-RT has important financial implications given higher total spending. Despite lower health care spending and reduced time, travel, and costs for patients, SC-RT was used in only 29% of patients by 2019. Whereas practice patterns may reflect site-specific case mixes, ensuring differences do not reflect competing financial incentives owing to reimbursement structures or opinions on evidence-based practice is important. Increases in spending were greatest for IMRT; therefore, regimen and technique should be considered for cost savings. Financial alignment with value-based practice irrespective of site of care is critical; future studies may help refine the balance between local control, patient treatment-related burden, and health care spending. Wider adoption of SC-RT may help reduce systemwide costs and optimize personalized RT decision-making. Limitations include lack of available clinical variables; despite efforts to limit the analysis to indolent lymphomas for which SC-RT is considered, more aggressive subtypes may have been included."}
{"text": "Bacteriophages are obligatory parasites propagating in bacterial hosts in a lytic or lysogenic/pseudolysogenic cycle. Phages are the most abundant biological particles in the world, being responsible for: (i) dissolved and particulate organic matter circulation via host cell lysis, (ii) regulation of numbers and biodiversity of populations, (iii) horizontal gene transfer (HGT) via transduction, or indirectly via transformation of bacterial DNA released during cell lysis, and, finally, (iv) lysogenic conversion by temperate phages. Therefore, phages greatly affect microbial diversification as an integral part of each ecological niche, including the human body. The tremendous dynamics of phage\u2013host interactions result in the continuous flow of genetic material, which drives the co-evolution of both entities.Viruses \u201cPhage\u2013Bacteria Interplay in Health and Disease\u201d focuses primarily on the regulation and functioning of human/mammal microbial ecosystems as the consequence of specific and non-specific virus\u2013bacteria interactions, bacterial defence against phages which can drive the outcome of the disease/infection, phages as human immunomodulators, and the description of innovative experimental techniques characterizing the phage traces in mammals.Advances in recent years in molecular biology, multi-omics, and bioinformatic approaches have contributed to the deeper insight into and functional analyses of a wide range of phage\u2013bacteria interactions at the individual and population levels, and their consequences on health and disease. The Special Issue of A total of 13 manuscripts, including 10 original and 3 review papers, have been published in this Special Issue. The collection contains recent papers that could be roughly classified into three themes comprising the following: (1) survival strategies of bacteria in response to phage infection as well as the impact of phages on bacterial virulence and pathogenicity ; (2) influence of lytic and temperate phages on the human microbiome as well as human virome composition ; (3) phages as potential antibacterial therapeutics . All papers reflect the impact of phage\u2013bacteria as well as phage\u2013bacteria\u2013host interactions on human health, disease, and economic activities. They also highlight the great importance and relevance of the topic addressed.Streptococcus pyogenes is a Gram-positive \u03b2-hemolytic pathogen that strictly infects humans and can cause superficial skin infections, pharyngitis, toxin-mediated diseases ), and invasive diseases of subcutaneous tissues [S. pyogenes to protect against Phage A1. Phage A1, as a temperate phage, was able to infect S. pyogenes strains, resulting in complete resistance against subsequent phage infections, most likely by providing superinfection immunity. Furthermore, the lysogenization did not influence the humoral host immune response or bacterial virulence and did not induce ampicillin tolerance to this common antistreptococcal antibiotic. The authors demonstrated that the type II-A CRISPR-Cas system of S. pyogenes acquires new spacers upon phage infection, which are increasingly detectable in the absence of a capsule. Finally, the authors showed that the number of infecting phages is limited through binding to released streptococcal outer membrane vesicles. The authors proposed a multistage interaction model between S. pyogenes and Phage A1 [ tissues . Bacteri tissues . Beerens tissues  describePseudomonas aeruginosa antiviral strategy based on outer membrane vesicles (OMVs) against the LPS-specific phages KT28 and LUZ7. To investigate the passive and active role played by OMVs towards these phages, the OMVs derived from the phage-sensitive wild-type PAO1 strain, and an LPS-deficient mutant (\u2206wbpl PAO1) resistant to both phages, were used. It turned out that naturally formed OMVs efficiently protected P. aeruginosa from phage infection. Next, it was verified whether OMVs derived from the wild-type PAO1 strain were able to sensitize the LPS-deficient mutant (\u0394wbpl PAO1) to the tested phages. The growth kinetic curves and one-step growth assay revealed no sensitization event [Outer membrane vesicles (OMVs) of Gram-negative bacteria are important virulence factors as decoys against a variety of antibacterials but are also an element in host\u2013bacteria interplay, and intra- or inter-species bacterial communication ,4. In thon event .Clostridioides difficile is one of the most common causes of antibiotic-related nosocomial infections with symptoms ranging from mild diarrhea to life-threatening pseudomembranous colitis and/or toxic megacolon [C. difficile phages, with a particular interest in phage CDHS-1 propagating on the hypervirulent 027 ribotype C. difficile R20291. The study focused on the transcriptomic takeover of phage CDHS-1 during infection, and analyses revealed that the majority of the bacterial genes connected with metabolism and toxin production were downregulated at the early phase of CDHS-1 replication, whereas genes related to DNA synthesis and ATP production were among those upregulated at this stage. The holin, endolysin, and structural genes were upregulated towards the mid-log and late phases of the phage replication. The retrieved phage-resistant clones and lysogens showed relatively low virulence in the larval model of Galleria mellonella compared to the wild-type strain. The data suggest that phage infection both reduces bacterial colonization and negatively impacts bacterial pathogenicity, supporting the therapeutic potential of the phage for human and animal use [egacolon . Therefoimal use .Enterococcus faecalis strains. The report indicated that vB_EfaS-271 can significantly decrease the number of viable E. faecalis cells in biofilms formed on catheters and in liquid cultures and revealed no considerable toxicity to mammalian cells, influencing neither their viability nor morphology. The efficiency of phage resistance development was especially significant under conditions of high MOI values; nevertheless, phage vB_EfaS-271 was considered promising for phage therapy [Topka-Bielecka et al.  describeStreptococcus agalactiae (GBS) clinical isolates to verify the prevalence of prophages versus the bacterial virulence potential. Based on the whole genome sequencing and PCR methods, the authors identified eight groups of prophages, amongst which the highest prevalence was observed for a prophage from group A (71%) and a satellite prophage from group B (62%). They observed that the prophage distribution did not differ between clinical and screening strains, but it was unevenly distributed in MLST (multilocus sequence typing) sequence types. This study implies that prophages could be beneficial for the host bacterium [Lichvarikov\u00e1 et al.  performeacterium .Siphoviridae, followed by Myoviridae, Podoviridae, Herelleviridae, and Inoviridae. The results pointed out that some prophages were present in cervicovaginal secretions of multiple participants, suggesting that prophages, and thus bacterial strains, are shared between adolescents. Shaping the biodiversity of bacterial populations, these viruses contribute to local fluctuations in the vaginal microbiome [Phages have an important role in shaping bacterial communities. Phages also impact human health by infecting bacteria forming human microbiomes in the gut, respiratory tract, skin, or vagina. Happel et al.  investigcrobiome .Lactobacillus (L.) spp. abundance and increased colonization of facultative anaerobes, such as Gardnerella spp. [L. crispatus and L. iners) as well as with known pathobionts (Gardnerella spp. and Atopobium vaginae) [Bacterial vaginosis (BV) is characterized by a reduction in lla spp. . The paplla spp.  related vaginae) .\u00ae) on the ex vivo human gut microbiome composition and function. Using a novel in vitro assay called RapidAIM as well as 16S rRNA gene sequencing and metaproteomic approaches, the authors documented that the ex vivo human gut microbiota composition and function were unaffected by BAFASAL\u00ae treatment, which proves the GRAS provision  [Mayne et al.  investigas safe) .At present, traditional antimicrobials are becoming ineffective against multidrug-resistant bacterial pathogens. Therefore, phages are increasingly beginning to be recognized as an alternative or supportive therapeutic agent .Staphylococcus aureus phage in the treatment of atopic dermatitis (AD), the most common inflammatory skin disease, in an atopic mouse model. As previously documented, phage SaGU1 can infect a broad range of S. aureus in AD patients, whereas it does not kill strains of the symbiotic bacterium S. epidermidis. In this work, the authors showed that administration of SaGU1 to the back skin of mice reduced both S. aureus counts and the disease exacerbation caused by these bacteria. Furthermore, the application of S. epidermidis in combination with SaGU1 inhibited the emergence of phage-resistant S. aureus, indicating that synergistic use of probiotics and phages can be promising and effective in the treatment of S. aureus-associated AD [Shimamori et al.  investigiated AD .E. coli lytic phages as a new tool for the investigation of phage interactions with cells and tissues. The deposition of an RFP-displaying phage in multiple murine organs ex vivo after various routes  of phage administration was verified. The most effective intravenous administration led to effective distribution kinetics with phage presence in the lymph nodes, lungs, and liver after 20 min, whereas in the muscles and spleen/lymph nodes, this occurred 30 min and 1 h after administration, respectively [Since interactions between phages and mammals strongly affect the possible applications of phages, tools to study how phages circulate in the body and can be deposited in tissues are highly desirable. Understanding this need, Ka\u017amierczak et al.  proposedectively .Iszatt et al.  providedS. aureus, Klebsiella pneumoniae, P. aeruginosa, and E. faecalis infection models. It also illustrated appropriate phage selection criteria, as well as recommendations for successful therapy. The main conclusions from the analysis of the selected literature were that phage applications (single or in cocktails) are effective and safe, can work synergistically in combination with certain antibiotics, and may induce the emergence of phage resistance [An interesting review of Al Ishaq et al.  addressesistance .Salmonella spp., E. coli, Campylobacter spp., Vibro spp., and P. aeruginosa, and other pathogens, such as Staphylococcus spp., Streptococcus spp., Klebsiella spp., Acinetobacter spp., and Mycobacterium sp. The varied therapeutic and immunomodulatory activities of phages on humoral and cellular immune response mechanisms were also discussed [In parallel with attempts to use phages against nosocomial human pathogens, there has been much work conducted on the use of these viruses as alternative methods to control zoonotic and foodborne pathogens. This problem was addressed in a review paper written by Alomari et al. . The autiscussed ."}
{"text": "Background: Phage therapy a promising antimicrobial strategy to address antimicrobial resistance for infections caused by the major human pathogen Staphylococcus aureus. Development of therapeutic phages for human use should follow pharmaceutical standards, including selection of strictly lytic bacteriophages with high therapeutic potential and optimization of their production process. Results: Here, we describe three novel Silviavirus phages active against 82% of a large collection of strains (n = 150) representative of various methicillin-susceptible and -resistant S. aureus clones circulating worldwide. We also investigated the optimization of the efficiency and safety of phage amplification protocols. To do so, we selected a well-characterized bacterial strain in order to (i) maximize phage production yields, reaching phage titres of 1011 PFU/mL in only 4 h; and (ii) facilitate phage purity while minimizing the risk of the presence of contaminants originating from the bacterial host; i.e., secreted virulence factors or induced temperate phages. Conclusions: In sum, we propose a quality-by-design approach for the amplification of broad-spectrum anti-S. aureus phages, facilitating the subsequent steps of the manufacturing process; namely, purification and quality control. Staphylococcus aureus is a major human pathogen responsible for a wide range of diseases [S. aureus is able to form biofilms that prevent the access of antibiotics and immune cells to bacteria, which are thus associated with therapeutic failures [S. aureus lytic Caudovirales bacteriophages, targeting a wide range of hosts, have been previously isolated [S. aureus phages belong to various families and genera, including Herelleviridae  and Podoviridae, with Silviavirus phages having the broadest activity spectrum [diseases . It is tdiseases ,3. In adfailures . In thisfailures ,6. Anti-isolated ,9,10,11.spectrum .To ensure the future success of phage therapy, phage production should comply with pharmaceutical standards, including a robust and safe process of phage amplification. Such standards still need to be established and validated by national and international drug agencies so that they can be adapted to the specific biological statuses of phages . HoweverS. aureus Silviavirus phages with broad activity against a large panel of S. aureus clinical strains. We also describe (i) the selection of optimal bacterial strains, aiming at limiting the production of bacterial contaminants upon phage amplification, and (ii) the optimization of experimental parameters for their optimal production, both in terms of amplification yields and safety.In the present study, we report the isolation and characterization of three novel anti-All bacterial strains included in the present study, both for host range assessment and for phage production, were obtained from the collection of the French National Reference Centre for Staphylococci . Clonal complexes of S. aureus strains were assigned using DNA microarray , following the manufacturer\u2019s instructions. Whole-genome sequencing was also performed for the screening of candidate strains for phage production. Illumina libraries were prepared using the Nextera XT or DNA Prep kits  and sequenced on a MiSeq or NextSeq 500 instrument (Illumina) using a 300 or 150 bp paired-end protocol, respectively. Detection of virulence- and resistance-associated markers was performed from assembled genomes using Abricate (v0.7) and an in-house nucleotide-based database, built mainly from the Resfinder database (version 2020-06-02) and covering the known S. aureus toxins, including enterotoxins, exfoliatins, toxic shock syndrome toxin TSST-1, and leukocidins. Accession numbers for genomes of selected strains are provided in Bacterial genomic DNA extraction consisted of an initial incubation with 40 \u00b5g of lysostaphin , 200 \u00b5g of lysozyme (Sigma-Aldrich) and 200 \u00b5g of RNaseA  for 1 h at 37 \u00b0C, followed by incubation with 12 mAU of proteinase K  for 20 min at 60 \u00b0C, then an extraction using the MaxwellThree phages  were isolated from three different wastewater samples in Lyon, France. Briefly, 5 mL of the filtered water samples was incubated with 500 \u00b5L of Tryptic Soy Broth (TSB) 10X  and 10 \u00b5L of overnight bacterial culture . Then, the culture supernatant was filtered using a 0.22 \u00b5m syringe filter and diluted in double-layer agars, pouring a mix of 100 \u00b5L of this supernatant, 250 \u00b5L of bacterial culture, and 5 mL of TSB-soft agar (TSB containing 0.75% agar) over a TSA plate . An individual plaque-forming unit (PFU) was further purified with five rounds of serial passages and eventually propagated in liquid medium. The obtained phage lysates were filtered using a 0.22 \u00b5m syringe filter and stored at +4 \u00b0C.g  over 3 h at +4 \u00b0C in a three-layer CsCl (Sigma-Aldrich) gradient with densities of 1.6, 1.5, and 1.3. After centrifugation, phages were collected between the 1.5 and 1.6 layers and dialyzed  twice in 3 L of DPBS buffer (Sigma-Aldrich) at 4 \u00b0C for 6 h, with one buffer change. Lastly, phage suspensions were filtered using a 0.22 \u00b5m syringe filter and stored at +4 \u00b0C.A volume of 9 mL of each crude phage lysate was purified by ultracentrifugation at 120,000\u00d7 g for 5 h and pellets were resuspended in 50 \u00b5L of NaCl 0.9%. Enzymatic treatment was then applied with 100 mU of benzonase\u00ae nuclease (Sigma-Aldrich) at 37 \u00b0C overnight to degrade extracellular bacterial DNA, followed by benzonase heat-inactivation at 95 \u00b0C for 30 min, a treatment with 4 \u00b5g of proteinase K (Sigma-Aldrich), and proteinase K heat-inactivation. Phage DNA was extracted using the DNA Extractor\u00ae WB kit  and sequenced on an Illumina NextSeq 500 instrument using a 150 bp paired-end protocol. Reads were trimmed  and the read mappings against the genome of the bacterial strain used for the amplification of phages  were removed. The remaining reads were assembled  and scaffolds smaller than 100 bp were removed. Taxonomic assignation was performed using kraken 1.1.1 with the minikraken database. Genomes were first annotated using PATRIC (v3.6.12) with parameters for bacteriophage annotation. The protein sequences of genes annotated as \u201chypothetical protein\u201d or \u201cphage protein\u201d were further analysed: a blastp was performed against the PHROGs database to improve annotation, considering annotated proteins with identity and query cover percentages higher than 90%. Finally, Abricate (v0.8.13) was used for resistance and virulence gene detection using all the databases available. The lytic nature of phages was assessed using the Phage AI repository (https://app.phage.ai/phages/ (accessed on 17 March 2022)). Phage genomes were deposited in GenBank under the accession numbers ON814134, ON814135, and ON814136 for V1SA19, V1SA20, and V1SA22, respectively.A volume of 6 mL of phages was centrifuged at 14,000\u00d7 S. aureus and S. argenteus strains genetically characterized using WGS and representative of clinical isolates collected in France between 2017 and 2020 , pH = 7, and then centrifuged again at 21,000\u00d7 g for 1 h and finally re-suspended in 100 \u00b5L of the same buffer. Phage suspensions were adsorbed on 200 Mesh Nickel grids coated with formvar-C for 10 min at RT. Then, grids were coloured with Uranyless  for 1 min and observed on a transmission electron microscope  equipped with a Orius\u00ae 1000 camera  and Digital Micrograph software.A volume of 1 mL of the phage suspensions obtained after ultracentrifugation was centrifuged at 21,000\u00d7 S. aureus or S. argenteus genomes from the French National Reference Centre for Staphylococci. First, strains harbouring genes encoding major virulence/resistance factors, such as enterotoxins, leukocidins, superantigens, and methicillin-resistance, were excluded. The remaining genomes were examined using prophage-prediction tools\u2014namely, PHASTER (https://phaster.ca/statistics (accessed on 10 January 2021)) and ProPHET (v0.5.1)\u2014allowing the exclusion of genomes harbouring intact prophages.Candidate strains for phage production were screened among a collection of more than 2000 \u22123 phage per bacteria in 10 mL of TSB. After 24 h of incubation, phage titres were measured.Using this in silico approach, only 10 candidate strains were selected for further experimental assessment. EOP ratios were determined for the three selected phages against these 10 strains as described above. Phage amplification yields were then assessed in small scale production conditions with a selection of the five strains presenting the highest EOP values for all phages and representative of each sequence type. For these experiments, bacteria in exponential phase and phages were mixed at two concentrations (multiplicity of infection (MOI)) of 1 and 10TM , and Superior BrothTM (Athenaes). To this end, bacteria were grown in 1 L of medium in 2.5 L Fernbach culture flasks with sided baffles  for 2 h until an exponential phase was reached. Then, phages were added at MOIs of 10\u22122 or 10\u22124. Phage titrations were performed at different time points during the incubation in order to follow the kinetics of phage amplification.Finally, the bacterial strain allowing the highest yield for each phage was then selected for production optimization in larger containers to test the impact of (i) the MOI or (ii) the medium used, comparing conventional media and animal protein-free media designed for pharmaceutical production of proteins, including TSB, LB Broth Lennox , Turbo Both\u22124 for graphical representation purposes. For phage production experiments, Student\u2019s t-test was used to compare the mean phage titres. A p-value < 0.05 was considered significant. Statistical analyses were performed and figures were generated using GraphPad Prism, version 8.0.0 .Phage activity variations between clonal complexes were assessed by comparing EOP distributions using the Kruskal\u2013Wallis test. Null EOP values were arbitrarily set to 10Herelleviridae family and the Silviavirus genus. Genome lengths were 138,507, 136, 919, and 133,701 bp for V1SA19, V1SA20, and V1SA22, respectively. The numbers of ORFs were 245, 234, and 225 for V1SA19, V1SA20, and V1SA22, respectively, including 74.6% to 76.4% of genes for which a putative function could not be attributed . Of note, V1SA20 was the only phage active against CC80 strains. Phages were poorly active against CC7, CC59, CC239, and CC398 strains.The host range of the three phages was determined using a collection of 150 strains chosen to be representative of (i) the major methicillin-susceptible (MSSA) or resistant (MRSA) clones circulating worldwide and (ii) the genetic and clinical diversity of fections . Phage Vfections A. Phage 10 PFU/mL  compared to conventional TSB or LB media were assessed. The Superior BrothTM yielded the maximal phage titres in shorter times compared to other media (10 to 1 \u00d7 1011 PFU/mL reached in 3 to 4 h). Of note, we observed significant mean phage titre drops, with all types of media, of 4 \u00d7 1010 and 7 \u00d7 1010 PFU/mL between 8 and 24 h of incubation for V1SA20 and V1SA22, respectively .The bacterial strains P2SA225 and P2SA237 were then selected for optimization of the amplification of phages V1SA19 or V1SA20 and V1SA22, respectively, in larger containers. Phage production yields were first assessed in conventional TSB medium at two MOI values: maximal phage titres were reached more quickly at MOI 10MOI 10\u22124 A,B. In aPhage therapy a promising alternative strategy to address antimicrobial resistance and improve prognoses in the treatment of staphylococcal infections. Although no marketing authorization has been issued for therapeutic phages as therapeutic products by the US Food and Drug Administration or the European Medicines Agency yet, several clinical trials are currently on-going and their current compassionate use, codified by the Declaration of Helsinki, is increasing very rapidly in diverse clinical settings ,24,25. TS. aureus phages belonging to the Silviavirus genus showing complementary activities: 82.0% of the bacterial collection were susceptible to one of the three phages, while 76.7% were susceptible to the most active phage V1SA20. Anti-S. aureus phages have previously been reported to have broad-spectrum activity within this bacterial species. However, among the numerous studies describing the isolation of such phages, very few of them have tested large, diverse, and clinically relevant genetically-characterized collections of clinical strains, ensuring the diversity of the tested strains, to more precisely describe the phage host range [S. aureus phages depended on the clonal complex (CC), which is in agreement with previous studies. This specificity has been linked to the presence of type I restriction-modification systems, which are also associated with the CC [In the present study, we characterized three novel, strictly lytic anti-st range ,27,28. IAlthough the status of phage products is not definitively established by the health authorities, the scientific community expects it to be adapted to the biological specificities of phages compared to conventional medicines. A quality-by-design approach has been recommended for the development of phage production in line with Good Manufacturing Practice (GMP) guidelines . It shouSilviavirus phages based on the simultaneous absence in their genomes of (i) a maximum of genes encoding virulence/resistance factors  and (ii) prophages. Very few other studies have previously addressed this topic [Staphylococcus xylosus strain, a low pathogenic staphylococcal species, for amplification of anti-S. aureus Kayvirus phages in order to limit the production of virulence factors and improve the safety of phage therapy [First, this implies the need to establish the possible contaminants of phage preparations that might affect the safety of the final product for phage therapy and to assess ways to control them . Indeed,is topic ,32. El H therapy . However therapy ,33. Thes therapy ,35. None therapy ,36.11 PFU/mL, and/or were reached more rapidly using these media compared to conventional TSB. Of note, shortening the time necessary for amplification could be of interest to limit the release of metabolites, notably bacterial DNA, which cannot be avoided despite the selection of optimal bacterial strains. These kinds of bacterial metabolites are known to be able to induce inflammation and their quantification is part of the quality controls that should be performed to assess the quality of phage therapy products [Consequently, propagation hosts must also allow the acquisition of high titres of phages. Interestingly, our study showed that high EOP ratios did not provide guarantees for a high level of phage amplification and, thus, that bacterial strains with high EOP ratios are not systematically the best candidates for phage production. Bacterial and phage inocula (MOI), the nutrient composition of culture media, stirring speed, oxygenation, and temperature strongly influence phage amplification yields . We notaproducts ,38.Staphylococcus aureus phages with large spectra of activity. We showed that it is possible to optimize phage production protocols to enhance amplification yields after the in silico selection of candidate strains, aiming at facilitating the subsequent steps of the manufacturing process\u2014namely, purification and quality controls\u2014in order to ensure patient safety.We reported the initial steps of the pharmaceutical development of three novel anti-"}
{"text": "K. pneumoniae emphasizes the urgent need of new therapeutic strategies for the control of this pathogen. There is growing interest in the use of bacteriophages (or phages) to treat K. pneumoniae infections, and newly isolated phages are needed. Here, we report the isolation and physical/biological/molecular characterization of a novel lytic phage and its efficacy in the control of MDR K. pneumoniae. The phage vB_KpnS_Uniso31, referred to hereafter as phage Kpn31, was isolated from hospital wastewater using K. pneumoniae CCCD-K001 as the host. Phage Kpn31 presents a siphovirus-like morphotype and was classified as Demerecviridae; Sugarlandvirus based on its complete genome sequence. The 113,444 bp Kpn31 genome does not encode known toxins or antimicrobial resistance genes, nor does it encode depolymerases related sequences. Phage Kpn31 showed an eclipse time of 15 min and a burst size of 9.12 PFU/host cell, allowing us to conclude it replicates well in K. pneumoniae CCCD-K001 with a latency period of 30 min. Phage Kpn31 was shown to be effective against at least six MDR K. pneumoniae clinical isolates in in vitro antibacterial activity assays. Based on its features, phage Kpn31 has potential for controlling infections caused by MDR K. pneumoniae.The worldwide increase in serious infections caused by multidrug-resistant (MDR)  Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., which have been integrated within the acronym \u201cESKAPE\u201d, are the leading cause of hospital-acquired infections throughout the world . After three rounds of purification on a K. pneumoniae CCCD-K001 lawn, the isolated phage produced clear and translucent plaques with diameters of \u22481.0 mm (13 PFU/mL) of phage Kpn31 were obtained for further assays, aiming at its characterization.One phage clear lysis plaque was selected among the clear and turbid plaques of the same size obtained from the enrichment of a wastewater sample (sewage) collected at Hospital UNIMED Miguel Soeiro, Sorocaba, S\u00e3o Paulo, Brazil, using  \u22481.0 mm . The isoSiphoviridae-like tail morphotype [The TEM photomicrograph  clearly rphotype  belonginK. pneumoniae is 57.5% [The genome of phage Kpn31 was sequenced and assembled, resulting in a contig of 113,444 bp. The contig had 141 bp direct terminal repeats, indicating that the assembled phage genome is complete. The GC content of the Kpn 31 genome is 45.3%, whereas that of the is 57.5% . The oveThe genome of phage Kpn31 encodes 14 tRNAs and 188 protein coding genes  . A compaDemerecviridae or Sugarlandvirus. Therefore, phage vB_KpnS_Uniso31 (phage Kpn31) can be assigned to the Sugarlandvirus genus. The taxonomic classification of Kpn31 agrees with the TEM analysis . Thus, within 10 min after phage-host mixture, ~90% of the phage particles were adsorbed to the bacterial cells , in the first 8 h of incubation, the inactivation factor was higher for MOI 1000 . At a MOI of 1000, after 6, 8, 10, and 12 h of incubation, the reduction in K. pneumoniae counts  was significantly larger  than that produced with MOI 1 . K. pneumoniae regrowth was observed after 6 h of incubation. However, at the end of the experiment, the rate of bacterial regrowth with a MOI of 1 and 1000  was significantly lower  than the one obtained with the bacterial control (p > 0.05)  a.p < 0.05) for MOI 1 and MOI 1000, respectively  increased 5.1 log CFU/mL a, whereaectively b.The effect of the MOI value was more pronounced during the first 8\u201310 h of bacterial inactivation a, with M13 virions/mL) were studied via UV\u2013VIS spectral scans and X-ray diffraction (XRD) analysis. Kpn31 = 2.2877 \u00d7 10\u221212 (PFU\u2019s/mL)\u22121 cm\u22121, allowing for calculating the phage particle concentration based on the resulting calibration curve . An EOP of 100% means that every phage particle adsorbing to a susceptible bacterial cell can inject its DNA and produce a lysis plaque under ideal conditions [K. pneumoniae phages (vB_KpnS_FZ10 and vB_KpnP_FZ12) were active against a high percentage of K. pneumoniae strains  [K. pneumoniae phages showed that phages of the Siphoviridae and Podoviridae families lysed about 7\u201315% of strains, and only one phage of the Podoviridae family was effective against 22% of the strains tested [Myoviridae family were active against 4\u201322% of K. pneumoniae strains [K. pneumoniae strains, which confirms its potential for prophylaxis and for the treatment of bacterial infections caused by this bacterium. In the future, new phages need to be isolated and tested together with phage Kpn31 in order to produce a cocktail with a broader spectrum of activity against K. pneumoniae and other human pathogenic bacteria.One (if not the major) advantage of antimicrobial phage treatment lies in the high specificity of phage particles, even though they should be able to promote the lysis of the majority of strains of a given bacterial species ,64,65,66s tested . However low EOP , indicatnditions . Zurabovctively) . Howeverctively) . In anots tested . Phages  strains . Phage K\u22128\u201310\u22129 mL/min. Approximately 90% of phage Kpn31 particles adsorbed to K. pneumoniae CCCD\u2014K001 cells after 10 min, and 100% of the phage particles adsorbed after 30 min, which confirms its potential for the treatment and prevention of bacterial infections. The adsorption rate of phage Kpn31 particles (1.700 \u00d7 10\u22129 PFU\u22121 CFU\u22121 mL\u22121 h\u22121) closely agrees with the results published by Zurabov et al. (2021) [K. pneumoniae phages .Before applying phages to control bacterial pathogens, the dynamics of phage\u2013host replication should be well characterized in vitro. The first step in the phage infection process is the adsorption of the phage virion onto a susceptible bacterial cell ,70,71,72. (2021)  for K. pK. pneumoniae CCCD-K001 with a short latency period (30 min). The latent period of phage Kpn31 correlates well with published data on K. pneumoniae phages [K. pneumoniae, but its effect only started after 4 h of incubation. K. pneumoniae was effectively inactivated by phage Kpn31, reaching a maximum inactivation of 5.6 log CFU/mL and 7.5 log CFU/mL after 6 h of incubation at MOI 1 and 1000, respectively. After this time, although some host cells were not inactivated by phage Kpn31, after phage treatment, the cell growth was much slower. Between 8 h and 24 h of treatment with phage Kpn31 at MOI 1 and 1000, the concentration of host cells was significatively lower than that observed for the control.The growth parameters of Kpn31 phage particles showed a burst size of 9.12 PFU/host cell, allowing to conclude that the phage replicates well in e phages ,69,77. Te phages . The bure phages , but low5 to 106 PFU/mL, 8 to 1012 PFU/mL, 5 to 1010 and from 105 to 109+ CFU/mL, respectively, during the first 4 h of treatment , the iK. pneumoniae clinical isolates. However, the spot test may produce false positives due to bacterial cell lysis, without them being infected by the phage [The phage Kpn31 host range was initially evaluated by spot test to assess if it was able to produce clear plaques on strains of different bacterial genera and of MDR he phage , either he phage ,87,88. Ihe phage .K. pneumoniae strains paves the way for new studies, especially in vivo studies, aiming at the development of strategies to control infections caused by K. pneumoniae. The results of this study highlight the importance of isolating, characterizing, and testing the effectiveness of new phages to inactivate bacteria in clinically relevant settings.The high bacterial inactivation efficiency of phage Kpn31 combined with the safety of the phage and its efficiency against"}
{"text": "Klebsiella pneumoniae.The development of alternative control measures, such as phage therapy or adjunctive therapy, is urgently needed to manage the dissemination of carbapenemase-producing K. pneumoniae clinical isolate in vitro in Kenya.This study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of carbapenemase-producing Klebsiella phages. The efficacy of individual bacteriophages and phage cocktails as well as their combination with antibiotics were determined for their inhibitory activity on carbapenemase-producing K. pneumoniae (KP20).The study was conducted from February 2021 to October 2021 at the Institute of Primate Research, Nairobi, Kenya. Phage cocktails were formulated based on the morphology and biological properties of precipitated Myoviridae, Siphoviridae and Podoviridae. Regarding the evaluation of the phage cocktails, the absorbances at 600 nm of the bacterial culture treated with the two-phage cocktail (2\u03c6 MA) ranged from 0.173 to 0.246 at 16 h and 20 h whereas it peaked from 2.116 to 2.190 for the positive control. Moreover, the results of the adjunctive therapy showed that the optical density at 600 nm of the bacterial culture treated with 2\u03c6 MA was 0.186 at 24 h post-incubation time while it was 0.099 with the bacterial culture treated with imipenem in combination with 2\u03c6 MA.The precipitated bacteriophages were members of K. pneumoniae growth in vitro.This study demonstrated that the two-phage cocktail in combination with imipenem was able to synergistically delay the increase in carbapenemase-producing  Klebsiella pneumoniae because of the decrease in the effectiveness of antibiotic drugs.2 Bacteriophage therapy has, therefore, been proposed as an alternative strategy in controlling multidrug-resistant (MDR) bacterial infections, including MDR K. pneumoniae clinical isolates.4 Some of the beneficial effects of bacteriophages over antibiotics include their abundance, host specificity and exponential replication.5 However, the narrowness of phage host range and the ever emergence of novel pathogen variants will at minimum represent some limitations for phage therapy.7 This challenge can be managed by formulating phage cocktails that contain different phages infecting one species or by combining phages with antibiotics, which may result in a broad spectrum of activity.8 The characterisation of therapeutic phages in terms of their biology and bactericidal activity is mandatory before any preclinical trials because the application of non-characterised lytic bacteriophages may cause undesirable virulence as an adaptive response to bacteriophage infection.9 In other words, the development of effective strategies, such as phage growth parameters, host range spectrum, the presence of lytic proteins, formulation of phage cocktail and phage synergistic interaction with antibiotic to enhance phage efficacy, is a key prerequisite for optimal treatment of MDR infections caused by K. pneumoniae. Several studies have reported the advantages and drawbacks of phage cocktails in treating MDR bacteria.11 Unfortunately, bacteriophages employed as cocktail or adjunctive therapy have not been well investigated in the African continent to effectively control MDR bacterial infections.12 This study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of CP K. pneumonia clinical isolate in vitro in Kenya.The development of alternative control measures is urgently needed to manage the dissemination of carbapenemase-producing (CP) This study was not human or animal research. However, clearance was issued by the Nairobi City Water & Sewerage Company Limited (NCWSC/HR/TRG.14/Vol.8/14/MMM/ak) for collection of waste water samples.K. pneumoniae (KP20) was obtained from the stock of the recently published data in Kenya.13 Bacteriophages were isolated from waste water samples of Dandora Estate Sewage Treatment Works (Ruai) and Rongai effluent, Nairobi, Kenya. They were purified from waste water samples through spot assay and double-layer plaque method as described, previously.15The study was conducted from February 2021 to October 2021 in the Phage Biology Research Laboratory at the Institute of Primate Research, Nairobi, Kenya. The clinical isolate of CP The minimum inhibitory concentrations of a panel of antibiotics including carbapenems for KP20 were determined using VITEK 2 Systems version 9.2 and AST-XN05 (bar code 1481424403205844) card , according to the manufacturer\u2019s instructions. The detailed procedure is described in online Supplementary Method 1.8 PFU/mL or 109 PFU/mL) was propagated in large volume, concentrated and cleaned up in the presence of sodium chloride and polyethylene glycol as described elsewhere.16Phage lysate  uranyl acetate on 3 mm carbon-coated copper grids of the precipitated Klebsiella phages was conducted at the University of Leicester and the bacteriophages were visualised with a transmission electron microscope .17 Bacteriophages were then classified according to their morphology.18The morphological characterisation of the precipitated 19 Bacterial strains tested in our study included reference strains and MDR bacterial strains reported by Michodigni et al.13 and described in online Supplementary Methods 2.A spot assay was conducted to determine the host range of precipitated bacteriophages and phage cocktails as described by Kutter.20 Exponential-growth-phase culture of CP K. pneumoniae (\u2248 2.98 \u00d7 108 CFU/mL) and phage precipitate (\u2248 109\u20131010 PFU/mL) were mixed with bacterial suspension to obtain a multiplicity of infection (MOI) of 0.1.14 The suspension was incubated at 37 \u00b0C in a shaking incubator for 10 min at 120 rotations per minute following with centrifugation. The pellet was then suspended in 7 mL of fresh tryptic soy broth and incubated at 37 \u00b0C in a shaking incubator at 125 rotations per minute. At 3-min intervals, from 0 min to 36 min, 500 \u00b5L of the suspension was taken and phage titres were estimated.21A one-step growth experiment for determination of latent period, and burst size of precipitated bacteriophages, was performed as described elsewhere, with some modifications.Klebsiella phages, designated as CPRSA, CPRSB and ESBLA, were used for the formulation of two-phage cocktail (2\u03c6 MA) and three-phage cocktail (3\u03c6 MB). The two-phage cocktail comprised CPRSA and CPRSB while the three-phage cocktail consisted of CPRSA, CPRSB and ESBLA. Bacteriophage cocktails consisting of two and three tested phage lysates were formulated by combining in equal ratios of 1:1 and 1:1:1. The evaluation of the phage cocktails\u2019 effectiveness was carried out based on the method described by Merabishvili with some modifications.22 Two-phage (2\u03c6 MA) and three-phage (3\u03c6 MB) cocktails were composed and their ability to inhibit the growth of KP20 at its exponential-growth-phase was evaluated. Subsequently, the efficacy of two individual bacteriophages, designated as Klebsiella phage CPRSA and CPRSB at MOI 1.0, 0.1 and 0.001, was also investigated. Only broth media (tryptic soy broth) and individual bacteriophages represented the negative controls. Bacterial culture with a final resulting concentration of \u2248 2.98 \u00d7 106 CFU/mL served as positive controls. The OD600 of the host bacterium was measured every 4 h for 24 h, and finally at 48 h, to assess the inhibitory activity of bacterial growth by both individual phages and phage cocktails. The different absorbances obtained at 600 nm were compared to the ones of the positive control sample. The different concentrations of individual bacteriophages and phage cocktails are summarised in online Supplementary Three 600 \u2248 0.6). The two antibiotics meropenem and tigecycline  were tested in combination with 2\u03c6 MA and 3\u03c6 MB. The phage cocktails 2\u03c6 MA and 3\u03c6 MB with optimal concentrations (MOI 0.1 and 0.001) were used in this experiment in combination with the single and double of the minimum inhibitory concentrations of imipenem and tigecycline. Sterile broth media (tryptic soy broth), bacterial culture treated with antibiotics, and bacteriophage cocktails represented the negative controls. Bacterial culture with a final resulting concentration of \u2248 2.98 \u00d7 108 CFU/mL served as a positive control sample. The growth of the host bacterium was monitored at 3-h intervals, between 0 h and 24 h, and at 48 h of incubation by measuring OD600. The optical densities obtained at 600 nm were compared to the ones of positive and negative control samples. The different concentrations of phage cocktails and antibiotics are indicated in online Supplementary The inhibitory activity of the phage cocktail alone, and in combination with imipenem or tigecycline was assessed on KP20 at its optimal exponential growth . A p < 0.05 was considered as significantly different.A two-tailed 13 The ESBL and carbapenemase genes produced by KP20 are indicated in The minimum inhibitory concentrations of imipenem and tigecycline to KP20 were 4 \u00b5g/mL and 1 \u00b5g/mL . We prevA transmission electron microscope showed that the bacteriophages included in this cocktail were all tailed phages . Among t11 \u00f7 2.98 \u00d7 108), 1 (2.294 \u00d7 108 \u00f7 2.98 \u00d7 108) and 10 (3.123 \u00d7 109 \u00f7 2.98 \u00d7 108) for Klebsiella phage CPRSA, CPRSB, ESBLA. Regarding their latent period, Klebsiella phages CPRSA and CPRSB had the same latent period (9 min) and the one of ESBLA was 18 min as shown in online Supplementary The burst sizes of the precipitated phages in the cocktail were 610  and the three-phage cocktail (3\u03c6 MB). The two-phage cocktail comprised CPRSA and CPRSB while the three-phage cocktail consisted of CPRSA, CPRSB and ESBLA. The two-phage cocktail was the combination of two bacteriophages belonging to the family Myoviridae while the three-phage cocktail was the combination of 2\u03c6 MA and one belonging to the family Podoviridae. The results of the host range analysis showed that both the individual phages and phage cocktails (2\u03c6 MA and 3\u03c6 MB) were active on bacterial strains belonging to Klebsiella species. The three carbapenem-resistant K. pneumoniae were susceptible to only one individual phage, the Klebsiella phage CPRSA. The two-phage preparations were also active on the three CP K. pneumoniae strains, in addition to K2, which was described as an extended-spectrum beta-lactamase producer in our previous study.\u2248 0.3 was determined at three different multiplicities of infection . It was observed in our study that there was an increase in CP K. pneumoniae growth to the individual phage CPRSB at its three different doses and to the phage CPRSA at MOIs 0.1 and 0.001 after 8 h and 16 h of incubation . At the most effective phage dose (MOI: 0.001), the absorbances at 600 nm of the bacterial culture treated with the individual phage CPRSA, and the two-phage cocktail (2\u03c6 MA), increased from 0.019 to 0.515, and 0.173 to 0.246 at 16 h and 20 h, while it rose from 2.116 to 2.190 for the positive control during the same interval of time (p = 0.12) at MOI 0.1 after 16 h of treatment time (p = 0.01) was observed between the two treatments at MOI 0.001.The ability of the mixture of two phages (MA) and three phages (3\u03c6 MB) to inhibit the growth of KP20 culture at an absorbance of cubation . The two to 24 h . This ba of time . For theent time . Signifi600 decreased from 0.294 (21 h) to 0.189 (24 h) for bacterial culture treated with tigecycline and the two-phage preparation at the lowest phage concentration, MOI 0001 (TG1 + 2\u03c6 MA3) while it increased slightly from 0.207 (21 h) to 0.331 (24 h) in the case of bacterial culture treated with tigecycline and the three-phage preparation (TG1 + 3\u03c6 MB3). The absorbances of the bacterial culture treated with tigecycline was 0.249 (21 h) and 0.314 (24 h) while it ranged from 1.212 (21 h) to 1.245 (24 h) for the bacterial culture treated with imipenem. The positive control sample exhibited an optical density of 1.166 (21 h) and 1.270 (24 h). The level of significance between the bacterial culture treated with 2\u03c6 MA and the bacterial culture treated with IMP2 + 2\u03c6 MA3 after 21 h of incubation are shown in p = 0.02). No statistical difference was observed between the results of the bacterial culture treated with a combination of phage and antibiotic and those of bacterial culture treated with tigecycline alone. A significant difference (p = 0.04) was observed between the bacterial culture treated with 2\u03c6 MA at MOI 0.001 and the adjunctive therapy .The in vitro studies showed that the same phage cocktails (2\u03c6 MA and 3\u03c6 MB) maintained their bactericidal activity after 24 h of incubation at the lowest phage concentration (MOI 0.001) . At diffMyoviridae with contractile tails consisting of a sheath and a central tube (25% of tailed phages), Siphoviridae with long and noncontractile tails (61%), and Podoviridae having short tails (14%).23Klebsiella tailed phages identified in our study belonged to the family of Myoviridae and Podoviridae.24 Previous studies reported similar observations regarding the morphological characters of Klebsiella phages.25This study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of the KP20 clinical isolate. On one hand, the phenotypic results revealed that the precipitated phages obtained in this study had lytic activity with high titers and they were related to tailed phages. The latter are generally divided into three families including Klebsiella phage had a relatively short latency period of 18 min and its burst size was ~220 phage particles per infected bacteria.20 Indeed, a high burst size is key to achieve a productive adsorption and replication of bacteriophages and to reach the benefits that phages could have in comparison with antibiotics while a short latency period is recommended from a phage therapy perspective.26On the other hand, this study showed that one of the precipitated phages (CPRSA) had the shortest latency period (9 min) and high burst size (610). Our findings were supported by Horv\u00e1th et al. in 2020. The authors reported that their isolated Klebsiella phages reported similar results.29 The major consequence of phage host specificity is that it demands an appropriate diagnosis of the bacteria involved in the infection before initiation of phage treatment.30 Nevertheless, this narrowness of phage host range to the strains of the same bacterial species could limit its lytic activity on microbiota.30 The phage host range is indeed affected by a number of factors including the absence of required accessory proteins for phage adsorption, restriction-modification and clustered regularly interspaced short palindromic repeats (CRISPR) systems.32 Despite their narrow host range, bacteriophages with lytic activity may still be useful in phage therapy and the use of bacteriophages as cocktails for adjunctive treatment can represent a highly attractive strategy.33Both the individual bacteriophages and phage cocktails were not susceptible to the reference bacterial strains tested in this study and, hence, displayed high specificity for their host bacteria, which were KP20 clinical isolates. Previous studies on host ranges of 22 Therefore, our study demonstrated that the mixture of two phages (2\u03c6 MA) was able to significantly delay the resurgence of bacterial cells in culture, as compared to the application of individual phage or the mixture of three phages (3\u03c6 MB) (p = 0.02). Our result was in line with the previous data published on phage cocktail efficacy.11 This synergistic activity of two-phage cocktail over the mixture of three phages in our study suggested that the individual phages might have employed different receptors to adsorb to the bacterial cells and, hence, effectively inhibited the bacterial growth. Some authors have reported that one phage in their two-phage cocktail used an outer membrane protein (OmpC) as a receptor, and another one employed a lipopolysaccharide component as its receptor to effectively control bacterial resistance.35 Moreover, the mixture of many phages may be less effective in the absence of identification of specific bacteriophage receptors because the same phages might share the same receptors and interfere with one another.37Besides the choice of bacteriophages with different bacterial cell wall receptor recognition sites in formulating a phage cocktail, the most important criterion for successful phage cocktail preparation also includes the compatibility of bacteriophages in mixtures.p = 0.04). At MOI 0.001, the two-phage cocktail combined with imipenem had significantly lysed the bacterial cell compared to the two-phage cocktail. This statistical difference might be related to the beneficial effect of bacteriophage treatment in adjunctive therapy. Our finding contradicted the study of Pacios et al., who indicated the inability of phage-imipenem combination to kill imipenem-resistant isolate harbouring OXA-245 b-lactamase.38 This divergence might be related to the use of a single lytic phage in adjunctive treatment instead of phage cocktail. Furthermore, an antibacterial effect was also observed between the phage cocktails in combination with the most effective antibiotic (tigecycline) without significant difference (p = 0.99). A number of studies have reported the positive effect of bacteriophages in combination with antibiotics.39 This synergistic activity between phage cocktail in combination with imipenem might have been due to the sensitivity of the bacterial strains to the select antibiotic after bacteriophage action. Indeed, it was reported in a previous study that phage-resistant bacterial strains are more susceptible to antibiotics and the rate of their growth is slower in comparison with wild bacterial strains.40 Our study also demonstrated the efficacy of phage cocktail in the absence of phage receptor analysis in formulating phage cocktails. Interestingly, this study pointed out the repurposing of imipenem using phage cocktail therapy, and further encourages the repurposing of the efficacy of Food and Drug Administration-approved antibiotics for acceptance of phage therapy worldwide, especially in Africa.The adjunctive treatment (IMP2 + 2\u03c6 MA3) significantly inhibited the growth of KP20 in vitro compared to the two-phage treatment alone  against carbapenem-resistant K. pneumoniae clinical isolate in vitro.This current study revealed the presence of lytic tailed"}
{"text": "Bacteriophages, also known as phages, are viruses that selectively target and infect bacteria. In addition to bacterial dysbiosis, dermatologic conditions such as acne, psoriasis, and atopic dermatitis are characterized by a relative reduction in the abundance of phages and the overgrowth of the corresponding bacteria. Phages often exhibit high specificity for their targeted bacteria, making phage-replacement therapy a promising therapeutic strategy for the control of pathogenic bacteria in dermatologic disease. Novel therapeutic strategies regulating pathogenic bacteria are especially necessary in light of growing antibiotic resistance. In this review, we aimed to review the medical literature assessing phage dysbiosis and therapeutic trials in dermatology. Ultimately, studies have depicted promising results for the treatment of acne, psoriasis, and atopic dermatitis but are limited by low sample sizes and the omission of control groups in some trials. Additional work is necessary to validate the efficacy depicted in proof-of-concept trials and to further determine optimal treatment vehicles, administration mechanisms, and dosing schedules. This review provides the necessary framework for the assessment of phage efficacy in future trials. Corynebacterium diphtheriae have contracted the ability to produce their pathogenic toxin from genetic material acquired by phages [Bacteriophages, also known as phages, are a type of virus that selectively target and infect bacteria . Phages y phages . Additioy phages . Since py phages . It has y phages , whethery phages . This iny phages . Among tTo date, the literature has extensively researched and reviewed the strong association between skin health and a healthy skin microbiome. However, the role of the phageome in regards to skin health has not been as substantially explained. Currently, among the studies that have researched the skin phageome, many have highlighted the lack of a diverse viral database that has been a prominent limiting factor for fully assessing the types of bacteriophages present on the skin ,8. RegarThe unique properties of phages have contributed to the recent re-emergence of interest in phage therapy. Phage therapy involves the administration of phages that selectively target pathogenic bacteria in order to alleviate associated health conditions . AlthougPrevious investigations have assessed microbial and phage compositions among patients with dermatologic disease. Reduced phage concentrations are typically associated with an abundance of the corresponding bacteria, depicting the capacity of phages to suppress their host bacteria . Figure Staphylococcus aureus strains. Furthermore, bacterial alpha diversity has been shown to be reduced in psoriatic lesional skin than in healthy control skin [Affecting almost 3% of the population in the United States, psoriasis is a chronic inflammatory disease characterized by plaques and scaly lesions . Althougrol skin ,15. As prol skin . In a 2020 study, psoriatic lesional skin was obtained from the elbow, forearm, knee, and scalp of sixteen patients with psoriasis . HealthyAcinetobacter phage Presley, Salmonella phage vB_SenS-Ent2, and Bacillus phage SP-10 were the most differentially abundant phages. In addition, the authors observed a significantly greater bacterial alpha diversity in the healthy skin than the lesional skin (p < 0.001) [The authors noted a significantly greater abundance of the top ten most-abundant phage species in the healthy skin compared to lesional skin . Acineto< 0.001) ,18. Acinetobacter phage Presley and Pseudomonas phage O4, and their corresponding bacterial genera. While the family control skin depicted the highest abundance of these two phages, an intermediate and low abundance was demonstrated by the contralateral non-lesional skin and lesional skin, respectively (p = 0.02 for each phage) [p = 0.03 for Acinetobacter; p < 0.001 for Pseudomonas), demonstrating the capacity of phages to suppress the abundance of their host bacteria [The authors further examined two phage species, h phage) . In addibacteria . This fiAcinetobacter baumannii and Pseudomonas aeruginosa, such as wound infections. A 2012 study found that the density of P. aeruginosa decreased in the presence of phages in human skin (ex vivo), suggesting that phage application can effectively alter and correct microbiota compositions [The results of the study conducted by Wang et al.  support ositions . In addiositions , supportInterestingly, the oral consumption of Ganga river water, reported to contain over two-hundred isolates of phages, demonstrated clinical benefits in psoriatic patients who were previously refractory to prescription medication . PatientA Likert Scale from 1\u201310 was used to categorize the severity of psoriatic lesions. Following the first treatment protocol, the subjects achieved a median improvement of 2.5, although with relapse during the rest period . SimilarIn addition, the phage display of anti-inflammatory peptides has been evaluated for psoriasis-like lesions in mice . Phage dCutibacterium acnes proliferation, further contributing to inflammation and worsening acne severity [C. acnes has long been regarded to contribute to the pathogenesis of acne, recent work has highlighted other bacteria such as Staphylococcus epidermidis [Affecting an estimated 85% of individuals within their lifetime , acne vuseverity . Althougdermidis . NeverthC. acnes and a reduced alpha diversity in comparison to non-inflammatory lesions of acne patients and healthy control subjects [Proteobacteria and Firmicutes overabundance and a reduction in the abundance of Actinobacteria [Staphylococci compared to non-lesional skin, and the proportions directly increased with acne severity. Cutaneous and gastrointestinal microbial dysbioses associated with acne have been previously reported. Acne inflammatory lesions are associated with an overabundance of certain strains of subjects . Similarbacteria . ComedonC. acnes phage abundance have been observed. A 2016 study found a greater relative abundance of C. acnes phages in healthy skin compared to the matched lesional skin of acne patients (p = 0.05) [C. acnes phage abundance and age, suggesting phage abundance may contribute to lower acne vulgaris prevalence among aging individuals. In addition to bacterial dysbiosis between healthy skin and that of acne patients, differences in  = 0.05) , therebyC. acnes bacterial abundance coupled with decreased C. acnes phage abundance in acne patients provides a theoretical framework for the therapeutic replenishment of C. acnes phages. However, it is necessary to note that significant gene conservation has been observed among C. acnes phages, despite the great diversity observed in essentially all other phage populations [C. acnes has evolved strategies to adapt to its cutaneous environment and survive host defenses, such as lipolytic activity, pH regulatory mechanisms, biofilm formation, and the potential activation of dormancy. Such adaptation has resulted in a reduced bacterial diversity, which may have contributed to the observed conservation among C. acnes-infecting phage genomes [C. acnes strains, phage homogeneity poses a therapeutic challenge in that phage host receptor mutations may rapidly confer broad resistance [C. acnes, allows phage-based therapy to be promising for future clinical studies in acne. Increased ulations . C. acne genomes . While bsistance . Still, sistance . FurtherC. acnes-induced inflammation in murine models [C. acnes suspension. While group A received no additional intervention (control), group B received an additional injection with C. acnes at four weeks, and group C received C. acnes and bacteriophages at four weeks. A decreased epidermal thickness and a decreased number and size of microcomedone-like cysts were observed in groups B and C compared to the control group. In addition, the inflammatory nodule size decreased to the greatest extent in group C, depicting the potential efficacy of bacteriophages to reduce C. acnes-induced inflammatory nodules [A 2019 study assessed the effect of dorsal bacteriophage injection on e models . Three g nodules . C. acnes infection in mice, as the antibiotic resistance of C. acnes is growing and poses a challenge for conventional acne treatment [C. acnes followed by phage injection on one side. The C. acnes injection resulted in bilateral inflammatory nodules, although the authors observed a significant reduction in inflammatory lesions on the side injected with the phage. In addition, a significantly decreased expression of the inflammatory marker IL-1\u03b2 and the apoptotic marker caspase-3 was observed with phage therapy [In addition, a 2021 study assessed the effect of lytic bacteriophage treatment on multi-drug-resistant reatment . Mice we therapy . C. acnes-associated inflammatory lesions, although proper storage is necessary for retaining the lytic activity. In addition, the authors assessed the phage activity of different phage formulations in hydroxyethyl cellulose (HEC) cream . Full lyC. acnes lysis from human skin microflora and formulated them into a cetomacrogol aqueous cream (2.5 \u00d7 108 plaque-forming units (PFU) per gram) [C. acnes lysis activity marked a necessary step in creating a topical phage-based acne therapy targeting pathogenic C. acnes. However, the formulation was not directly assessed in human subjects. Similarly, a 2016 study isolated ten phages capable of er gram) . A retaiC. acnes-associated inflammatory nodules in murine models, an additional study evaluated the efficacy and safety of a topical phage formulation among human subjects diagnosed with mild-to-moderate acne [C. acnes with low susceptibility of commensal species. The BX001 cocktail was formulated into an aqueous topical gel. Ex vivo analysis on human skin depicted no irritation effects at any of the studied concentrations, leading the researchers to evaluate the formulations among seventy-five female acne patients stratified into three groups: control, low dose, and high dose. Those receiving the high-dose topical formulation depicted a significantly reduced C. acnes bacterial load compared to vehicle control at day 35 (p = 0.036), although a bacterial reduction was not observed in the low-dose group [C. acnes bacterial load upon the topical application of the phage. However, the authors did not assess the effect of the topical gel on clinical measures of acne, such as inflammatory lesion count or size. Additional research is necessary to assess the clinical efficacy of oral, topical, or intradermal phage formulations among human subjects with acne, although the described studies depict phage replacement to be a promising therapeutic strategy. Whereas the prior studies assessed the effect of phage therapy in the reduction in ate acne . The autse group . While bStaphylococcus aureus [S. aureus growth [Atopic dermatitis (AD) is a chronic inflammatory dermatologic condition commonly present during childhood. Clinically, it presents with patches of intense dryness, scaling, erythema, and pruritus that usually affects the face, neck, scalp, and flexor surfaces of the skin . Typicals aureus . The flas growth . S. aureus growth on the skin [S. aureus bacteria and does not disrupt the beneficial Staphylococcus epidermidis bacteria present on skin. The in vitro experiment involved assessing the effects of S. aureus growth in the presence of SaGU1. During the first 9\u201313 h, S. aureus levels began to decline in the presence of SaGU1; however, they gradually began to increase after 14 h. This finding suggested that it was probable that S. aureus began to become phage-resistant. A separate experiment involved determining the effects of SaGU1 on S. epidermidis growth, and the results showed that SaGU1 did not affect its growth. Interestingly, the researchers found that when both SaGU1 and S. epidermidis were supplied to S. aureus cultures, the regrowth of S. aureus after 14 h did not occur, and the levels remained suppressed. Therefore, a combination therapy of the phage SaGU1 and S. epidermidis may be beneficial in controlling S. aureus overgrowth. A study conducted in 2020 on atopic mouse models found promising results for the use of a phage in controlling the skin . The stuS. epidermidis or SaGU1 and in combination [S. aureus growth on the skin of atopic mice. However, when the combination therapy was compared to the phage SaGU1 treatment alone, there was no statistically significant difference in terms of effectiveness on S. aureus growth suppression. This suggested that phage therapy alone may be more effective in vivo than in vitro. Overall, these results are extremely promising for the field of phage therapy in treating AD. Although much more intensive research must be performed to determine if phage therapy is clinically useful, this study provided a unique treatment approach that warrants more investigation. During the in vivo phase of the study, atopic mouse skin was treated individually with bination . Each trPhage-based therapeutic strategies appear promising for the treatment of a variety of dermatologic conditions. There are many theoretical attributes of bacteriophages that make them ideal therapeutic candidates for reducing the presence of pathogenic bacteria implicated in disease: phages can be bactericidal, increase in number over time, minimally disturb commensal microflora, depict efficacy against antibiotic-sensitive and antibiotic-resistant bacteria, can potentially disrupt bacterial biofilms, and exhibit low toxicity . HoweverCertain phage characteristics predict their therapeutic success. First, in order to allow optimal bacterial lysis, virulent phages must be selected in contrast to temperate phages. Similarly, phages characterized by a poor killing potential against desired bacteria are sub-optimal for therapy. Toxin-carrying phages should be avoided in addition to those depicting a high transduction potential . Lastly,Additional factors mediate phage-based treatment efficacy, including the phage-to-target bacteria ratio, environmental conditions, accessibility to target bacteria, administration mechanisms, and bacterial resistance . First, Secondly, pH has been shown to affect phage titers, with decreasing pH associated with reduced titers. In addition, phages cannot freely diffuse across membranes and thus require a delivery method to reach target cells and bacteria. Administration techniques include oral, topical, intraperitoneal, intravenous, and intranasal administration . HoweverPseudomonas aeruginosa with resistant O-antigen deletion mutants [Lastly, experimental data has demonstrated the presence of phage resistance in up to 80% of studies targeting the intestinal microbiome . Bacteri mutants . The phaUltimately, phage therapy is not without limitations, including the development of resistance, environmental factors, and the necessary consideration of the optimal administration mechanism. Perhaps such limitations have contributed to the fact that only a few countries have accepted the use of therapeutic phages in humans thus far . HoweverBacteriophage dysbiosis has been observed on the lesional skin of psoriasis and acne patients. Experimental confirmation of the suppressing capacity of phages on their corresponding bacteria suggests that phage dysbiosis may contribute to the microbial dysbiosis characteristics of each dermatologic condition. In addition, phage deficiency with associated bacterial abundance suggests that phage replacement, either topically or orally via phage \u201ccocktails,\u201d may be an effective therapeutic strategy to mitigate the phenotypic signs and symptoms of disease. Few studies assessing phage-based therapies have been promising for the treatment of psoriasis, acne, and atopic dermatitis, both in murine models and human subjects. However, the current evidence is limited by low sample sizes and the omission of controls in some cases. Future research is necessary to assess the efficacy of phage replacement in large scale studies in addition to determining the optimal treatment vehicles, administration mechanisms, and dosing for particular purposes."}
{"text": "Xanthomonas euvesicatoria. Phages were isolated from the rhizosphere of tomato plants with symptoms of bacterial spot. The plaque morphology of all isolates was determined on a X. euvesicatoria lawn via a plaque assay. Three of the isolates were attributed to the family Myoviridae based on TEM micrographs. All phages showed good long-term viability when stored at 4 \u00b0C and \u221220 \u00b0C. Three of the phage isolates possessed high stability at very low pH values. Fifty-five-day persistence in a soil sample without the presence of the specific host and a lack of lytic activity on beneficial rhizosphere bacteria were found for the phage isolate BsXeu269p/3. The complete genome of the same isolate was sequenced and analyzed, and, for the first time in this paper, we report a circular representation of a linear but circularly permuted phage genome among known X. euvesicatoria phage genomes.Bacteriophages have greatly engaged the attention of scientists worldwide due to the continuously increasing resistance of phytopathogenic bacteria to commercially used chemical pesticides. However, the knowledge regarding phages is still very insufficient and must be continuously expanded. This paper presents the results of the isolation, characterization, and evaluation of the potential of 11 phage isolates as natural predators of a severe phytopathogenic bacterium\u2014 Xanthomonas euvesicatoria pv. euvesicatoria (formerly known as Xanthomonas euvesicatoria), X. euvesicatoria pv. perforans (formerly known as Xanthomonas perforans), Xanthomonas hortorum pv. gardneri (formerly known as Xanthomonas gardneri), and Xanthomonas vesicatoria) has cyclic peaks. It is an economically important disease and a severely limiting factor for fruit yield in these crops worldwide [Bacterial spot in tomato and pepper  that infect tomato and two species (X. vesicatoria and X. euvesicatoria) that infect pepper have been described in Bulgaria. The natural population of pathogens is heterogeneous by symptoms, species, phenotypic and genotypic characteristics, pathotype, and races [To date, three species  [X. vesicatoria (an Inoviridae phage [Myoviridae phage [Podoviridae phage) [X. perforans (unidentified phages) [X. gardneri  phage) [Xanthomonas phages, with only 134 fully sequenced genomes compared to 133 for Escherichia coli phages, 543 for Acinetobacter phages, 231 for Erwinia phages, etc. (NCBI). Out of these 134 fully sequenced genomes, only five are from Xanthomonas phages associated with bacterial spot xanthomonads: one X. euvesicatoria phage (access. number NC_054460); one X. campestris pv. vesicatoria phage (access. number MH206183); and three X. vesicatoria phages .Deep and extensive studies on the nature of bacteriophages and their role in combating severe bacterial plant diseases have been carried out in the last 20 years or so. Hopeful results regarding phage biocontrol have been reported for several devastating bacterial plant diseases, such as: potato soft rot , bacteri species . There hicatoria ,23,24; p Serbia) ,26; phagae phage , a Myoviae phage , and onee phage) ; phages  phages) ; and pha) phage) . MoreoveRalstonia solanacearum, Xanthomonas oryzae pv. oryzae, X. campestris pv. vesicatoria, X. euvesicatoria, and X. perforans [X. euvesicatoria phage K\u03a61 to different pH values, storage temperatures, and UV radiation in vitro has been reported [X. euvesicatoria, X. vesicatoria, and X. gardneri [It is known that the rhizosphere and leaf surfaces are the main areas for the application of phage-based biopesticides. Studies reporting phage survival after application in soil and the phyllosphere have been reported for phages infecting erforans ,32,33,34erforans ,23,35. Treported . The samgardneri . PerhapsX. euvesicatoria-specific bacteriophages and the determination of several main and specific characteristics concerning their biology and biocontrol potential. The aim of this paper was the isolation of X. euvesicatoria strains, including the type strain, regardless of their pathotype, race, and source of isolation (host and region). The strains of the remaining four species of phytopathogenic bacteria  were resistant to the studied phages  around the center. Slight differences were observed only in plaque dimensions. The diameter of the clear center ranged from 1 to 3.5 mm, with an average value of 2.3 mm, and the diameter of the whole plaque  ranged from 3 to 5 mm, with an average value of 4 mm . Myoviridae family (phages with long contractile tails), based on the classification of Ackermann [The transmission electron microscopy of the three selected phages showed that the virions had icosahedral capsids (approx. 50 nm) and long contractile tails (approx. 100 nm) . These rckermann  and Bradckermann .Xanthomonas phages such as K\u03a61 (KPhi1) . The fiPure phage lysates were stored under the chosen temperature conditions for a period of 11 months. The phage titers at 0h were determined and used for subsequent comparison with the titers obtained during the storage period. The concentration of viable phage particles was determined by point testing  . The obtX. euvesicatoria strains 105t and 269p) was investigated. We found that an inhibitory effect was only observed when applying undiluted buffers with pH 13.0 for both strains, as well as with pH 2.0 for strain 105t , and 7.5, respectively. Incubation at pH 9.0 and 11.0 caused a slight titer decrease of about 1 lg units only for phage isolate BsXeu269p/3 . A stron10 pfu/mL). No viable phages were detected on the 62nd day.The ability of phage isolate BsXeu269p/3 to retain its lytic activity in soil was studied  mainly related to the nitrogen cycle in the soil. For this purpose, the total number of these groups was previously determined in the soil sample used and is presented in ectively .X. euvesicatoria. This study was a continuation of our previous work [Bacterial spot disease in tomato and pepper plants is spread worldwide especially in countries with warm and humid climates. Disease management is difficult due to increasing bacterial resistance to conventionally used copper-based pesticides ,11. Therous work .X. euvesicatoria  and then characterize them according to parameters that are relevant for their further study, with a view to future application as biocontrol agents. In this paper, 11 bacteriophages isolated with the bacterial host X. euvescatoria are reported. The host range of four of them was determined, and the results showed that they could infect all strains of the species, regardless of the host and the region from which they were isolated. According to the widely accepted concept of host range determination, these phage isolates could be defined as broad-host-range phages [X. euvesicatoria in Serbia [X. euvesicatoria has been mainly reported as a bacterial spot pathogen on pepper [X. euvesicatoria-specific phages (XESP). This difference in the source of XESP isolation could be explained by the fact that in Bulgaria this pathogen infects tomatoes too [X. vesicatoria and X. gardneri [In this study, the main objective was to isolate and propagate phages specific to e phages . To our n Serbia ,26. The n Serbia . So far,toes too . In our gardneri .X. euvesicatoria phages by Gasic et al. [X. euvesicatoria, as member of the Xanthomonas genus, formed extracellular polysaccharide xanthan [Erwinia amylovora phage f-Ea1h [Pseudomonas aeruginosa [Escherichia coli K12 [Rhizobium trifolii [Xanthomonas phages forming clear plaques without a halo have also been reported [The morphology of plaques is an essential characteristic. All our phage isolates formed approximately identical plaques, as the differences were observed only in plaque size. The formation of clear plaques is typical for lytic phages . The prec et al. . X. euve xanthan , and thee f-Ea1h , Pseudomruginosa , Eschericoli K12 , and Rhitrifolii . Howeverreported .Myoviridae family had relatively small capsids (approx. 50 nm), but the plaques they formed were relatively big (4 mm). Some publications have claimed that the plaque size and capsid size are inversely related. Phages with a smaller capsid formed a large plaque in . The autMyoviridae family, such as T4-like and P1-like viruses [X. vesicatoria phage phiXaf18 [Although the genome of BsXeu269p/3 was best represented as a circular molecule, this phage has a physically rather linear, although circularly permuted genome, as in other representatives of the  viruses . CirculaphiXaf18 . The iniphiXaf18  were invXanthomonas phages [Xanthomonas arboricola phages [Different strategies have been studied for the long-term storage of pure or purified phage lysates , but the efficacy of each method varies and depends on the phages themselves . Generals phages ,29,53. Ha phages . Deep fra phages , but we Xanthomonas phages is between 5.0 and 11.0, with a few exceptions whereby phages have shown resistance to lower pH values (pH 4.0). To a large extent, our results were consistent with those reported by the latter authors. However, data demonstrating phage tolerance to pH 2.0 could also be found for other Myoviridae phages specific to P. aeruginosa \u0438 E. coli [Among the main strategies for the application of phage-based biopreparation in agriculture is administration via the rhizosphere or phyllosphere of plants. The phyllosphere is the part of the plant that is exposed to the action of a number of environmental factors such as UV radiation, wind, rain, and drought. These factors have been reported to affect the survival of phages on the leaf surface, thereby reducing their efficiency ,35. In t E. coli .X. euvesicatoria. The experiment was thus designed to determine the ability of the phage to remain viable under the conditions described. Furthermore, X. euvesicatoria is not a soil-borne phytopathogenic bacterium, so it can only persist in the soil for a limited period of time. Thus, we conducted an experiment aiming to more closely simulate natural soil conditions. The results obtained gave us reason to conclude that this phage would be suitable for soil application, since under these conditions it remained viable for 55 days. Similar results have been previously reported for several phages specific to E. amylovora, R. solanacearum, X. perforans, X. euvesicatoria, and X. oryzae, which can migrate from roots to the aerial parts of plants when applied to the rhizosphere [Another important feature of phages intended for soil application is their long-term persistence. In this study, we selected the phage isolate BsXeu269p/3, which was inoculated into a soil sample under in vitro conditions in the absence of its host, zosphere ,32,34,55Rhizosphere microorganisms play a key role in plant growth. Therefore, it is important that their structure and function are not negatively affected during the application of plant disease control practices in agriculture. Copper-based preparations kill not only the target phytopathogenic bacteria, but also many microorganisms that are beneficial to the plant. Bacteriophages are considered safe in this regard due to their high specificity towards the target microorganism. However, before a bacteriophage is proposed as a biocontrol agent, its impact on non-target microbiota should be investigated to demonstrate its safety. In our study, we demonstrated the safety of one phage isolate (BsXeu269p/3) against four groups of rhizosphere bacteria. X. vesicatoria, X. euvesicatoria, X. gardneri, and P. syringae pv. tomato) were used in this study  were used as host microorganisms for bacteriophage isolation , X. vesicatoria (14 strains), X. gardneri (3 strains), and P. syringae pv. tomato (1 strain)) and five types of cultures, shown in Pure bacteriophage cultures were obtained in LB broth with their target bacterium, according to the procedure described previously by Kizheva et al. , and stoX. euvesicatoria strains 105t and 269p were used as host bacteria for 11 phage isolates, 5 of which were designated as BsXeu105t and 6 as BsXeu269p. The analysis was performed by measuring the diameter of the plaques and the determination of the type of plaque center and edges.The morphology of the plaques produced by the phages on the surface of the semi-solid agar medium inoculated with the target bacterium was determined by DAOPA. 9 pfu/mL) was used for preparing the negatively stained formvar-coated grids for observation by TEM [The virion morphology of three phage isolates  was investigated by transmission electron microscopy (TEM). The bacteriophages were propagated on a semi-solid agar medium (Nutrient Agar (NA), Merck KGaA, Darmstadt, Germany) by DAOPA along with their target bacterium. Fresh phage suspensions were prepared in phage buffer , accordin by TEM . The obs\u00ae Syringe Filter 28 mm, pore size 0.22 \u03bcm, Sartorius, 37079 Goettingen, Germany) to completely remove residual bacterial cells; then, it was used for the extraction of phage genetic material by a NORGEN Phage DNA isolation Kit . The sequences were obtained after whole-genome sequencing by Illumina NovaSeq PE150 in Novogene (Novogene (UK) Company Limited, Cambridge, CB4 0FW, United Kingdom).Fresh phage suspension was obtained after the propagation of the phage isolate BsXeu269p/3 with its specific host on NA medium by DAOPA. Five milliliters of phage buffer  was pourwww.clcbio.com, accessed on 12 May 2022). The genome annotation was performed manually and using the online platform RAST [Raw sequences were assembled into contigs (contig 1 and 2) using Unicycler  . Two priorm RAST .The determination of phage survival at different storage temperatures (4 \u00b0C and \u221220 \u00b0C) was carried out using pure phage lysates, obtained after propagation in LB medium. Glycerol solution  was used as a cryoprotectant for phage lysates stored at \u221220 \u00b0C. Phage titers were determined by a spot-testing assay, and the determination was performed at various time intervals for the different phage isolates. The experiments were carried out in triplicate.X. euvesicatoria strains 105t and 269p were used as host strains. In order to obtain a pure phage suspension, 10 mL of each buffer solution was poured on the surface of the obtained phage cultures in petri dishes and incubated on a rotary shaker at room temperature for 1.5 h. At this step, only viable phage particles passed from the medium surface into the buffer solutions. The resulting phage suspensions were filtered through a membrane filter (pore size 0.22 \u03bcm) to completely remove the residual bacterial cells. The effect of the pH on phage viability was evaluated based on the difference in phage titer at 0 h, and that at 48 h was determined by a spot-testing assay, as follows: a series of tenfold dilutions of the phage suspensions were prepared, and then 10 \u00b5L of each dilution was spotted on the surface of the semi-solid agar (NA) medium along with bacterial hosts, followed by cultivation at 28 \u00b0C for 24 h. The sensitivity of the host bacteria to different pH values (controls) was determined using the same buffer solutions but in the absence of phage particles. A series of tenfold dilutions of the buffer solutions were prepared, and 10 \u00b5L of each dilution was spotted on the solid agar medium surface inoculated with the host bacterium (100 \u00b5L\u2013108 cfu/mL). All experiments were performed in duplicate. The design of the analysis is illustrated in Basic buffer solution with pH 5.92 was prepared as described by Msimbira et al. . It was The soil sample was collected from the rhizosphere of a healthy tomato plant in 2018 from a private vegetable garden in the village of Novo Panicharevo, Primorsko municipality, Burgas region, southeastern Bulgaria. The sample was stored in a sterile plastic jar at 4 \u00b0C and immediately transported to the laboratory, where it was analyzed. X. euvesicatoria-specific phages. Pure phage lysate was obtained for the selected phage isolate (BsXeu269p/3) in LB medium. The initial titer (1010 pfu/mL) of the resulting phage culture was determined by a spot-test assay. Ten milliliters of the phage lysate was added to a 30 g soil sample (in triplicate) in a sterile plastic jar and incubated at room temperature for a period of 62 days. The persistence of the viable phage particles was studied by DAOPA (host\u2014X. euvesicatoria strain 269p), and the phage titers on the 55th and 62nd day of incubation were compared to the initial phage titer.To determine the time limit of phage survival in soil, we used a soil sample free of naturally occurring \u22127) of the soil suspension in saline were prepared, and an aliquot of 100 \u00b5L of each dilution (in triplicate) was plated on the surface of the appropriate culture media  of beneficial rhizosphere bacteria  was calculated in a native soil suspension prepared in saline . The media, methods, and cultivation conditions applied in the analyses are listed in re media . The num10 pfu/mL was used. The analysis was carried out by two methods\u2014DAOPA and MPN. We used DAOPA to determine the ability of the phage isolate to form clear plaques on a lawn of previously enriched soil heterotrophs (in NAII medium) and nitrogen-fixing bacteria (in nitrogen-free broth medium)  or NFM agar (0.45% agar) for heterotrophs and nitrogen-fixing bacteria, respectively, was poured onto the surface of the respective agar media in previously prepared petri dishes. The phage lysate was diluted up to 10\u22127, and an aliquot of 10 \u00b5L of each dilution was spotted on the surface of the prepared double agar plates, which were cultivated at 28 \u00b0C for 24 h. The formation of clear plaques after cultivation indicated the presence of heterotrophic and nitrogen-fixing bacteria susceptible to the phage isolate. The MPN method was applied to study the ability of the phage isolate to reduce (by lysis) the amount of soil microorganisms in liquid media (\u22127), 100 \u00b5L pure phage lysate (1010 pfu/mL), and 10 mM CaCl2 was added to each test tube containing liquid media. To determine the influence of the phage isolate BsXeu269p/3 on the beneficial rhizosphere microorganisms, a pure lysate with a titer of 10 medium) . A mixtuid media . A mixtuX. euvesicatoria phages. This, along with the other characteristics possessed by this phage , makes it a potential candidate as a biocontrol agent against bacterial spot disease in tomato and pepper.Our study emphasized the necessity of studying bacteriophages as promising candidates for combating bacterial infections. We believe that our results for the genome organization of phage BsXeu269p/3 and the virion morphology of two more phages will contribute to the study of phage biology in general. Moreover, for the first time in this paper, we provided evidence of the circular permutation of a linear genome among the known"}
{"text": "The endocannabinoid system is involved in physiological and pathological processes, including pain generation, modulation, and sensation. Its role in certain types of chronic orofacial pain (OFP) has not been thoroughly examined. By exploring the profiles of specific salivary endocannabinoids (eCBs) in individuals with different types of OFP, we evaluated their use as biomarkers and the influence of clinical parameters and pain characteristics on eCB levels. The salivary levels of anandamide (AEA), 2-arachidonoyl glycerol (2-AG), and their endogenous breakdown product arachidonic acid (AA), as well as the eCB-like molecules N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), were assessed in 83 OFP patients and 43 pain-free controls using liquid chromatography/tandem mass spectrometry. Patients were grouped by diagnosis: post-traumatic neuropathy (PTN), trigeminal neuralgia (TN), temporomandibular disorder (TMD), migraine, tension-type headache (TTH), and burning mouth syndrome (BMS). Correlation analyses between a specific diagnosis, pain characteristics, and eCB levels were conducted. Significantly lower levels of 2-AG were found in the TN and TTH groups, while significantly lower PEA levels were found in the migraine group. BMS was the only group with elevated eCBs (AEA) versus the control. Significant correlations were found between levels of specific eCBs and gender, health-related quality of life (HRQoL), BMI, pain duration, and sleep awakenings. In conclusion, salivary samples exhibited signature eCBs profiles for major OFP disorders, especially migraine, TTH, TN, and BMS. This finding may pave the way for using salivary eCBs biomarkers for more accurate diagnoses and management of chronic OFP patients. Chronic orofacial pain (OFP) is a debilitating condition that is associated with the structures innervated by the trigeminal nerve . It is one of the most common pain conditions, with a reported prevalence of 7\u201311% in the general population . The difDue to the nature of pain as a sensory experience that cannot be directly quantified or measured , subjectSensation to the orofacial tissues, such as teeth, facial skin, TMJ, and adjacent musculature, is mainly supplied by branches of the trigeminal (V) nerve . Pain paN-arachidonoylethanolamide (anandamide) (AEA) and 2-arachidonoyl glycerol (2-AG)); and (3) enzymes, which are responsible for the biosynthesis/inactivation of the ligands. Furthermore, AEA is a member of the N-acylethanolamines (NAEs) family, which includes the eCB-like compounds N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA) [In this context, recent research found that the endogenous cannabinoid (endocannabinoid (eCB)) system is essential in pain pathophysiology , and thede (OEA) . N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) catalyzes AEA biosynthesis, whereas 2-AG is catalyzed by sn-1-specific diacyl-glycerol lipase (DAGL). The released eCBs are retrieved by a membrane transporter. AEA is degraded by fatty acid amide hydrolase (FAAH), whereas monoglyceride lipase (MAGL) is the main 2-AG hydrolase [eCBs are synthesized and released locally by enzymatic cleavage of membrane phospholipids in response to physiological and pathological stimuli . N-acylpydrolase . 2-AG isydrolase . AEA alsydrolase . ECS comydrolase , which bydrolase . Based oydrolase , it is rWhereas blood serum/plasma is the most frequent source of measurable biomarkers, saliva has many advantages over blood as a collectible bio-fluid, such as being easy to collect in a non-invasive manner and safer to handle. Many substances enter saliva from the blood via intercellular spaces due to transcellular or paracellular diffusion, and most substances found in the blood are also present in saliva, reflecting physiological and pathological states, making it a useful source of biomarkers for pain research .A recent study conducted by our group demonstrated significantly reduced levels of salivary eCB in chronic OFP disorders, which were categorized by etiology  . This avA total of 126 individuals participated in the current study, 83 of which had OFP, including TMD (25.3%), migraine (28.9%), TTH (6%), PTN (13.4%), TN (13.4%), and BMS (8.5%). A total of 4.8% were defined as \u201cothers\u201d. Of the 126 participants forming the cohort, 83 (66%) had OFP, while 43 (34%) were pain-free and formed the control group. Of the OFP group 21 (25.3%) had TMD, 24 (28.9%) had migraine, 5 (6%) had TTH, 11 (13.4%) had PTN, 11 (13.4%) had TN, 7 (8.5%) had BMS, and the remaining 4 (4.8%) were defined as \u201cothers\u201d.The primary headaches group represents the sum of migraine  and TTH, while the neuropathic group included PTN, TN, and BMS. Considering the vast amount of data collected and the many comparisons performed between the clinical factors and patient pain characteristics for each of the five measured eCBs, we decided to only present the statistically significant results. It is important to note that for some of the diagnoses, e.g., BMS and TTH, there were not enough patients for within-group comparisons.The limited understanding of the etiology and pathogenesis of OFP disorders, the subjective nature of current pain assessments, and the limited efficacy of existing treatment options highlight the urgent need for objective data to assist with chronic pain evaluation and management. The identification of mechanistic biomarkers is crucial since it would not only improve our understanding and ability to diagnose pain disorders accurately but also facilitate the development of disease-modifying drugs . Over thOur data demonstrated significantly lower salivary PEA levels in the migraine group compared with the controls. No other significant differences between these groups were found. PEA, an endogenous fatty acid amide signaling molecule, is synthesized on demand as a protective response to tissue injury or stress as part of homeostatic mechanisms. It has anti-inflammatory, pain relieving, and neuroprotective actions . PEA\u2019s dOne of the most important anti-inflammatory effects of PEA is the inhibition of mast cell activation , which iN-acylethanolamine acid amidase (NAAA), which catabolize PEA and AEA at significantly different rates [Interestingly, in contrast to our findings, Sarchielli et al.  found thnt rates , it is pnt rates .Our data also demonstrated low salivary 2-AG levels in the TTH group compared with the controls. Although TTH is the most prevalent type of headache, its pathophysiology remains unclear . InterreWe also found significantly lower levels of salivary 2-AG in the TN group. TN is a chronic neuropathic condition that affects one or more divisions of the trigeminal nerve . Devor eN-arachidonoyl-dopamine. Additionally, the intrinsic efficacy of AEA on TRPV1 is lower than at the CB1 receptor, implying that in the presence of CB1, AEA action on TRPV1 may be attenuated [Surprisingly, we found an elevation in salivary AEA levels for BMS compared with the controls. No other significant differences between these groups were found. BMS is a chronic neuropathic pain disorder characterized by an oral mucosal burning sensation and is frequently associated with xerostomia and dysgeusia . The etitenuated . AEA is tenuated . Indeed,tenuated . Therefore, despite AEA being a known anti-nociceptive eCB and its stimulation of cannabinoid receptors producing analgesic effects, it may act as an antagonist in the presence of full cannabinoid agonists . Taken together, we hypothesize that the significant elevation of salivary AEA in BMS with no concomitant elevations of full eCB agonists, such as 2-AG, reflects a faulty mechanism of the ECS in these patients. The elevated AEA accompanied by altered epithelial tongue cell eCB receptor expression  implies In addition to the correlations between specific pain disorder subgroups and salivary eCB levels, we also found a correlation between ECS activity and patient characteristics in three pain disorder groups: TN, PTN, and migraine. There were significantly lower salivary AA levels in women than men in the PTN group and this trend was also noted for OEA and PEA levels in the migraine group. These findings are consistent with Cupini et al. , who detInterestingly, our data indicated a significant negative correlation between the VPS and salivary OEA and PEA levels in the migraine group. This is congruent with previous studies, where chronic administration of PEA reduced pain behaviors and counteracted spinal neuronal hyper-excitability in murine models of persistent pain ,41.HRQoL reflects the perceptions and reactions of an individual to their health status and the non-medical aspects of their lives and assesses their psychological functioning and, to a smaller extent, physical functioning . We founWe found that a BMI above 30 was significantly related to higher salivary 2-AG levels in the migraine group. This is consistent with previous studies, where levels of circulating 2-AG positively correlated with BMI, total body fat, and intra-abdominal adipose tissue ,43. ThisIn the TN group, a positive correlation was found between sleep awakenings and salivary AEA, OEA, and PEA levels. Significant OEA elevation was shown in human CSF after 24 h of sleep deprivation . It is pFinally, our data demonstrated a significant positive correlation between pain duration and salivary AA levels in the TN group. This may have been due to pain-management-related symptom relief over time. This assumption is strengthened by the inverse correlation between pain intensity and salivary eCB levels .There were some important limitations to this carefully designed study. First, we only measured salivary eCB levels from samples taken at a single point in time, gathering samples over more time points may reveal other important information, such as correlations between treatment outcomes and eCB levels or their correlation with long-term clinical symptoms. Second, there were several therapeutic treatments with different efficacies, which could have affected the salivary eCBs concentrations. Third, serum eCB levels were not measured in our study. Thus, a correlation between salivary and circulating eCBs could not be evaluated. Fourth, we had relatively small sample sizes for each subgroup, which limited the comparative statistical analysis.The study was approved by the Ethical Committee of Hadassah Medical Center, request no. 0662-17-HMO. All data were fully anonymized; informed consent was waived according to the ethical committee\u2019s instructions. The medical records of 83  OFP patients meeting our inclusion criteria attending the Orofacial Pain Clinic at the Hebrew University-Hadassah School of Dental Medicine between 2017 and 2018 were reviewed. Forty-three  pain-free participants formed the control group.Inclusion criteria: over 18 years of age, definite diagnosis of chronic OFP for at least 3 months according to the IHS or ICOP ,48, and Exclusion criteria: background illnesses, including cancer or diseases affecting the salivary glands, such as Sj\u00f6gren syndrome; alcohol use; patients who did not sign the consent form; and patients whose saliva sample was unusable, e.g., too foamy or bloody.The pain patients were divided based on their diagnoses:Temporomandibular disorders (TMD) according to the diagnostic criteria for temporomandibular disorders (DC/TMD) , which aPrimary headaches , including migraine  and tension-type headache (TTH), according to the ICHD-3 . Trigeminal neuralgia (TN) , which iPost-traumatic trigeminal neuropathy (PTN) , which iBurning mouth syndrome (BMS), which is a chronic pain condition characterized by a moderate-to-severe sensation of burning from the oral mucosa, especially from the dorsum of the tongue with no clinical signs . TN, PTN, and BMS formed the \u201cneuropathic\u201d group. Other diagnoses that were rare and non-specific (8 patients) formed the \u201cothers\u201d group.Primary and resultant data were recorded on the standard intake form used in our clinic ,53. DemoUnstimulated saliva was collected for 10 min as described previously ,59 into Saliva samples were centrifuged at 3500\u00d7 g for 10 min at 2 \u00b0C to remove insoluble materials, cell debris, and food remnants. The supernatant fraction was aliquoted into polypropylene tubes and immediately stored at \u221280 \u00b0C. The running order of the samples was cycles of 2 samples from pain patients with the same diagnosis and then a control sample with the same gender and general BMI. 3:MeOH  and then washed with ice-cold chloroform three times. The samples were then dried under a thin stream of nitrogen and reconstituted in MeOH. Analysis using LC-MS was performed on an AB Sciex  Triple Quad 5500 Mass Spectrometer and a Shimadzu  UHPLC System, while the liquid chromatographic separation was acquired via a Kinetex (Phenomenex) column . Sample levels of AEA, 2-AG, AA, PEA, and OEA were measured against standard curves and then expressed as fmol/mg protein.The extraction, purification, and quantification of saliva eCBs were performed using stable isotope dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) as previously described . BrieflySPSS version 25 software was used for all calculations. To examine the differences in eCB levels for nominal and categorical background variables, t-tests and one-way analysis of variance were performed, and when significant differences were found, additional post hoc Scheffe tests were performed. A Spearman coordinator was used to examine the specific categories that made up the differences. The differences between the eCB types and specific background variables were examined for each diagnosis using the Kruskal\u2013Wallis test.Our findings suggested that the ECS played a significant role in the pathogenesis of OFP disorders. In addition, the non-invasively gathered salivary samples exhibited signature eCB profiles for prominent OFP disorders. Therefore, the profile of salivary eCBs and eCB-like molecules may be used as biomarkers to aid in the diagnosis and management of these patients. Future research is needed to evaluate whether salivary eCB levels correlate with serum levels in chronic OFP disorders, to understand the underlying mechanisms of altered ECS found in these patients, and to evaluate the therapeutic use of cannabinoids possibly as part of a personalized medicine approach for the management of chronic OFP disorders."}
{"text": "Strike action in healthcare has been common over the last several decades. The overarching aim of this systematic review was to synthesise and analyse the empirical literature that examines the impact of strike action on patient morbidity, that is, all patient outcomes except mortality. After conducting a search and apply eligibility criteria, 15 studies were included in this review. These articles included a variety of outcomes from hypertension control to rates of chlamydia. Strikes ranged from 13 to 118\u00a0days, with a mean strike length of 56\u00a0days. A textual narrative synthesis was employed to arrange studies by whether they had a positive, mixed or neutral or negative impact on patient morbidity. Results suggest that strike action has little impact on patient morbidity. The majority of studies reported that strike action had a neutral or mixed impact of strike action on patient morbidity. One study reported positive outcomes and three studies reported negative outcomes, however in both cases, the impact that the strike had was marginal. Strike action is widely debated with a major concern related to patient outcomes, including patient morbidityStrike action has little impact on patient morbidity, almost all studies reported no adverse outcomesStrike action by healthcare staff can be carried out safely in relation to patient morbidity Strikes have occurred on almost every continent, for a range of reasons. They have been carried out over a matter of hours to hundreds of days. While healthcare strikes raise a range of issues, one of the most pressing that is almost always raised relates to the impact that the action could have on patient wellbeing. That is, most debates centre on the impact that strike action could have on patients, with those arguing both for and against such action citing patient safety as a major concern.Over the years there has been a growing body of evidence that has examined the impact of strike action on the health and wellbeing of patients. The majority of this evidence has examined patient mortality, with evidence suggesting that generally, strikes do not significantly change patient mortality in\u2010hospitalThe overarching aim of this review is to synthesise and analyse the empirical literature that examines the impact of strike action on patient morbidity, that is, all patient outcomes except mortality. This reviews seeks to (1) understand if strike action has an impact on morbidity and if so (2) what factors related to the strike, or the health of patients in particular impact these outcomes.22.1A systematic review was employed to identify and synthesise all relevant literature in relation to the above research questions. PRISMA and ENTREQ reporting guidelines were followed.2.2The following electronic databases and time periods were searched: EMBASE (1980\u20132021), MEDLINE (1946\u20132021), CINAHL (1982\u20132001), BIOETHICSLINE (1972\u20131999), EconLit (1969\u20132021), WEB OF SCIENCE (1960\u20132021). In addition, grey literature was searched through OPEN GREY, and SIGMA REPOSITORY. Search terms were developed to capture the core concepts related to the form of intervention we were interested in  and the populations in question . The final search terms were: strike OR \u2018industrial action\u2019 OR \u2018industrial dispute\u2019 OR \u2018collective action\u2019 AND doctor OR physician OR clinician OR \u2018medical practitioner\u2019 OR nurs* OR \u2018health profession*\u2019 OR healthcare OR \u2018health care\u2019 OR \u2018pharmac*\u2019 OR \u2018dentist\u2019 OR \u2018midwi*\u2019 OR dieti* OR \u2018occupational therap*\u2019 OR \u2018paramed*\u2019 OR \u2018physiotherap*\u2019 OR \u2018radiograph*\u2019 OR \u2018psycholog*\u2019 OR \u2018health worker\u2019 OR \u2018hospital\u2019. There were no publication dates or language restrictions. Where complete data for a relevant outcomes was not available we contacted authors to request data. In addition, we conducted a manual search of the reference lists of eligible studies.2.3The initial search returned 5728 results, which were imported into Endnote where duplicates were removed. This left 4415 articles. The title and abstract of these articles was scanned and articles not meeting the inclusion criteria were removed. After the initial screen, 392 articles remained and a second full\u2010text screen was undertaken and reference lists were searched. A further four papers were found and all 396 articles were assessed against the below eligibility criteria, leaving 15 articles  or patient mortality.They examined patient mortality during strike action.Papers were excluded if:2.4Data from the included studies was extracted by RE, checked by SMW and categorised according to the source, country of where the research took place, study aims and objectives, research methods/design, the context of the study, nature of the strike, main outcomes, and quality appraisal scores and issues .2.6Studies were combined to summarise descriptive statistics of the study characteristics, followed by a textual narrative synthesis. This approach arranges disparate study types into more homogenous sub\u2010groups which aids in the synthesising of different types of evidence and in this case, answering research questions which can be informed by multiple methodological approaches.3The 15 articles included measured a variety of outcomes from hypertension control to rates of chlamydia. The articles also included substantial geographic diversity with studies from Europe, North America, Africa and Asia. Six studies examined strikes by doctors, four examined strikes by nurses, with the remainder of studies examining strikes by ambulance staff, \u2018non\u2010professional employees\u2019, government employees and multiple healthcare staff from a mental health centre. Strikes ranged from 13 to 118\u00a0days, with a mean strike length of 56\u00a0days. These results are summarised in Table\u00a03.1Thirteen of the papers included in this review were reviewed against the criteria set out in the NOS. Two papers were excluded as they did not utilise observational designs.3.2Papers were categorised into whether they had a negative, mixed or neutral or positive impact on patient morbidity. Studies were categorised as having a negative impact when they reported worse patient outcomes during a strike. Studies were categorised as neutral when they reported no substantial impact on patient outcomes during a strike. And studies were categorised as positive when they reported an improvement in patient outcomes during a strike. Studies were categorised as mixed when they reported a mixture of positive, negative or neutral results. There were three studies that reported a clear negative impact. These studies varied substantially. There were four studies that reported a mixed impact of strike action. Amongst these studies, three were conducted in Canada and one in Israel. One Israeli study reported a positive impact of strike action. There were no obvious patterns that linked these outcomes to the nature of the strike, for example, the length of strike, when or where the strike occurred.3.3There were three studies that reported a clear negative impact. These studies varied substantially. These were carried out in Kenya, Denmark and the UK. One involved ambulance staff, the two involved nursing staff. The strike in Kenya lasted 100\u00a0days, the strike in Denmark lasted 60\u00a0days, while the strike in the UK last 35\u00a0days. The studies examined the impact on immunisation services, diabetic control and more general outcomes from a day hospital.Taking a closer look at the first of these studies, Njuguna3.4There were four studies that reported a mixed impact of strike action. Amongst these studies, three were conducted in Canada and one in Israel. Strikes ranged in length from 31 to 118\u00a0days. Each study examined a different group of workers; doctors, nurses, \u2018non\u2010professional\u2019 health workers and sexual health programme workers. The outcomes examined include hypertension control, mental health, caesarean birth rates and prevalence of Chlamydia.In one of the earliest studies Norman and MallaThere were seven studies that reported a neutral impact of strike action. These studies were again diverse conducted in the UK, Canada, India, Spain, Finland and two in the US. Strikes varied in length from 13\u00a0days in Finland to 69\u00a0days in India. Five studies examined strikes that involved doctors; two studies examined strikes that involved ambulance workers and healthcare staff. The outcomes examined included paediatric pneumonia, high\u2010risk deliveries, nosocomial infection, appendectomies. Three studies examined more general mental and general health outcomes or multiple outcomes.Looking more closely at these studies, Crocker et\u00a0al.3.5One study reported a positive impact of strike action. Sigal et\u00a0al.4This paper sought to synthesise and analyse the empirical literature on the impact of strike action on patient morbidity in an effort to understand if strike action has an impact on morbidity and if so what factors related to the strike or patients impacted these outcomes. As a whole, the literature suggests that strike action has little impact on patient morbidity. The majority of studies reported that strike action had a neutral or mixed impact of strike action on patient morbidity. One study reported positive outcomes and three studies reported negative outcomes, in each case however and with the exception of NjugunaThe studies included in this review were relatively heterogeneous in the outcomes they examined and the context in which they occurred, so there is a general need for caution in how these results are interpreted. Furthermore, we have been deliberately broad in regards to the studies included here. Some overlap substantially with the provision of services. For example, it is arguable that some studies measure disruption to service rather than patient outcomes. For example, while NjugunaOver the last few years and since the COVID\u201019 was declared a global pandemic strike action appears to have become increasingly common across the globe.The authors declare that they have no competing interests.Ethics approval was not sought or required for this study."}
{"text": "The design and manufacturing technology of interference-absorbing short-wave filters based on a layered composition of Si\u2013SiO on a sapphire substrate of various shapes was developed. A transition layer of SiO was applied to the surface of the substrate, alternating with layers of Si\u2013SiO with an odd number of quarter-wave layers of materials with high (Si) and low refractive indices (SiO), and the application of an outer layer of SiO as an appropriate control of the materials\u2019 thickness. The optical properties of the infrared light filter were studied. It was established that the created design of the light filter provides the minimum light transmission in the visible region of the spectrum from 0.38 to 0.78 \u00b5m and the maximum in the near infrared region from 1.25 to 5 \u00b5m and has stable optical indicators. A method for studying the stress\u2013strain state and strength of a multilayer coating of a light filter under the action of a local arbitrarily oriented load was developed. For simplicity in the analysis and for obtaining results in the analytical form, the one-dimensional model of the configuration \u201cmultilayer covering\u2014firm substrate\u201d constructed earlier by authors was used. From a mechanical point of view, the upper protective layer of the multilayer coating was modeled by a flexible plate, and the inner operational composite N-layer was subjected to Winkler\u2019s hypothesis about the proportionality of stresses and elastic displacements. Devices equipped with various designs of optical filters  for optiAccording to the ISO 20473:2007 standard, infrared radiation is divided into three ranges: near infrared radiation\u2014from 0.780 to 3 \u03bcm; mid-infrared radiation\u2014from 3 to 50 microns; far infrared radiation\u2014from 50 to 1000 microns.As optical elements for the near-infrared region of the spectrum, multilayer interference-absorption short-wave cut-off filters are used as part of powerful optical devices (exit window). These filters adjust the spectrum of the broadband emitter, passing infrared radiation and absorbing/reflecting visible radiation. It is known that the reflection coefficient of the light filter increases with the increase in the number of layers, and an odd number of layers exerts a greater influence on the reflective capacity of the interference coating than an even number.The Doublet Metalens  was deveThe design of an ultra-broadband and highly efficient beam splitter based on a quasi-continuous metasurface in the near-infrared range, proposed in , consist3) and yttrium oxide (Y2O3) dielectric, with a critical defect of the high-temperature superconductor, yttrium-barium-copper oxide. It is a superconductor-based multichannel photonic crystal optical filter tunable in visible and telecommunication windows at cryogenic temperature. In [The authors  developeture. In , an inte2O5) and based on alternating layers with a high and low refractive index, respectively. The researchers [The study  proposedearchers  developeWhen designing filters, researchers mainly take into account recommendations ,12. They2 oxide films with Sn improves their photocatalytic properties, and carbon with palladium nanoparticles provides it with electrocatalytic properties [Computer modelling is widely used to predict photovoltaic properties , phase coperties ,26.During the construction of light filters at the stage of design and technological preparation of production, it is advisable to take into account technological heredity  to ensurResearchers  proposedWear-resistant coatings are formed by thermovacuum evaporation on piezoceramic materials and silicon probes for atomic force microscopy ,36. To iOne of the modern trends in the development of methods for assessing the strength and durability of modern structures is the improvement of interference-optical methods of the mechanics of a deformable solid body. The essence of these methods is that transparent materials become optically anisotropic under the influence of mechanical stresses . The useCalculation of thin-walled layered shells under radial load str given in works ,48, and There are well-known methods for solving problems of thermal conductivity ,55,56 anAs the above review shows, researchers have not paid due attention to the study of the optical and physico-mechanical properties of layered coatings under extreme conditions of high temperatures, under the action of arbitrarily oriented local mechanical loads, and the defectiveness of their structure.This study aims to investigate the optical properties and develop an engineering method for calculating the stress state of the layered coating of the interference-absorption filter under an arbitrarily oriented local mechanical load.producing experimental samples of interference light filters of various configurations;investigating their optical properties at different lengths of the light flux;developing an engineering methodology for calculating the stress state of the layered coating of the interference-absorption filter under an arbitrarily oriented local mechanical load and to investigate the stress state of the composition.To achieve the goal, the following tasks should be solved:First, the workpiece of the sapphire substrate was cut and mechanical processing was carried out\u2014diamond grinding and polishing. After that, washing, degreasing, and drying were carried out. The substrate blank prepared in this way was installed in the chamber of the device for forming light filter layers, the chamber was evacuated, and the sapphire substrate was preheated. On the flat surface of the substrate, a transition layer of SiO was deposited (by resistive method) and its thickness was controlled; a layer of Si was deposited (by electron beam method sputtered) and its thickness was controlled; a layer of SiO was then deposited and its thickness was controlled; and again a layer of Si was deposited and its thickness was controlled, etc., until the given was an odd number of light filter layers  has a high value of thermal conductivity, while meeting one of the important requirements\u2014effective heat removal under high temperature loads during use in powerful infrared optics. High-resistance silicon and silicon monoxide fraction 2 were used for sputtering the layers. All the materials used were particularly clean in terms of purity. Si and SiO materials have high adhesive characteristics (bond strength) and satisfactory resistance to the formation of residual thermal stresses both to the sapphire substrate and to each other, which will ensure high optical and mechanical characteristics of the wear composition and a long service life in extreme conditions.\u22123 Pa. The VU-1A vacuum sputtering unit was equipped with a UELI-1 electron-beam evaporation source and a resistive heater, as well as a SFKT-751 thickness photometric control complex . The SFKT-751 photometric control complex , St.-Petersburg, Russian Federation) was designed to control the optical thickness of films that form a coating during their application on VU-1A type vacuum units. The self-writing two-coordinate device N-307  was designed for registration in rectangular coordinates in linear or logarithmic scales of the functional dependence of two measured values, presented in the form of electrical signals of electric current voltage. The materials for sputtering the Si layer were placed in a crucible, and the SiO layer in a boat. The accelerating voltage was 12 kV, and the current was up to 250 mA. Silicon layers were sputtered by electron beam method, and silicon monoxide was deposited by resistive method. Control of the spraying process of coating layers was carried out experimentally by the photometric method using the SFKT-751 system and the N-307 device. The control wavelength was 690 nm. The measurement system consisted of the following main parts: a radiation source, a monochromator, a photoreceiver, an amplification unit, and a registration unit. The device uses the phenomenon of interference: under monochromatic illumination, the intensity of light falling on the sample increases (decreases) to \u00bc the optical thickness of the film, and then begins to decrease (increase) to \u00bd the optical thickness. Thus, the thickness of the film can be determined by the formula: n\u2219d = m\u2219(\u03bb/4), where n is the refractive index; d\u2014thickness; m is the number of maxima or minima; \u03bb is the wavelength of monochromatic light. The two-coordinate self-recording device N-307 registers extremes of light intensity.For conducting the research, samples of interference light filters of a round shape with a diameter of 30 mm and a rectangular shape with a length of 150 mm and a width of 80 mm were made with a thickness of sapphire substrate of 3 mm. The Si\u2013SiO interference multilayer coating was applied to a specially prepared sapphire substrate preheated to 300 \u00b0C using thermal and electron beam evaporation methods in a VU-1A vacuum unit under an excess vacuum of 3\u00b710The total number of Si\u2013SiO layers is chosen depending on the functional purpose of the light filter. The produced interference light filter contained a transition layer with a low refractive index (SiO), an odd number of quarter-wavelength optically thick layers of materials with high (Si) and low refractive indices (SiO), the layers of which alternate, and an additional outer layer with a low refractive index (SiO), which has a larger optical thickness .To study the optical properties of light filters, optical transmittance studies were conducted for the ultraviolet, visible, and infrared regions of the spectrum, using round and rectangular samples.-Optizen 3220 UV  double-beam spectrophotometer, which is designed for measuring transmission coefficients, optical density and scanning transmission or absorption spectra in a given wavelength range of ultraviolet and visible radiation from 190 nm to 1100 nm;-Spectrometers Spectrum One FT-IR  in the wavelength range from 1.25 \u03bcm to 10 \u03bcm.Optical transmission tests of light filter samples were performed on:Measurements were performed with a scanning step of 1 nm.The structural elements of the interference-absorption filter  consist In an effort to obtain the final result in an analytical form, in this paper we developed a one-dimensional stress analysis proposed earlier for two-layer compositions ,78,79. TMechanically, we considered the upper protective SiO-layer as a tensing and bending plate interacting with the multilayered filter. At the same time, the operational compositional N-layer Si\u2013SiO set meets Winkler\u2019s hypothesis on the proportionality of tangential and normal stresses to respective elastic displacements. To simplify our analysis, we assumed that the sapphire substrate was absolutely rigid, and that the mechanical contact between the components on the layer interfaces was ideal. Moreover, we assumed the plane deformation state as Bd2uxLet us define the coefficients of substrate rigidity for the multi-layer depth-inhomogeneous filter as the values inversely proportional to the total compliance of series-connected layers:The forces and moments vanish at infinity:Thus, the boundary problem (1), (2) describes the required field of displacements of the coating-substrate/plate on the elastic layered substrate.The technological features of round and rectangular light filter manufacturing are very different, especially when their geometric dimensions are increased. The asymmetry of the sample shape can affect the inhomogeneity of the functional characteristics of the product\u2019s layered coating on the plane. Therefore, samples of light filters of various shapes and sizes were produced, and their optical and mechanical characteristics were investigated. The general appearance of light filter samples with Si\u2013SiO interference coatings formed on round and rectangular sapphire substrates, manufactured according to the developed technology is presented in The analysis of the photos  shows thA review of the pictures presented in The results of the studies of the optical transmission of light filters, conducted in different ranges of radiation wavelengths from 0.19 \u03bcm to 1.1 \u03bcm and ranges from 1.25 \u03bcm to 10 \u03bcm, are presented for round samples in As we can see from In order to study the optical properties of the large-sized light filter, we conducted a study of optical transmittance for different surface areas of rectangular samples in the ultraviolet, visible, and infrared regions of the spectrum. The measurement results are presented in The analysis of the results of the study of the transmission capacity of light filters for the ultraviolet, visible, and infrared regions of the spectrum  shows thSince the developed light filters are proposed for use in optical pyrometers, where the entire surface is used during operation, the uniformity of optical transmission over the entire area of the sample will affect the accuracy of measuring the temperature of objects. The uniformity of the thickness of the applied coating layers on the surface of the large-sized light filter samples is ensured by the use of special equipment installed inside the chamber of the vacuum-spraying installation.The analytical solution to the boundary-value problem (1), (2) is built in the following form:The corresponding force and bending moment in the coating are obtained using Equation (3) for the displacements:The stresses in the coating are linearly distributed across the thicknessIn particular at the bottom base of the coating \u2013(8) were analyzed for the composition containing the silicon oxide coating with the parameters: As we can see on represented graphs, the most dangerous place is the point The admissible loadings Taking into account that the theoretical strength of The design of an interference-absorbing short-wave filter based on a layered composition of Si\u2013SiO on a sapphire substrate has been developed. The light filter manufacturing technology includes the application of a transition layer of SiO to the surface of the substrate, alternating Si\u2013SiO layers , and low refractive indices (SiO), alternating layers), completing the process of forming the light filter coating with the application of an outer SiO layer with appropriate control of the layers\u2019 thickness.Based on the results of the optical properties study, it was established that the light filter provides minimum light transmission in the visible part of the spectrum from 0.38 to 0.78 \u00b5m and maximum light transmission in the near-infrared region from 1.25 to 5 \u00b5m and has stable optical indicators over the entire surface area of the samples.The main feature of the proposed technique of mechanical analysis is the use of strength criteria for all components of a partially homogeneous layered structure. For the layered Si\u2013SiO composition on a sapphire substrate, the stress\u2013strain state and the allowable load were evaluated and their dependence on the angle of inclination of the applied load was analyzed. The calculation method proposed by the authors will allow engineers to control, in the analytical form, the influence of the ratio of geometric and mechanical characteristics of nanolayers on the stress state and the limit equilibrium of the interference-absorbing filter depending on the magnitude and orientation of the localized load.Studies have shown that the most dangerous stresses are in the coating, which should be taken in account in the strength calculation, and the worst force inclination angle is the case of perpendicular loading.In the future, the effect of heating on the change in the stress\u2013strain state of the layered light filter will be investigated."}
{"text": "Left side traumatic diaphragmatic hernias (DH) are very rare and usually present acutely. They might represent after years of minor trauma, and they should be considered among differentials to avoid complications. We present a 28-year-old female coming with acute epigastric pain radiating into the chest with dyspnea and vomiting. Her history was negative for trauma and other than very minor trauma two years earlier. Chest X-ray showed atelectasis with mild pleural effusion. Computed tomography scan showed several cavities, filling the left chest with a gaseous liquid level. Surgery was performed that demonstrated DH and the abdominal viscera were returned to the abdomen without any complications. Traumatic DHs can be easily overlooked with the absent of recent major trauma. They can represent years after the original trauma with acute symptoms, which can make it hard to diagnosis if not considered. Traumatic injury to the diaphragm is relatively uncommon, with an incidence between 0.8 and 8% . We presA 28-year-old housewife who presented with dyspnea and vomiting for one week, with epigastric pain radiating into the chest. She had no fever, cough or hemoptysis. She had no significant history for cardiac or pulmonary disease, and she was a non-smoker. She did not take any medication, including oral contraceptive pills. No significant family history other than diabetes and high blood pressure was found. The patient recalled carrying heavy weights around a year ago without a previous trauma story, and she had a subsequent dyspnea with a compressive intermittent chest pain that increased after eating at that stage, which all resolved spontaneously after a few days. She had a history of three Caesarean sections, with the last operation being four years ago.2. Other systems were also normal.On examination, vitals were within normal range. However, high-pitched sounds were noticed in the left hemithorax. Cardiovascular and abdominal examination were otherwise remarkable. Her BMI was 23.4\u00a0kg/m3\u2009=\u200920). Chest x-ray (CXR) of the chest was not able to visualize the left hemidiaphragm with a hollow viscus in the left thoracic cavity and a right shift of the mediastinum  and respiratory alkalosis  . The claDiaphragm defects are not easily detected because there are often no direct signs and symptoms. Moreover, most accompanying symptoms are nonspecific .High clinical suspicion is required to make the diagnosis of diaphragmatic rupture; CXR is usually the first hint regarding the possibility of a diaphragm injury , 6. In tIn this case, when the woman was presented, there was a large hernia with much protruding viscera, which led to a deterioration in the general condition, and thus a significant increase in the risk due to the size of the hernia being 3\u00a0cm. Therefore, the patient was rapidly managed due to the concerns of developing incarcerated hernia where mortality rates are very high . Such prCXR might give a misleading diagnosis as it might resemble a hydatid cyst or pneumothorax, which might lead to mismanagement such as inserting a chest tube, which might cause a perforation in the stomach and colon; therefore, great care should be taken when dealing with other similar cases .Traumatic diaphragmatic hernias are rare and can be caused by relatively minor trauma that might be direct or indirect. This can occur even years after the initial trauma. Early diagnosis and management of symptomatic causes is crucial to avoid serious complications."}
{"text": "HPV was detected in nearly all (82%) samples, , and nearly all HPV-positive samples included types in multiple genera (88%). A total of 560 HPV detections  were made. The most frequently detected HPV types were alpha , beta , and gamma . High-risk alpha types  were not common. A novel gamma type was identified  along with 90 unclassified types. This pilot study demonstrates the utility of the eWGS assay for broad-spectrum type detection and suggests a significantly higher type diversity in males compared to females that warrants further study.Most human papillomavirus (HPV) surveillance studies target 30\u201350 of the more than 200 known types. We applied our recently described enriched whole-genome sequencing (eWGS) assay to demonstrate the impact of detecting all known and novel HPV types in male genital samples ( Papillomaviridae that includes 49 genera. More than 200 HPV types that infect cutaneous and mucosal surfaces have been found in five of these genera, with most in alpha, beta, and gamma genera. While most infections clear, persistent infection with some alpha types  is a risk factor for anogenital and oropharyngeal cancers [Human papillomaviruses (HPV) are DNA viruses in the family  cancers , the ass cancers . Individ cancers . Gamma HCurrently, most studies have used PCR assays with degenerate primers  to detect and type HPV within specific genera ,13. Epidn = 50) from external genital swabs in specimen transport medium collected from US males between 2010 and 2011 (IRB determination\u2014Exempt). The vaccination history was unknown, but collection was prior to the US recommendation of routine HPV vaccination for males. DNA extracts were prepared using a Chemagic gDNA kit with an automated Chemagic MSM1 extractor  and stored at \u221280 \u00b0C for 63\u201368 months prior to use in the eWGS assay. The DNA concentration of the thawed extracts was determined using a Qubit dsDNA HS assay .We used residual anonymized DNA extracts , HPV-negative placental DNA was added to bring the amount of DNA/sample to 100 ng prior to shearing . Librarihttps://pave.niaid.nih.gov/) (as of 24 February 2022). A sample was considered positive for an HPV type if there were at least 100 mapped reads that covered at least 20% of a classified or unclassified reference HPV genome. This cut off is more stringent than recommended (minimum 10 reads and 10% genome coverage) for determining positive signal in HPV whole-genome studies [The sequence data were analyzed using CLC Genomics Workbench . Read de-multiplexing, quality control, alignment, and HPV type determination were performed as described previously , except  studies .2 and 995 K/mm2, with a total of 30, 935, and 33,083 mega base (Mb) sequence data, respectively. All samples with DNA generated an average of 10.5 million reads/sample with a mean Q score of 36.2; 93.4% of the bases had a Q score \u2265 30. Results from analysis of these data are presented below. The 13 control samples gave the expected results (Sequencing libraries 1 (samples 1\u201331) and 2 (samples 32\u201363) generated similar cluster densities of 962 K/mm results . Of 50 male genital samples, 41 (82%) were HPV eWGS-positive. There were 560 total HPV detections of two-hundred thirty-seven unique HPV types in four genera: forty-six alpha, fifty-five beta, one-hundred thirty-five gamma, and one mu types. Within genera, the types were spread across forty-two species: fourteen alpha, five beta, and twenty-three gamma species. The number of HPV types per sample ranged from 1 to 85 (mean = 13.6 types per sample). Of the 560 HPVs detected, 72% (402) were officially classified and 28% (158) were unclassified in the PAVE database. Gamma HPV detections dominated , followed by beta , alpha , and mu genomes . Among the 122 alpha HPV detections, 46 unique types were identified in 32 samples. The number of reads/alpha genome was 88,996 \u00b1 29,619 and >80% of the genome was sequenced in over 75% (94/122) of these detections. Among the 179 beta HPV detections, 55 unique beta types were identified in 35 samples. The number of reads/beta genome was 17,020 \u00b1 4769, and >80% of the genome was sequenced in over 57% (102/179) of the detections. Among the 257 gamma HPV detections, 135 unique types were identified in 36 samples. The number of reads/gamma genome was 36,585 \u00b1 9738, and >80% of the genome was sequenced in nearly 42% (107/257) of the detections. The two mu papillomavirus signals were mapped from two samples  to the same unclassified novel type, HPV-md01c06. 2 = 1) with the depth of coverage (p = 0.0057) and gamma genomes . The GC content (%) was significantly higher in alpha genomes  compared to both beta  and gamma genomes   .The number of alpha types detected per sample ranged from 1 to 21 (mean = 4/sample). While the most common alpha types were HPV90, HPV43, HPV74, and HPV84 A, the grThe number of beta types detected per sample ranged from 1 to 26 (mean = 5/sample). While the most common beta types were HPV types 115, 195, 120, 22, 23, 24, and 120 A, the grThe number of gamma HPV types detected per sample ranged from 1 to 53 (mean = 7/sample). While the most common gamma types were HPV134, HPVmSD2, HPV 50, HPV179, HPVmKN1, and HPV147 A,B, the The overall prevalence of male genital HPVs belonging to alpha, beta, and gamma genera is presented in Interestingly, of the 237 unique HPV types identified by eWGS, there were no RNA baits in the enrichment hybridization for ninety-seven types  and eighty-three of these types were unclassified. The study also identified a novel HPV genome (GenBank Accession # MW535770), that has subsequently been officially classified by the International HPV Reference Center, Sweden, as gamma HPV type 229.Papillomaviridae genera recognized to have HPV types   . For thrIt is interesting to note that of the five most prevalent high-risk types in a meta-analysis of data from women with normal cervical cytology in the pre-vaccine era  , HPV 58 Alpha types, the major genus detected in cervicovaginal samples, were not the most frequently detected in male genital samples, although they were present at a higher copy number (averaging two to five times more reads/sample than beta and gamma genomes) and had the highest percentage  of complete genomes. It is also striking that the HPV diversity in male genital samples in our study was greater than that reported in cervical samples tested with the same eWGS assay ,25. PrioPrior studies of male genital samples using multiple assays (linear array followed by sequencing of PCR products of PGMY 09/11 and FAP59/64 primer systems) found fewer than 20 unique beta types largely in one beta species (beta 2) . HoweverWe identified 55 of the 98 types within gamma, the genus with the largest number of types with official classification. Of the unclassified types detected in our study, the largest number  were in this genus. Among gamma types, the prevalence was highest for HPV-mSD2 and HPV 134, each at 14%, followed by HPV-mKN1, HPV 50, and HPV147, each at 12% in this study. HPV-mSD2 and HPV134 were previously identified as the most prevalent gamma types in oral rinse samples . This pilot study has several limitations. It used residual samples without demographic information, and results cannot be considered representative of any population. Storage may have compromised sample quality, although quality control suggested this was not an issue. HPV vaccination status was unknown, although the dates of the original sample collection were before widespread male vaccination. Enrichment methods, particularly those that rely on PCR, may bias the observed type distribution. As eWGS uses a limited number of PCR cycles and maintains a strong linear relationship between target copies and signal strength , this coThis pilot study demonstrates the usefulness of eWGS for broad-spectrum detection of HPV genomes belonging to multiple genera. Systematic studies using eWGS will generate a more complete view of HPV type distribution, to better understand their biology, tissue tropism, and potentially new disease associations."}
{"text": "Obesity is a highly prevalent chronic disease that is associated with the development of other metabolic comorbidities. Its etiology is complex and multiple risk factors have been reported. In women, weight gain during pregnancy and the effect of pregnancy on subsequent weight gain are important events in women\u2019s history. Both pregnancy and postpartum are critical periods for the development of obesity.To identify sociodemographic and reproductive risk factors associated with obesity in women in their fourth decade of life.Cohort study conducted on women born from June 1978 to May 1979 in Ribeir\u00e3o Preto, Brazil. Sociodemographic, clinical, and obstetric data were collected by interview and clinical evaluation. Univariable and multivariable binomial logistic regression models were constructed to identify the risk factors of obesity and the adjusted relative risk (RR) was calculated.The cohort included 916 women and 309 (33.7%) of them were obese. Obesity was associated with low educational level  and teenage pregnancy . There was no association of obesity with the other covariates studied.Obesity is associated with years of schooling and teenage pregnancy. In adul30\u00a0kg/m2 , 2.It has been increasing in epidemic proportions in both adults and children , 4. The Many chronic conditions that reduce longevity and quality of life are caused or affected by obesity, including diabetes mellitus, systemic arterial hypertension, dyslipidemia, metabolic syndrome (association of these previous conditions), and cardiovascular diseases , 7. The Different scholars mention a lot of predisposing factors which vary depending on geography, social conditions, political and economic factors, and human genetics. In aggregate, the commonest factors were sociodemographic, behavioral, genetic, and living in obesogenic environment. Many of the risk factors are known; however, a major gap in the scientific literature is how these factors are interrelated . KnowingThere are multiple factors and processes that lead to obesity. In fact, the traditional view is usually that the main cause is the excess energy stored in fat cells than the energy the body used. In addition, more and more etiologies or defects that lead to obesity can be identified under the lights of social, genetic, epigenetic, environmental and microenvironment issues .While there has been a plethora of articles about obesity published worldwide , 2, 7, 8Women tend to be more overweight and obese than males around the world and studies have shown that some NCDs have a predilection for women . This woThe primary objective of the present study was to identify sociodemographic and reproductive risk factors associated with obesity in women in their fourth decade of life, from a birth cohort that has been analyzed since 1978/79. The secondary objectives were to identify reproductive risk factors of obesity in women with previous pregnancies, as well as to describe the frequency of obesity and other chronic diseases  in women in their fourth decade of life.This is an analytical, observational cross-sectional study (nested in a cohort study). The sample of the present study consisted of women from the 1978/79 birth cohort conducted in the city of Ribeir\u00e3o Preto, State of S\u00e3o Paulo, Brazil . This isRibeir\u00e3o Preto is a city in the State of S\u00e3o Paulo, a rich and industrialized region with a Human Development Index (HDI) of 0.800 in 2010. Its population was 604,682 inhabitants in 2010 . It is oFor the cohort study, the records and charts of the eight maternity hospitals (three public and five private) that attended 98% of all deliveries in the municipality  from JunThis research has been performed in accordance with the Declaration of Helsinki and has been approved by the Research Ethics Committee of the School of Medicine of Ribeir\u00e3o Preto, University of S\u00e3o Paulo. All stages of this cohort were submitted to the Research Ethics Committee. Before the review of records and charts of the eight maternity hospitals in 1978/79, permission was obtained from all clinical directors and the registry offices. The interviewed mothers from whom data and data of their children were collected provided oral consent and were released by their responsible physicians . The lasWomen who participated in the 2016/17 assessment were included in the present study (n\u2009=\u2009929). The participants were invited to come to the research center on a scheduled day and time. Eligible participants received information about the objectives of the study and were invited to sign the free informed consent form. Data collection was started only after the participants had signed the form.2 may not reflect the diagnosis of obesity depending on the gestational age.Women who were pregnant at the time of assessment (n\u2009=\u20092), women who did not undergo collection of the anthropometric data (n\u2009=\u20096), and women who did not answer the questionnaire about obstetric history (n\u2009=\u20095) were excluded. Pregnant women were excluded because the BMI cut of 30\u00a0kg/mQuestionnaires including demographic, social, clinical, and reproductive data were applied during the interviews. Sociodemographic and reproductive health-related variables were included in this study. The following sociodemographic variables were collected: race/ethnicity (white and others), paid work (to be engaged in a paid activity at the time of application of the questionnaire), socioeconomic class, stable marital status , educational level , smoking , alcohol misuse , and ilSocioeconomic status was defined according to the ABEP categories . The estThe participants underwent body composition assessment, anthropometry, blood collection, and blood pressure measurement. Trained health professionals collected the data. Information on chronic diseases was obtained by interview. An existing condition was defined when the participant had a diagnosis or was being treated for systemic arterial hypertension, diabetes mellitus, and dyslipidemia. The criteria defined by the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III), revised by the American Heart Association and the National Heart, Lung and Blood Institute (AHA/NHLBI) , were coHeight was measured using a stadiometer graduated in centimeters and fixed to a smooth wall, with the patient standing erect, barefoot, with arms extended along the body, head positioned in the Frankfurt plane  with eyes fixed on a point at eye level, legs parallel and heels, calves, buttocks, shoulder blades and the back of the head  against the stadiometer or wall. Weight was measured using a Welmy \u00ae digital anthropometric scale, with a capacity of 200\u00a0kg and accuracy of 100\u00a0g. The patient was positioned in the center of the scale, barefoot, erect, arms extended along the body, with feet together and in such a way that the weight was distributed symmetrically, avoiding the support more firmly on one of the legs and with the gaze fixed. at a point on the straight line.2) and classified according to the World Health Organization [Based on measured weight and height measurements, BMI was calculated according to the formula BMI\u2009=\u2009weight (kg)/height  was used for all statistical analyses. A level of significance of 5% (p\u2009<\u20090.05) was adopted. Missing data were excluded from the analysis.The stage used in this study had 1759 individuals who attended for data collection. Of these individuals, those of the male gender were excluded (n\u2009=\u2009846), allowing the analysis of a total of 929 women. Women who were pregnant at the time of assessment (n\u2009=\u20092), women who did not undergo collection of the anthropometric data (n\u2009=\u20096), and women who did not answer the questionnaire about obstetric history (n\u2009=\u20095) were excluded. Then, 916 women were analyzed.Most of them (80.2%) were white, had paid work (90%), lived with a companion (67.2%), had more than 12 years of schooling (45.9%), belonged to socioeconomic class A/B (68%), had a previous pregnancy (77.7%), were non-smokers (88.8%), were not abusive alcohol drinkers (78.4%), and did not use illicit drugs (96.6%) (Table\u00a02) and obesity was 34.7% and 33.7%, respectively. The prevalence of clinical comorbidities was 11.2% for diabetes mellitus, 19.5% for systemic arterial hypertension, 18.3% for dyslipidemia, and 31.2% for metabolic syndrome  continued to be associated with obesity in women with a previous pregnancy when compared to age at first pregnancy over 30 years . The other covariates were not associated with obesity. Table\u00a0The population consisted of women aged 37 to 39 years from a cohort study conducted in the city of Ribeir\u00e3o Preto. Participants were recruited by telephone, advertisements through media and on social networks, and searches in a digital environment, strategies that may have been unable to reach marginalized groups. Thus, only 1,775 of 6,973 women were available for data collection. This sample corresponds to 25% of the initial sample, which means a loss of 75% of individuals. This loss can bring an important bias to the results of this study.A significant proportion of the population was overweight (68.4%) and approximately one-third was obese (33.7%). Although different methods were used for data collection, these percentages are much higher than the Brazilian estimates obtained in the same year (2016) by the VIGITEL survey (Surveillance System for Risk and Protective Factors for Chronic Diseases by Telephone Survey), in which the prevalence of overweight was 53.7%, with 17.7% of obese adults and 19% of obese women aged 35 to 44 years .The high prevalence of obesity found here agrees with North American estimates, which were slightly higher than the Brazilian rates. Data from the National Health and Nutrition Examination Survey (NHANES) showed a prevalence of obesity of 40% among adults aged 20 to 39 years in the United States; when stratified by age, there were 40.3% of obese men in this age group and 39.7% of obese women . The higThe difference between the national prevalence of obesity and the prevalence found in this study may be due to some limitations of this cohort follow-up, including the recruitment process, the number of women from the initial cohort who attended the data collection, and the fact that the data were from a single city.This percentage of women who were not followed may have brought some bias to the study, since it may determine a more frequent profile in the study. For example, women with more comorbidities may be more interested in health studies than women without chronic diseases, which would increase the prevalence of obesity in the sample.The characteristics of the women participating in the 2016/17 assessment rendered the population more homogeneous. The majority of these women were white, had more than 8 years of schooling, lived with a companion, had paid work, and belonged to higher socioeconomic classes; these conditions may also have brought some bias to the study. The fact that the study gathered a more homogeneous and biased population, perhaps due to the greater demand for participation in the study by women with chronic diseases, such as obesity, the primary outcome of the study, may have hindered the statistical analysis. For example, this difficulty may have limited the association of obesity with previous pregnancy. Besides, the question that the data were from a single Brazilian city can limit the generalizability of the present findings.The women were divided into two groups according to the presence or absence of obesity to identify factors associated with this disorder. The only associated sociodemographic factor was a low educational level (less than 8 years of schooling compared to more than 12 years) and the only reproductive health-related factor was teenage pregnancy (<\u200920 years). In the case of women with higher educational level, possible explanations are greater social pressure and better access to weight control and weight loss programs, regardless of whether or not they are healthy , 25. On In contrast to expectations, this study did not find an association between other reproductive factors and obesity. Pregnancy and postpartum are critical periods for the development of obesity; however, although the relationship between maternal pregnancy weight and the risk of becoming obese has been the focus of studies in recent years , 30, theSeveral Brazilian studies aimed to estimate the prevalence of obesity and to identify associated factors. One study reported a prevalence of obesity of 16.8% among men and of 24.4% among women in 2013/2014. Advanced age (over 50 years), low educational level (no schooling or incomplete elementary school), African descent, and living with a partner were risk factors for obesity. Leisure-time physical activity and the habit of watching more than 4\u00a0h of television per day had significant effects in both sexes. Regarding morbidity, obese people were more likely to have a diagnosis of hypertension, diabetes, or non-communicable chronic diseases .A study conducted in 2011 in Rio de Janeiro revealed a higher prevalence of abdominal obesity (waist circumference\u2009>\u200980\u00a0cm) among women older than 35 years with two or more children. When the analyses were stratified by BMI category, parity and age were no longer significantly associated with abdominal obesity . AnotherOne major strength of this study is its cohort design. Birth cohort studies have been a top priority on the research and technology agenda of developed countries . The assIn summary, the present results are in line with the global scenario of obesity being a highly prevalent disease in adults. In women, obesity is associated with a low educational level and teenage pregnancy. Primary prevention strategies are necessary, and attention must be paid to women with low educational level and to pregnant adolescents. During prenatal care of teenagers, interventions that promote appropriate weight gain are vital to prevent postpartum weight retention because excess gestational weight gain is a strong predictor of maternal overweight and obesity after pregnancy ."}
{"text": "Yinlan Tiaozhi capsule (YLTZC) has been widely used to treat hyperlipidemia (HLP). However, its material basis and underlying pharmacological effects remain unclean. The current study aimed to explore the mechanisms involved in the treatment of YLTZC on HLP based on network pharmacology, molecular docking, and experimental verification. Firstly, UPLC-Q-TOF\u2013MS/MS was used to comprehensively analyze and identify the chemical constituents in YLTZC. A total of 66 compounds, mainly including flavonoids, saponins, coumarins, lactones, organic acids, and limonin were characterized and classified. Simultaneously, the mass fragmentation pattern of different types of representative compounds was further explored. By network pharmacology analysis, naringenin and ferulic acid may be the core constituents. The 52 potential targets of YLTZC, including ALB, IL-6, TNF, and VEGFA, were considered potential therapeutic targets. Molecular docking results showed that the core active constituents of YLTZC (naringenin and ferulic acid) have a strong affinity with the core targets of HLP. Lastly, animal experiments confirmed that naringenin and ferulic acid significantly upregulated the mRNA expression of ALB and downregulated the mRNA expression of IL-6, TNF, and VEGFA. In sum, the constituents of YLTZC, such as naringenin and ferulic acid, might treat HLP by regulating the mechanism of angiogenesis and inhibiting inflammatory responses. Furthermore, our data fills the gap in the material basis of YLTZC. The main drugs currently used in the treatment of HLP include statins, fibrates, and cholesterol absorption inhibitors, which have caused many side effects for patients during treatment, such as gastrointestinal tract issues, myopathy, and rhabdomyolysis3. Therefore, it is necessary to find alternative medicines for the treatment of HLP.Hyperlipidemia (HLP) is one of the most common disorders of lipid metabolism, which is closely related to metabolic syndrome, diabetes, obesity, and arteriosclerosis, and is a major risk factor for cardiovascular and cerebrovascular diseasesCitri Grandis Exocarpium, Ginkgo Folium, Gynostemma pentaphyllum, and propolis7. The clinical trials of new drug approval (2012L01011) have been successfully admitted by National Medical Products Administration (NMPA), and is currently in phase II clinical stage. Data from previous studies revealed that the YLTZC showed obvious anti-HLP activity and could regulate lipid levels in various animal models of experimental HLP, including mice, rats, and New Zealand rabbits. The experimental results showed that YLTZC remarkably lowered the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-c) in HLP rabbits, mice, and rats, while increasing the levels of high-density lipoprotein cholesterol (HDL-c), and the anti-HLP effect of YLTZC might be related to inhibiting PXR expression, promoting bile acid excretion and RCT processes, and enhancing TG hydrolysis7. In addition, in the treatment of type 2 diabetes mellitus, YLTZC was performed to prohibit the FA \u03b2-oxidation, synthesis of cholesterol and phospholipids, gluconeogenesis, and inflammation level, as well as promote TG hydrolysis, glycolysis, and blood circulation8. However, there are still some problems in the related research of YLTZC. On the one hand, its pharmacodynamic substance basis and quality control system are still unclear. On the other hand, most of the current reports are limited to a single constituent and single target, while for the treatment of HLP, YLTZC plays a role primarily by acting on multiple ingredients, targets, and pathways, which means that its potential pharmacological effects are complex. The above-mentioned problems limited the systematic understanding of the mechanism of action of YLTZC. Therefore, it is necessary to establish a rapid and effective method to investigate the material basis in YLTZC, as well as the relationship and mechanism of action between the core active constituents and the core targets.Yinlan Tiaozhi capsule (YLTZC) is a classic traditional Chinese medicine (TCM) prescription and patent formula. It has been clinically used for treating HLP and consists of 9. With the rapid and continuous development of bioinformatics, network pharmacology has become an important approach in the study of TCM, which provides a basis for systematically understanding the complexity between drug action and disease and elucidating potential therapeutic mechanisms11.Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF\u2013MS/MS), with high resolution, high sensitivity, and short analysis time, has become a powerful tool for the qualitative analysis in complex systems of TCMIn this research, we revealed the effective ingredients and predicted the potential targets and signaling pathways of YLTZC for the treatment of HLP by integrating the chemical profile, network pharmacology, molecular docking, and experiment verification. The detailed flowchart is shown in Fig.\u00a0Methanol, formic acid, and acetonitrile of MS grade were purchased from Thermo Fisher Scientific (United States), and laboratory-deionized water generated by a Milli-Q system (United States) was used in all experiments.YLTZC (Batch No. 20211101) was provided by Guangdong Efong Pharmaceutical Co. Ltd. . Reference substances including vicenin-2, luteolin, naringenin, isomeranzin, ferulic acid and pinocembrin were all purchased from Chengdu Herbpurify Co., Ltd . Gallic acid, protocatechuic acid, rutin, naringin, ginkgolide A, ginkgolide B, limonin, and chrysin were all purchased from National Institutes for Food and Drug Control . Triton WR-1339 was purchased from Sigma-Aldrich .YLTZC was ground into powder, 0.25\u00a0g of powder was accurately weighed and extracted with 50\u00a0ml 50% aqueous ethanol under ultrasonication for 30\u00a0min. The extract solution was centrifuged at 12,000\u00a0rpm for 10\u00a0min at 25\u00a0\u00b0C. And then, the supernatant was filtrated through 0.22\u00a0\u03bcm filter membrane and 1.0\u00a0\u03bcl of filtrate was injected into UPLC-Q-TOF\u2013MS/MS of analysis. 14 reference standards  were weighed in appropriate amounts, dissolved in methanol and filtered by 0.22\u00a0\u03bcm filter membrane to prepare 0.3\u00a0mg/ml stock solution, respectively.18 column  at 30\u00a0\u00b0C, with mobile phases A (0.1% formic acid) and B (acetonitrile). The flow rate was 0.3\u00a0ml/min, and the gradient profile was showed as follows: 0\u20133\u00a0min, 8\u201325% B; 3\u201312\u00a0min, 25\u201347% B; 12\u201325\u00a0min, 47\u201379% B; 25\u201332\u00a0min, 79\u201395% B; 32\u201335\u00a0min, 95% B; 35\u201336\u00a0min, 95\u20138% B. The sample injection volume was 1\u00a0\u03bcl.Liquid chromatography analysis was conducted by using a SHIMADZU ExionLC system (Japan). Chromatographic separation was used Waters ACQUITY BEH Cm/z 50\u20131000. Data were collected in information-dependent acquisition mode, and the instrument was recalibrated every four hours in order to exclude dynamic background. All data collected and analyzed by SCIEX OS v2.1 software .The MS analysis was acquired using an AB SCIEX X500R Q-TOF\u2013MS/MS system (United States) with an electrospray ionization (ESI). The results of the mass spectrometry optimization conditions are as follows: ion voltage: \u2212\u20094.0\u00a0kV and +\u20095.5\u00a0kV, Gas1 (nebulizer gas): 55 psi; Gas2 (heater gas): 55psi; curtain gas: 35 psi; declustering potential voltage: 80\u00a0V; ion source temperature: 500\u00a0\u00b0C; collision energy: 60\u00a0V; collision energy spread: 15\u00a0V; scan range: https://www.cnki.net/), SciFinder (https://scifinder.cas.org/) and literature searches, etc. Then, a self-built database of YLTZC chemical constituents containing component names and molecular formula information was established  was used for analyzing active constituents with absorption, distribution, metabolism, and excretion (ADME) properties and druglikeness evaluation to screen for active constituents with potential therapeutic effects. We applied in this study screening criteria of (1) pharmacokinetics \u201chigh\u201d and (2) druglikeness (DL) with more than two \u201cyes\u201d12.The SwissADME tool (http://www.swisstargetprediction.ch/), and targets with probability greater than 0 were selected. The HLP-related targets were selected from CTD (http://ctdbase.org/) and GeneCards (https://www.genecards.org/) database using the keywords \u201chyperlipidemias\u201d and \u201chyperlipidemia\u201d. Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/) was used to identify the overlapped targets between YLTZC-related targets and HLP-related targets. The common targets were entered into the STRING database (https://string-db.org), and biological species was set to Homo sapiens and the confidence >\u20090.413. Cytoscape 3.7.1 was used to construct the component-target-disease (C-T-D) network and Protein\u2013Protein Interaction (PPI) Network14.In this research, the targets of active constituents in YLTZC were searched by Swiss Target Prediction database (https://david.ncifcrf.gov/), and were selected for \u201cOFFICE_GENE_SYMBOL\u201d and \u201cHomo sapiens\u201d17. The GO enrichment analysis included the following 3 categories: biological process (BP), cellular component (CC), and molecular function (MF). The top 10 enriched GO entries and the top 20 enriched KEGG pathways were selected, and uploaded to the Bioinformatics (http://www.bioinformatics.com.cn/) cloud platform for data visualization. Cytoscape 3.7.1 was used to construct the component-target-pathway (C-T-P) network, and the core active constituents were obtained through the C-T-P network.The GO and KEGG pathway enrichment analysis of the common targets involved in the PPI network were performed using the DAVID 6.8 database (https://www.rcsb.org/) and the 3D structure of compounds were acquired from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/)18, and docking validation after hydrogenation and dehydration was carried out by AutoDockTools (version 1.5.6) and AutoDock Vina (version 1.1.2). Proteins should be selected from human proteins with one or more cocrystallized ligands and crystal structures with small \u201cresolution\u201d value19. Due to the still unclean functional binding sites of HLP-related targets to ingredients of YLTZC, all binding sites were restricted to the docking pockets region predicted by DeepSite software (https://www.playmolecule.com)20. Lastly, the molecular docking results were visualized into Pymol (2.5.0) software.The core targets and the core active constituents were obtained from the PPI and C-T-P network for molecular Docking. The protein PDB files of the core targets were obtained from the RSCB PDB online platform , and all animal experiments were performed in accordance with relevant ARRIVE guidelines. The 30 male KM mice weighing between 18 and 22\u00a0g were obtained from the Guangdong Medical Laboratory Animal Center . All animals were housed in barrier system at standard room temperature and a 12\u00a0h light/dark cycle conditions and fed normal food and water. The food was supported by Guangdong Medical Laboratory Animal Center . The experiment mice were divided into the following five groups with six mice in each: control group, model group, fenofibrate group (26\u00a0mg/kg), ferulic acid group (50\u00a0mg/kg), and naringenin group (50\u00a0mg/kg). The administration groups were given corresponding drugs by gavage, once a day, for 5\u00a0days. On the third day of administration, triton WR-1339 (480\u00a0mg/kg) was administered intramuscularly to all groups except the normal control group to establish a model of acute HLP. On the fifth day, one hour after gavage administration, all mice were anesthetized with isoflurane, sacrificed by inner canthus artery exsanguination, and the organs were retained for analysis.The serum sample was obtained by centrifugation of blood at 3000\u00a0rpm at 4\u00a0\u00b0C for 10\u00a0min and preserved at \u2212\u200980\u00a0\u00b0C until analysis. TC, TG, HDL-c and LDL-c levels of serum were measured by Microplate Reader  with assay kits were purchased from Nanjing Jiancheng Bioengineering Institute .\u2212\u0394\u0394Ct method, and Gene-expression data were normalized to that of the internal control GAPDH. The primer sequences are shown in Table About 50\u00a0mg of liver was weighed and transferred into a 1.5\u00a0ml grinding tube. After adding 500\u00a0\u00b5l Trizol reagent  and 2 grinding beads to the grinding tube, the liver was crashed by a freezing grinder . The grinding liquid was centrifuged  and the supernatant (about 400\u00a0\u00b5l) was transferred into a 1.5\u00a0mL centrifuge tube. 100\u00a0\u00b5l chloroform was added to the centrifuge tube and they were fully mixed by sharking. After centrifuging at 12,000\u00a0rpm at 4\u00a0\u00b0C for 10\u00a0min, the supernatant (about 200\u00a0\u00b5l) and the equivalent isopropyl alcohol were added into a 1.5\u00a0mL centrifuge tube and stored overnight in a \u2212\u200920\u00a0\u00b0C refrigerator. The crushed total RNA was settled at the bottom of the centrifuge tube after centrifugation at 12,000\u00a0rpm at 4\u00a0\u00b0C for 10\u00a0min. After washing with 75% ethanol 2 times, the pure total RNA was gained and dissolved in 50 \u00b5l RNase-free water. The content of total RNA was measured by UV and 500\u00a0ng total RNA was reverse transcribed by using the Evo M-MLV RT Premix for qPCR . RT-qPCR reactions were performed on iQ5 Multicolor Real-Time PCR detection system  with SYBR Green Dye detection. The relative gene expression was determined by the 2p\u2009<\u20090.05 was considered statistically significant.Results were expressed as the mean\u2009\u00b1\u2009standard deviation (SD), and analyzed by SPSS 20.0 software. Statistical significance was assessed using one-way analysis of variance (ANOVA). The value of 32. The main chemical constituent cluster was composed of 42 flavonoids, 6 saponins, 4 coumarins, 4 lactones, 9 organic acids and 1 limonin. Meanwhile, the identified constituents were attributed and classified based on the sources of their medicinal materials. Among them, 14 constituents  were determined by comparing with the reference standards. An additional 52 constituents were identified by comparing relevant literatures and mass fragmentation patterns.The mass spectrum data of YLTZC were gained by UPLC-Q-TOF\u2013MS/MS. As shown in Table Citri Grandis Exocarpium and Ginkgo Folium, in the form of free or glycoside, and etc. The identification process was analyzed by taking peak 5, peak 12, and 55 as examples. In negative ion mode, peak 5 had [M\u2009\u2212\u2009H]\u2212 ions at m/z 593.148 9, its MS/MS fragment ions at m/z 473.105 3 [M\u2009\u2212\u2009H-C4H8O4]\u2212, m/z 383.073 8 [M\u2009\u2212\u2009H-C4H8O4-C3H6O3]\u2212, m/z 353.063 4 [M\u2009\u2212\u2009H-2C4H8O4]\u2212, and m/z 353.063 4 [M\u2009\u2212\u2009H-C4H8O4-C3H6O3-CH2O]\u2212 were further pointed out by comparing with the reference standard and literature21. Thus, peak 5 was deduced as vicenin-2, the detailed fragmentation pathway of peak 5 is shown in Fig.\u00a0m/z 327.085 9 [M\u2009\u2212\u2009H]\u2212, with m/z 253.047 9 [M-C3H5O-H2O]\u2212, m/z 209.060 2 [M-C3H5O-H2O-CO2]\u2212, m/z 181.065 8 [M-C3H5O-H2O-CO2-CO]\u2212, m/z 185.060 5 [M-C3H5O-H2O-C3O2]\u2212, m/z 165.070 9 [M-C3H5O-H2O-2CO2]\u2212 being the main fragment ions in negative ion mode. Based on the reports in literature24, peak 55 was deduced as pinobanksin-3-O-propionate, the detailed fragmentation pathway of peak 55 is shown in Fig.\u00a0A total of 42 flavonoids  were identified from YLTZC, and the flavonoids mainly came from propolis, Gynostemma pentaphyllum. These compounds mainly exist in the form of anion [M\u2009+\u2009HCOO]\u2212, and the cracking law is mainly manifested as loss of \u2013C2H2O, \u2013C4H4O and glycosidic. For instance, the mass loss of 162\u00a0Da, 146\u00a0Da and 132\u00a0Da represents the loss of glucose (Glc), rhamnose (Rha), and xylose (Xyl), respectively. Peak 53 had [M\u2009\u2212\u2009H]\u2212 and [M\u2009+\u2009HCOO]\u2212 ion at m/z 799.482 6 and m/z 845.489 4 in negative ion mode. The fragment ions at m/z 637.431 1 [M\u2009\u2212\u2009H-Glc]\u2212 and m/z 475.379 9 [M\u2009\u2212\u2009H-2Glc]\u2212 were formed by the [M\u2009\u2212\u2009H]\u2212 ion following succession to lose of Glc residue. Based on the reports in literature, it was tentatively characterized as gypenoside LXXIV, the detailed fragmentation pathway of peak 53 is shown in Fig.\u00a0\u2212 and [M\u2009+\u2009HCOO]\u2212 ion at m/z 783.488 2 and m/z 829.492 8 in negative ion mode. The fragment ions at m/z 621.438 0 [M\u2009\u2212\u2009H-Glc]\u2212 and m/z 459.382 0 [M\u2009\u2212\u2009H-2Glc]\u2212 were formed by the [M\u2009\u2212\u2009H]\u2212 ion following succession to eliminate Glc residue. Hence, it was tentatively characterized as ginsenoside-F2, the detailed fragmentation pattern of ginsenoside-F2 is shown in Fig.\u00a022.Six saponins  were detected from YLTZC, all from Citri Grandis Exocarpium, and most coumarins have stronger response in the positive ion mode. Peak 38 exhibited [M\u2009+\u2009H]+ ion at m/z 261.111 3M\u2009+\u2009H]+, with m/z 189.053 9 [M\u2009+\u2009H-C4H8O]+, m/z 159.044 8 [M\u2009+\u2009H-C4H8O-CH2O]+, m/z 131.049 1 [M\u2009+\u2009H-C4H8O-CH2O-CO]+ as the fragment ions in positive mode, by comparing with the reference standard, peak 38 was deduced as isomeranzin. Peak 20, and 38 are a group of isomers, based on the reports in literature, peak 20 was deduced as meranzin. In positive ion mode, peak 66 had [M\u2009+\u2009H]+ ions at m/z 299.164 7, and the main fragment ions at m/z 163.040 0 [M\u2009+\u2009H-C10H16]+, m/z 145.103 8 [M\u2009+\u2009H-C10H16-H2O]+, m/z 135.042 6 [M\u2009+\u2009H-C10H16-CO]+, m/z 107.050 0 [M\u2009+\u2009H-C10H16-2CO]+, by based on the reports in literature33, peak 66 was inferred as auraptene. The detailed mass fragment pathway of peak 66 is shown in Fig.\u00a0Four coumarins  were identified from YLTZC, all of which were derived from 9 M\u2009+\u2009H-CH8O+, m/zGinkgo Folium. Ginkgolides have similar cleavage pathways, and the typical cleavage pathways is the opening of the lactone ring, with continuous loss of CO, CO2, and H2O. Peak 22, and peak 23 were identified as ginkgolide A, and ginkgolide B by comparing with reference standards. Taking peak 22 as an example in detail to illuminate the MS fragmentation pattern of these diterpenoid lactones23. Peak 22 had [M\u2009\u2212\u2009H]\u2212 ions at m/z 407.134 1 in negative ion mode. And then [M\u2009\u2212\u2009H]\u2212 ion continuous loss of CO, CO2, and loss of H2O to form m/z 351.143 6 [M\u2009\u2212\u2009H-2CO]\u2212, m/z 333.132 1 [M\u2009\u2212\u2009H-2CO-H2O]\u2212, m/z 319.144 0 [M\u2009\u2212\u2009H-2CO2]\u2212, m/z 307.160 3 [M\u2009\u2212\u2009H-2CO-CO2]\u2212, m/z 289.143 3 [M\u2009\u2212\u2009H-2CO-CO2-H2O]\u2212, m/z 273.148 1 [M\u2009\u2212\u2009H-2CO2-CO-H2O]\u2212, m/z 245.154 0 [M\u2009\u2212\u2009H-2CO-H2O-2CO2]\u2212. Therefore, the compound was identified as ginkgolide A. The detailed mass fragment pathway of peak 22 is shown in Fig.\u00a0Four lactones  were detected from YLTZC, all of which were derived from \u2212 ion at m/z 153.018 9 [M\u2009\u2212\u2009H]\u2212, with m/z 109.028 5 [M\u2009\u2212\u2009H-CO2]\u2212, m/z 91.018 9 [M\u2009\u2212\u2009H-CO2-H2O]\u2212 as the fragment ions in negative mode, by comparing with the reference standard24, peak 2 was deduced as protocatechuic acid, the detailed mass fragment pathway of peak 2 is shown in Fig.\u00a0A total of nine organic acids  were identified. Taking peak 2 as an example, peak 2 exhibited [M\u2009\u2212\u2009H]We performed an in-depth assessment of the absorption, distribution, metabolism, and excretion-related properties of 66 constituents in YLTZC using the online tool SwissADME. A total of 38 constituents in YLTZC were screened from the SwissADME tool  KEGG pathways were shown in Fig.\u00a0Gene Ontology (GO) and KEGG pathway enrichment analysis were undertaken on the 52 common targets mentioned above using the DAVID 6.8 database. The top 10 GO enrichment analysis results listed in BP, MF and CC are shown in Fig.\u00a034. Naringenin and ferulic acid had good affinity with the core targets, which was consistent with the results of literature reports, indicating that naringenin and ferulic acid have good anti-HLP effects. Based on these data, we suggested that the core active constituents have a good affinity for the core targets, which also demonstrated that YLTZC exerted its efficacy through multi-target combination.Based on the PPI and C\u2013T\u2013P network, we selected molecular docking between the 2 core active constituents and the top 5 core targets. The docking score and local structure of the results are presented in Table p\u2009<\u20090.01) were significantly increased in model group compared with the control group, and the levels of HDL-C (p\u2009<\u20090.05) was decreased. Compared with the model group, the levels of serum TC, TG, and LDL-c  were reduced in the naringenin and ferulic acid group, and the levels of serum HDL-C (p\u2009<\u20090.01) was significantly increased. These results suggested that naringenin and ferulic acid from YLTZC have good effect on improving blood lipid levels.Firstly, we investigate ferulic acid and naringenin on serum lipid levels in acute hyperlipidemia in triton WR-1339-induced mice. As shown in Fig.\u00a0p\u2009<\u20090.01), while naringenin and ferulic acid treatment markedly reversed these key targets. As expected, naringenin and ferulic acid treatment significantly increased ALB mRNA expression and decreased IL6, TNF and VEGFA mRNA expression compared to the model group.To clarify the molecular mechanism of YLTZC treatment for HLP, the mRNA expressions of the targets predicted above were measured by RT-PCR. As shown in Fig.\u00a0In conclusion, these results demonstrated that naringenin and ferulic acid from YLTZC may modulate the mechanism of angiogenesis and inhibiting inflammatory responses by regulating the expression of ALB, IL-6, TNF-\u03b1, and VEGFA to alleviate HLP.1. In our previous study, YLTZC showed a strong hypolipidemic effect, the results showed that YLTZC significantly decreased the levels of serum TC, TG, and LDL-c, and enhanced the level of serum HDL-c in HLP mice7. However, previous studies provided clues for the current study, but were not comprehensive, and relevant studies on the material basis and related mechanisms of action of YLTZC in the treatment of HLP are still lacking. This limits the further clinical studies and quality control and evaluation of YLTZC. Therefore, this study fills these shortages by developing a comprehensive research method combining chemical profile with network pharmacology, molecular docking, and experimental verification.HLP is one of the leading risk factors for the development and progression of cardiovascular diseases, characterized by elevated TC, TG, LDL-c, and decreased HDL-cIn recent years, along with the generalization of systems biology, network pharmacology has become an important method to clarify the potential mechanisms of multiple constituents, targets, and pathways of TCM. In this study, the chemical constituent of YLTZC was comprehensively characterized by UPLC-Q-TOF\u2013MS, and a total of 66 constituents, including naringenin, and ferulic acid, were identified. The results of the study provide more information on the chemical substance basis for further studies of YLTZC. According to the C-T-P and PPI network analysis, naringenin and ferulic acid were discovered to be the core active constituents associated with the most targets, and the HLP-related core targets of YLTZC were ALB, TNF, IL6, and VEGFA. In this study, enrichment analysis of GO and KEGG pathways was performed on 52 common targets. GO function enrichment analysis results showed that YLTZC treatment of HLP may involve the following BP: lipid metabolic process, positive regulation of smooth muscle cell proliferation, positive regulation of MAPK cascade, and positive regulation of inflammatory response. We hypothesized that the response to lipid metabolic process and inflammatory may be the most important BP in the treatment of HLP by YLTZC. KEGG pathway enrichment analysis results demonstrated that HIF-1 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, PPAR signaling pathway, PI3K-Akt signaling pathway, IR, and TNF signaling pathway were highly involved in YLTZC treatment of HLP. These pathways are highly associated with the regulation of angiogenesis and anti-inflammation systems, which may be significant pathways for the alleviation of the symptoms of HLP. Consequently, our findings suggest that the modulating the mechanism of angiogenesis and inhibiting inflammatory are potential therapeutic strategies of YLTZC for the treatment of HLP.36. It has been shown that naringenin prevents HLP, IR, and atherosclerosis by decreasing TC, TG, and LDL and increasing HDL37. In addition, naringenin inhibited fibroblast activation and inflammatory cell recruitment. The mRNA and protein expression levels of TNF\u2011\u03b1, IL\u20111\u03b2, IL\u20116, and TGF\u2011\u03b21 were downregulated following naringenin treatment38. Meanwhile, some studies have found that the effects of naringenin are related to the activation of PPARs, which can regulate hepatic lipid metabolism at the transcriptional level in human and rat by activating PPAR\u03b1, PPAR\u03b2, or PPAR\u03b3, respectively, and reduces serum lipids in HLP rats40. Ferulic acid has been shown to have anti-HLP, antioxidant, and anti-inflammatory effects. Compared with the placebo, the ferulic acid supplementation showed a statistically significant decrease in TC, LDL-c, and TG, and increased HDL-c42. In addition, ferulic acid could inhibit the expression of several pro-inflammatory cytokines including IL-1\u03b2, TNF-\u03b1, IL-10, and IL-6, and also inhibit angiogenesis by reducing the expression of VEGFA mRNA45. In our study, we established a triton WR-1339-induced HLP mice model, supplied with core active constituents of YLTZC for confirming its hypolipidemic effect. The results showed that naringenin and ferulic acid treatment significantly decreased the levels of serum TC, TG, and LDL-c, and enhanced the level of serum HDL-c in HLP mice. Therefore, naringenin and ferulic acid as the two core active constituents may be the potential material basis for YLTZC to alleviate HLP.Naringenin has an antioxidative activity to relieve oxidative stress, alleviate IR, and inhibit the production of inflammatory mediators46.On the one hand, YLTZC may treat HLP by regulating the mechanism of angiogenesis. VEGFA was found to be a significant factor in the regulation of vascular endothelial cells. Vascular remodeling during atherosclerosis was also associated with the expression of VEGFA, which has the effect of inducing angiogenesis, promoting their survival, and enhancing vascular permeability47. IL6 is also a pro-inflammatory cytokine that plays a crucial role in inflammation, atherosclerosis, and thrombosis, and can influence the rate of lipid metabolism, specifically the metabolism of TCs and TGs48. ALB is an important substance for maintaining plasma colloid osmotic pressure, and studies have found that ALB levels are closely related to cardiac function, and play a critical role in regulating the osmotic pressure and metabolic processes49. Meanwhile, studies have also shown that ALB is the most important carrier/transporter protein in vivo and plays an essential role in plasma antioxidant activity. In addition, in the HLP model, the decreased levels of ALB may be closely associated with the decline of liver function leading to reduced liver production51. Therefore, we speculate that ALB is mainly used as a drug transport platform to treat HLP by YLTZC. PPARs play a vital role in regulating the systemic inflammatory response, and they also modulate several biological processes that perturbed obesity, including inflammation, lipid and glucose metabolisms52.On the other hand, YLTZC can alleviate HLP by regulating inflammatory and oxidative stress targets. For example, TNF-\u03b1 is one of the most significant pro-inflammatory mediators and a critical factor in IR, which is involved in the pathophysiology of various CVDs53. Activation of the AGE-RAGE signaling pathway can trigger the production of tissue factors, and inflammatory factors54. Moreover, the PPAR signaling pathway plays an essential part in cholesterol metabolism and cholesterol efflux55. IR is associated with hyperinsulinemia and HLP56. These pathways are highly involved in YLTZC treatment of HLP, among which the PI3K-Akt signaling pathway, PPAR signaling pathway, and IR play critical roles in insulin secretion and lipid metabolism. Furthermore, fluid shear stress and atherosclerosis, the HIF-1 signaling pathway, and the TNF signaling pathway are highly participated in angiogenesis, pro-inflammatory factor secretion, and vascular tone regulation48.The HIF-1 signaling pathway is primarily involved in maintaining the steady state of oxygen in the body, and is also engaged in regulating angiogenesis and inflammationp\u2009<\u20090.01), and decreased the mRNA expression of ALB (p\u2009<\u20090.01) in the model group. However, compared with the model group, the naringenin and ferulic acid groups inhibited the mRNA expression of IL-6, VEGFA, and TNF-\u03b1 (p\u2009<\u20090.01), and promoted the mRNA expression of ALB . Therefore, we reasoned that YLTZC can effectively alleviate HLP by modulating the mechanism of angiogenesis and inhibiting inflammatory responses. The results of molecular docking and experimental verification were consistent with the predicted results of network pharmacology, indicating the accuracy of this method in screening the active constituents and action targets of YLTZC.In summary, we identified 2 core active constituents in YLTZC and the potential targets and pathways underlying the effects of YLTZC in HLP using the network pharmacology method. The results identified some pathways and biological processes that could be related to the lipid-lowering effects of YLTZC. To further validate the feasibility of network pharmacology analysis, IL-6, TNF-\u03b1, VEGFA, and ALB were selected as candidate targets of YLTZC against HLP. The molecular docking verified that the core active constituents have good binding properties with the core targets. In vivo experiment, compared with the control group, the mRNA expression of IL-6, VEGFA, and TNF-\u03b1 mRNA were significantly increased (In this study, we revealed the therapeutic effect and underlying mechanism of YLTZC against HLP based on network pharmacology, molecular docking, and experimental validation. 66 chemical constituents of YLTZC were rapidly characterized by UPLC-Q-TOF\u2013MS/MS. Based on the network pharmacology approach, the core active constituents including naringenin and ferulic acid, as well as core targets such as ALB, TNF, IL6, and VEGFA were screened for YLTZC for the treatment of HLP. Functional enrichment analysis through GO and KEGG pathways demonstrated the regulation of lipid metabolism, angiogenesis, and anti-inflammation systems, which may be important pathways in alleviating HLP. Molecular docking verified the possibility of the core active constituents binding to the core targets. In vivo experiments further showed that the hypolipidemic mechanisms of YLTZC were associated with the down-regulation of TNF-\u03b1, IL6, and VEGFA mRNA expression levels, and the up-regulation of ALB mRNA expression levels. Collectively, this work demonstrated that YLTZC may act against HLP by modulating the mechanism of angiogenesis and inhibiting inflammatory responses, and provided an efficient way to understand the active constituents and underlying mechanisms of YLTZC.Supplementary Figure S1.Supplementary Table S1."}
{"text": "Background: Thyroid metastases (TMs) are a rare entity, ranging between 0 and 24% in the autopsy series. In the assessment of the best management, the discrimination between a primary and a metastatic thyroid lesion is crucial. In this regard, fine needle aspiration cytology (FNAC) is likely to play a crucial role especially when ancillary techniques  and molecular testing) are carried out. Methods: We searched for all the TMs diagnosed using FNAC and analyzed between 2014 and 2023. The cases were processed with liquid-based (LBC) and ICC and molecular testing performed on LBC-stored material. Results: We reported 2.2% (19 cases) of TMs out of 1022 malignancies. TMs included: 1 larynx carcinoma (LX-Ca), 1 melanoma, 2 breast carcinomas (B-Ca), 3 lung carcinomas (LG-Ca), 4 gastro-intestinal carcinomas (GI-Ca), and 8 clear cell renal carcinomas (CCRC). All patients had a previous cancer history, between 300 and 2 months from the primary cancers. The morphological features were supported by ICC, which were contributive in 100% of cases. All TMs cases were characterized by multiple thyroid nodules except the melanoma case. Four cases underwent total thyroidectomy  whilst 15 TMs were treated with radio-chemotherapy. Conclusions: FNAC empowered the diagnostic workup of patients with TMs avoiding useless surgery. The low sensitivity of cytology might be reinforced by the application of ancillary techniques. We found a predominant rate of kidney metastatic carcinomas, followed by lung and breast. TMs are frequently multifocal and in a context of a systemic disease so a tailored therapy seems to be the best treatment. In these last decades, fine needle aspiration cytology (FNAC) has clearly shown its crucial diagnostic role in the evaluation of thyroid nodules ,2,3,4,5.In the spectrum of cytological diagnoses, the majority of thyroid nodules are diagnosed as primary thyroid entities using FNAC even though a possible metastatic localization to the thyroid gland should be always kept in mind especially when the morphological features are not univocally in favor of a primary thyroid entity. Not only should the lesions be associated to a previous or current history of malignancy, but they are likely to represent the first evidence of the disease ,8,9,10. Some papers, especially those related to autopsy series, documented that the estimated incidence of thyroid metastatic lesions ranges between 0 and 24%. Furthermore, as emerged from a multi-institutional study of 62 secondary neoplasms to the thyroid gland, the most frequent metastatic diseases seem to be colon, kidney, breast, and lung cancers ,14,15,16Despite the fact that thyroid metastases (TMs) may represent an unexpected finding, either cytological or histological series have assessed that TMs account for 1.4% to 2.5% of all thyroid cancers ,19,20,21In 2014, our group published the results of a TMs series, diagnosed in the period between 2000 and 2013 [TM . Each FNAC was performed with two passes for each lesion, using 25 to 27 G needles without any rapid adequacy assessment of the material.We conducted a retrospective, computerized search of all metastatic thyroid cytological cases recorded in the files of the Division of Anatomic Pathology and Histology of the Catholic University, \u201cAgostino Gemelli\u201d Hospital of Rome  between January 2014 and February 2023. A global cohort of 19 (1.8%) cases out of 1022 total thyroid malignancies were diagnosed as positive for malignancy (PM), favoring a metastatic localization, using liquid-based cytology (LBC). We searched for cases with a known history of malignancies or primary suggested diagnoses of TMs with FNAC. In our institution, all the nodules were evaluated under sonographic guidance (US) by pathologists, surgeons, and endocrinologists. All the cases were processed with the LBC method Thin Prep 2000The size of nodules ranged from 0.7 cm to 7.0 cm and they were all found during routine US thyroid check-ups performed in the Centre for Thyroid Diseases of the Departments of Endocrinology and Endocrine Surgery of the Catholic University. Regarding the use of the LBC method, all the patients had been appropriately informed for processing their aspiration samples and a written informed consent was signed by each of them. Our study followed the tenets of the Declaration of Helsinki and we received the internal ethics approval for the study.TM solution at room temperature for 3 to 4 months to be used for eventual additional investigations or preparation of additional slides. These ancillary techniques can be performed when the remaining material is at about 2 mL eluted in 5 mL of PreservCyt solution. The adequacy was defined, according to the British RCPath classification, by six groups of epithelial cells within the submitted slides, each of them with at least 10 well-visualized epithelial cells [Concerning the LBC method, the aspirated material was processed according to the manufacturer\u2019s suggestions and following the standard method that we have been using since the introduction of LBC in our institution in 2000. The resulting slide was fixed in 95% ethanol and stained with Papanicolaou while the remaining material was stored in the PreservCytal cells .At the beginning, the cytological cases were classified according to the Italian Working Group SIAPEC-IAP classification and then reclassified for scientific purposes, according to the Thyroid Bethesda System. Albeit the differences in classification systems, the selection of metastatic lesions did not incur any discrepancies, and they all belonged to the malignant category regardless of the different classification systems ,18.Patients with lymphomas and malignant lesions that invade the thyroid to contiguity were excluded from our series. We excluded any thyroid lymphoma because, as for the previous paper, we wanted to compare the epithelial metastases. Furthermore, we did not include any thyroid infiltration from a contiguous malignancy because we wanted to analyze the thyroid metastatic lesions mimicking a thyroid primary neoplasm instead of analyzing an infiltrative process to the thyroid. In our institution, all the cytological and histological sections were reviewed by two expert cytopathologists and those cases whose interpretation was equivocal were submitted to diagnostic judgment until a final agreement was achieved. In this specific series, there was not any disagreement that needed to be re-evaluated. The follow-up included a period between 2 and 120 months.Immunocytochemical stainings were carried out with the avidin-biotin peroxidase complex using a selection of specific antibodies mostly based on the morphological diagnostic hypotheses. The immunocytochemical method was standardized according to the manufacturer\u2019s protocol adopted in our institution and previously published ,22. PosiThe surgical specimens were fixed in 10% buffered formaldehyde, embedded in paraffin and the 5 micron-thick sections were stained with haematoxylin-eosin. The concordance of immunohistochemistry between the primary surgical carcinomas and TM cytological samples was 100%. All the fibro-adipose tissue close to the thyroid gland was included for lymph node research and evaluation.p value less than 0.05 were considered significant.Statistical analysis was performed by using a commercially available statistical software package  for iOS . The comparison of categorical variables was performed by Fisher\u2019s exact test when appropriate. A During the reference period, between January 2014 and February 2023, we found 19 metastatic thyroid FNACs out of 30,006 thyroid FNACs (0.06%). Those metastatic lesions represented 1.8% of 1022 thyroid malignant diagnoses. The series comprised 13 (68.4%) female and 6 (31.6%) male patients with a median age of 65 years old . No signIn In p for the CCRC. In fact, the current series is characterized by a high number of CCRCs, followed by colon adenocarcinoma. Notably, the gap between the primary RCC and the metastatic localization ranged from 20 to 30 years.The conclusive diagnosis resulted from a combination of morphological features, IIC yields, and clinical history in all our 19 cases. Notably, none of the cases was positive for Thyroglobulin , confirmThe main purpose of the current paper is to evaluate our institutional experience in the diagnosis of TMs, with reference to the period between 2014 and 2023. Furthermore, we correlate the yields with our previous results referring to a published series of TMs in the period between 2000 and 2013 [Unexpectedly, in the first series, we found a predominant number of gastrointestinal metastatic adenocarcinomas and only one clear cell renal carcinoma. Based on those data, we decided to compare them with our further cases from 2014 to 2023 and to discuss similarities and/or differences between the two series.Data from the autopsy series have documented that TMs range between 0 and 24% of thyroid nodules, whilst according to the clinical series, mostly published by Cheung et al. and Montero et al. ,8, the rAccording to our results, we found 2.2% TMs in the first series versus 1.8% in the second reference period. Apart from slight differences, these two figures showed an overlap in the numbers of TMs, with quite stable figures in the different decades. In fact, these percentages, belonging to two consecutive decades, have confirmed the trend that the thyroid gland represents a rare metastatic site for many different primary carcinomas despite its rich vascular structure and supply ,29,30,31Few papers have discussed the role of FNAC in the diagnosis of TM, mostly suggesting that the crucial step is represented by the ability to obtain the adequate amount of diagnostic neoplastic cells ,9. Both p < 0.0001) [In a series by Wysocka et al., the authors found TMs and nodal metastases (NM) in 57 patients . The authors assessed that their frequency was higher in the group of patients with a known history of primary cancer compared with the group of unknown primary lesions  .A revision of literature that referred to the years before 2014, reported a medium number of TMs ranging from 14 and 22 cases over a period between 55 years and 7 years . Both ouDespite the fact that different papers highlighted an equal gender distribution of TMs, we have to underline a slightly higher incidence in the female population. In agreement with the majority of the series, we have also documented a presentation in the sixth or seventh decade of life. Nonetheless, concerning the TMs from the current series, we found some significant discrepancies with the results obtained in the first period. In detail, the current series supported the evidence that CCRC is the most frequent thyroid metastasis 42.1%) representing a cause of common pitfalls. In a previous multicenter study, including 7 United States and European medical centers, also involving our institution, 3 out of 9 CCRC cases were misdiagnosed as follicular neoplasms [% represeHence, in our series, as also documented in other publications, only one case diagnosed as a metastatic melanoma was defined by a single solitary nodule, characterized by single-patterned and pleomorphic cells, which might lead to several differential diagnoses, including anaplastic thyroid carcinoma. Despite the fact that 100% of our cases were diagnosed as malignant-favoring a metastatic localization, the diagnosis of the primary carcinoma/adenocarcinoma cannot be further classified by the morphological evaluation alone. Regardless of the previous clinical history, the support of ancillary techniques, especially ICC, performed on LBC-stored material, is useful for making the definitive diagnosis ,26,27,28The last point is dedicated to the management of thyroid metastases, which is, however, not univocally defined by specific surgical guidelines. Whilst some papers suggest a lobectomy in cases of solitary thyroid metastasis, others propose total thyroidectomy in a multifocal metastatic process ,32. NotwIn conclusion, for those cases with a primary known cancer, FNAC shows high accuracy in the diagnosis of TM, especially when it is combined with the application of ancillary techniques which may facilitate to tailor the best and appropriate management (including chemotherapy and radiotherapy) for a metastatic patient ,34,35,36"}