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+{"text": "Vif functions to counteract an anti-retroviral cellular factor in non-permissive cells named APOBEC3G. The current mechanism proposed for protection of the virus by HIV-1 Vif is to induce APOBEC3G degradation through a ubiquitination-dependent proteasomal pathway. However, a new study published in Retrovirology by Strebel and colleagues suggests that Vif-induced APOBEC3G destruction may not be required for Vif's virus-protective effect. Strebel and co-workers show that Vif and APOBEC3G can stably co-exist, and yet viruses produced under such conditions are fully infectious. This new result highlights the notion that depletion of APOBEC3G is not the sole protective mechanism of Vif and that additional mechanisms exerted by this protein can be envisioned which counteract APOBEC3G and enhance HIV infectivity.The viral infectivity factor, Vif, of human immunodeficiency virus type 1, HIV-1, has long been shown to promote viral replication  Retrovirology article [In contrast to most animal viruses, infection with the human and simian immunodeficiency viruses results in prolonged, continuous viral replication in the infected host. Remarkably, viral persistence is not thwarted by the presence of apparently vigorous, virus-specific immune responses. Several factors, including the evasion of an innate cellular anti-viral defense by HIV-1 as discussed in a recent  article . Interes article -5. Earli article ,6.APOBEC3G is a virion-encapsidated cellular protein that deaminates dC to dU in minus-strand viral cDNA during reverse transcription -10. The vif+ virus are not known; but, current observations are that APOBEC3G confers a major deleterious effect to the HIV-1 genome when the Vif protein is absent. Historically, Vif has been known to play a dramatically important role in HIV-1 infectivity [vif-defective virus can replicate in some permissive cells such as Jurkat and SupT1 cells, but cannot replicate in other non-permissive cells such as macrophages, primary human T cells, and some restrictive T cell lines [The effects of APOBEC3G and its G-to-A deaminase activity on the survival of wild type HIV-1 ectivity ,13. Vif ectivity -17. HIV-ll lines -20. For ll lines .Following on the heels of that initial observation, an enormous amount of effort emerged from several laboratories directed at elucidating how Vif mechanistically counteracts APOBEC3G in order to protect HIV-1 . These results strongly support a new mechanistic function of HIV-1 Vif protein, complementing the model where Vif counteracts the inhibitory effects of APOBEC3G by enhancing its degradation via ubiquitin-proteasome pathway  may indicate an alternative protective mechanism used by HIV-1 to eliminate innate cellular immunity. Nevertheless, we cannot exclude that HIV-1 uses Vif to exert multiple mechanisms to synergistically and more effectively inhibit the anti-viral activity of APOBEC3G.m pNL-A1 . Even thytoplasm ,46. If AIn conclusion, the existence of two different mechanisms may represent two faces of the same coin, with the common goal of inhibiting APOBEC3G's anti-viral activity. As is often the case with new findings, new questions are posed. The link between APOBEC3G's enzymatic function, its degradation pathway, and its incorporation into virions in the presence of Vif is certainly to require additional attention. Answers to these questions are likely to keep many of us busy for the foreseeable future.None declared.The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; Vif, Viral Infectivity Factor; SIV, simian immunodeficiency virus; NC, nucleocapsid protein; APOBEC3G, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G; APOBEC3F, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3F PBMC, peripheral blood mononuclear cells; Cul5, Cullin type 5; SCF, skp1-cullin-F-box protein ligase."}
+{"text": "Anyone who uses a word processor is likely thankful for the spell checker program. But that autocorrect function can introduce errors, \u201ccorrecting\u201d the spelling of words to fit its stored repertoire, which is decidedly limited. Take that one step further and imagine a rogue program that destroys the coherence and meaning of your prose by swapping out one letter for another throughout the document. That's the situation retroviruses like the human immunodeficiency virus (HIV) face during the course of their infectious cycle, when a protein encoded by the host genome slips into the virus, mutates the virus's genetic material, and alters the viral genome.APOBEC3G, belongs to a family of primate genes that produce enzymes  that \u201cedit\u201d DNA and RNA, by slipping into viral particles and inducing mutations that replace one base (cytosine) with another (uracil) as the virus undergoes reverse transcription in the host cell's cytoplasm. The edited virus fails to replicate. HIV, in turn, generates a protein called Vif that binds to the APOBEC3G enzyme and targets it for degradation, thereby eliminating its antiviral activity.The gene, Since the protein-binding regions that govern these interactions have a direct effect on the fitness of both virus and host, one would expect to see the proteins angling for advantage, with Vif maximizing its ability to recognize APOBEC3G and APOBEC3G doing its best to evade Vif. Such battles are thought to result in frequent mutations that alter the amino acids involved in the interaction; the perpetuation of such advantageous mutations is called positive selection.PLoS Biology, Sara Sawyer, Michael Emerman, and Harmit Malik investigate the genetic roots of this battle for evolutionary advantage and find something surprising. As predicted, the APOBEC3G gene is under strong positive selection. But that selection appears to predate the existence of HIV-type viruses.In this issue of APOBEC3G evolution, Sawyer et al. analyzed the gene from twelve primates\u2014New World monkeys, Old World monkeys, and great apes, including humans\u2014spanning 33 million years of evolution. Most of the primate lineages showed evidence of positive selection, indicating that the gene has been under pressure to adapt throughout the history of primate evolution. But viruses like HIV have been found in only five of the primates studied\u2014three African monkeys, chimpanzees, and humans\u2014and appear to be at most one million years old. And HIV infection in human populations is too recent to account for the positive selection of APOBEC3G in humans\u2014so what has been fueling APOBEC3G's rapid evolution?To characterize the selective pressures on APOBEC3G has been constant over the course of primate evolution suggests that another force is also acting on the gene. Sawyer et al. propose that this force is most likely occurring in germline cells (sperm and egg precursors), which also produce high levels of APOBEC3G and can pass mobile genetic elements on to the next generation. Despite being non-infectious, these elements increase their own copy number in the host genome, moving from one part of the genome to another. The human genome is littered with such \u201cretrotransposons,\u201d and it is these mobile genetic elements, the authors conclude, that likely antagonize APOBEC3G.APOBEC3G and Vif interact in T-cells, but the fact that selective pressure on One class of retrotransposons, called human endogenous retroviruses, acts in many ways like foreign retroviruses. A retrovirus emanating from one's own genome poses less of an immediate threat than a retrovirus like HIV. But the constant efforts of the endogenous retrovirus to \u201cjockey for evolutionary dominance,\u201d the authors conclude, could eventually take a toll and would be expected to provoke efforts to contain it. And it may be that this ancient intragenomic conflict endowed APOBEC3G with the means to do battle with foreign retroviruses like HIV.APOBEC human genes appear to be engaged in similar conflicts. Combined with the finding that rodents have only one APOBEC3G gene and that five out of the six human APOBEC3 genes have been under positive selection, these results suggest that this gene family expanded in mammalian evolution as a means of defending the germline from the promiscuous intrusions of mobile genetic elements.Sawyer et al. also found evidence that five other"}
+{"text": "The human immunodeficiency virus Vif protein overcomes the inhibitory activity of the APOBEC3G cytidine deaminase by prohibiting its packaging into virions. Inhibition of APOBEC3G encapsidation is paralleled by a reduction of its intracellular level presumably caused by the Vif-induced proteasome-dependent degradation of APOBEC3G.In this report we employed confocal microscopy to study the effects of Vif on the expression of APOBEC3G on a single cell level. HeLa cells dually transfected with Vif and APOBEC3G expression vectors revealed efficient co-expression of the two proteins. Under optimal staining conditions approximately 80% of the transfected cells scored double-positive for Vif and APOBEC3G. However, the proportion of double-positive cells observed in identical cultures varied dependent on the fixation protocol and on the choice of antibodies used ranging from as low as 40% to as high as 80% of transfected cells. Importantly, single-positive cells expressing either Vif or APOBEC3G were observed both with wild type Vif and a biologically inactive Vif variant. Thus, the lack of APOBEC3G in some Vif-expressing cells cannot be attributed to Vif-induced degradation of APOBEC3G. These findings are consistent with our results from immunoblot analyses that revealed only moderate effects of Vif on the APOBEC3G steady state levels. Of note, viruses produced under such conditions were fully infectious demonstrating that the Vif protein used in our analyses was both functional and expressed at saturating levels.Our results suggest that Vif and APOBEC3G can be efficiently co-expressed. Thus, depletion of APOBEC3G from Vif expressing cells as suggested previously is not a universal property of Vif and thus is not imperative for the production of infectious virions. Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T cell lines is dependent on the expression of a functional Vif protein. In the absence of Vif, virus replication is restricted by a host factor that was recently identified as CEM15 (now referred to as APOBEC3G)  but whosVif is a 23-kDa basic protein that is expressed late during infection in a Rev-dependent manner . ImmunocLike Vif, APOBEC3G is a cytoplasmic protein. In fact, co-immunoprecipitation analyses demonstrated an interaction of Vif and APOBEC3G in transiently transfected cells ,27,29-32The current study aims at characterizing in more detail the effects of Vif on the expression of human APOBEC3G on a single cell level. The study was initiated because of the apparent discrepancy between the drastic effects of Vif on APOBEC3G reported by Marin et al and our own finding of only moderate effects of Vif on APOBEC3G expression in transiently transfected cells. In our study, Vif was expressed from a subviral construct in a Tat- and Rev-dependent manner while APOBEC3G was expressed either in a Tat-dependent manner from an HIV-1-LTR-based vector or independently from a CMV-promoter-based expression vector. The Tat-dependent APOBEC3G expression vector was used to restrict APOBEC3G expression to cells also expressing Tat (and thus Vif).Confocal microscopic analysis of HeLa cells transiently transfected with Vif and APOBEC3G expression vectors revealed significant variations in the number of double-positive cells in identical samples ranging from as low as 40% to as high as 80% of transfected cells depending on fixation method and antibodies employed. Importantly, the appearance of cells expressing only Vif or APOBEC3G was observed both with wild type Vif and a biologically inactive variant and thus cannot be explained by Vif-induced degradation of APOBEC3G. Finally, despite the efficient co-expression of Vif and APOBEC3G, viruses produced in these cultures were fully infectious. We therefore conclude that the Vif-induced exclusion of APOBEC3G from virus-producing cells reported by Marin et al  does notA number of previous studies reported the efficient Vif-dependent degradation of APOBEC3G by cellular proteasomes ,8,28. HoAn earlier study investigating the coexpression of Vif and APOBEC3G by confocal microscopy concluded that APOBEC3G was virtually excluded from Vif-expressing COS7 cells . To veriIn the experiment shown in figure In addition of measuring the context-dependent expression of APOBEC3G, we also wanted to determine the influence of the fixation procedure on the efficiency of Vif:APOBEC3G co-staining. It is well known that the choice of fixative can affect the ability of a given antibody to recognize a specific epitope on its target protein. Frequently, epitopes are masked because of the folding properties of a protein in vivo or because of pre-existing protein-protein interactions that may compete for antibody binding. To test this possibility we compared a formaldehyde fixation procedure employed previously  with theHeLa cells were transfected with pNL-A1 and pHIV-APO3G at a 1:1 molar ratio. Cells were fixed 24 hr later either with methanol (MeOH) as in figure For a more quantitative analysis and to determine possible effects that arise from the use of different antibodies, we extended the experiment shown in figure To quantify the results, multiple optical fields were analyzed n = 5\u201310) with a total of at least 100 transfected cells for each parameter. As can be seen in figure \u201310 with Under optimal conditions, wild type Vif and APOBEC3G were coexpressed in about 80% of transfected cells . Experiments are ongoing to study the differential effects of Vif expressed from pNL-A1 and Vif expressed from a codon-optimized vector on APOBEC3G stability. However, these results could suggest that the effect of Vif on APOBEC3G steady-state levels may be influenced by the context in which Vif is expressed. At any rate, despite our inability to observe Vif-dependent cellular depletion of APOBEC3G, we were invariably able to recover fully infectious HIV under conditions were the intracellular levels of APOBEC3G were only moderately affected. We therefore conclude that (i) Vif has the ability to rescue viral infectivity even in the presence of APOBEC3G and (ii) that intracellular depletion of APOBEC3G and rescue of viral infectivity may be functionally separable activities of Vif.For now, the reason for the differences in the sensitivity of APOBEC3G to Vif noted by us versus other research groups remains unexplained. APOBEC3G can form oligomeric structures and is able to interact with Vif. It is therefore possible that such complexes undergo conformational changes that can mask epitopes thus limiting the access of antibodies used in the experiments. Thus, the discrepancy between our findings of the coexpression of Vif and APOBEC3G in the majority of cells and the virtual exclusion of APOBEC3G from Vif-expressing cells reported by Marin et al.  may be aThe inability of Vif expressed from pNL-A1 to deplete APOBEC3G is consistent with our previous inability to observe APOBEC3G degradation in kinetic studies . More reExpression of Vif and APOBEC3G in our experimental setup does not lead to the elimination of APOBEC3G from Vif expressing cells. In fact, more than 80% of successfully transfected cells efficiently co-expressed both proteins. Similar results were observed when a biologically inactive Vif variant was co-expressed with APOBEC3G suggesting that the absence of APOBEC3G in some of the Vif-positive cells is not due to Vif-mediated APOBEC3G degradation but reflects a general characteristic of the transient expression system. Moreover, APOBEC3G expression levels were very similar for Vif-positive and Vif-negative cells as judged from the immunostaining consistent with the only modest reduction in APOBEC3G steady-state levels observed in our immunoblot analyses. Nevertheless, viruses produced under such conditions were fully infectious in the presence but not in the absence of Vif attesting to the biological activity of all the proteins involved and demonstrating that Vif was expressed at saturating levels. We conclude that production of infectious viruses from APOBEC3G expressing cells is dependent on Vif but does not necessitate APOBEC3G exclusion from virus-producing cells.gag and pol products. A vif-defective variant of pNL-A1, pNL-A1vif(-) was constructed by deletion of an NdeI/PflMI fragment [The full-length molecular clone pNL4-3  was usedSerum from an HIV-positive patient (APS) was used to detect HIV-1-specific capsid (CA) proteins. A monoclonal antibody to Vif (MAb #319) was used for all immunoblot analyses and some of the immunohistochemical analyses as indicated in the text and was obtained from Michael Malim through the NIH AIDS Research and Reference Reagent Program ,35,37 FoHeLa cells were propagated in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS). LuSIV cells are derived from CEMx174 cells and contain a luciferase indicator gene under the control of the SIVmac239 LTR . These c2 flasks to about 80% confluency. Cells were transfected using LipofectAMINE PLUS\u2122  following the manufacturer's recommendations. A total of 5\u20136 \u03bcg of plasmid DNA per 25 cm2 flask was used. Cells were harvested 24 hr post-transfection. Transfection efficiency in our analyses was generally 30% to 40%.For transfection of HeLa cells, cells were grown in 25 cmVirus stocks were prepared by transfecting HeLa cells with appropriate plasmid DNAs. Virus-containing supernatants were harvested 24 hr after transfection. Cellular debris was removed by centrifugation  and clarified supernatants were filtered (0.45 \u03bcm) to remove residual cellular contaminants. For determination of viral infectivity, unconcentrated filtered viral supernatants were used for the infection of indicator cells. For immunoblot analysis of viral proteins, virus particles (7 ml) were concentrated by ultracentrifugation through 4 ml of 20% sucrose in PBS as described before [7 cells), and mixed with an equal volume of sample buffer . Proteins were solubilized by boiling for 10 to 15 min at 95\u00b0C with occasional vortexing of the samples to shear chromosomal DNA. Residual insoluble material was removed by centrifugation . Viral proteins were obtained by boiling concentrated viral pellets in a 1:1 mixture of PBS and sample buffer. Cell lysates and viral extracts were subjected to SDS-PAGE; proteins were transferred to PVDF membranes and reacted with appropriate antibodies as described in the text. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies  and visualized by enhanced chemiluminescence .For immunoblot analysis of intracellular proteins, whole cell lysates were prepared as follows: Cells were washed once with PBS, suspended in PBS (400 \u03bcl/105) in a 24-well plate total volume 1.2 to 1.4 ml. Cells were incubated for 24 hours at 37\u00b0C. Cells were then harvested and lysed in 150 \u03bcl of Promega 1x reporter lysis buffer . To determine the luciferase activity in the lysates, 50 \u03bcl of each lysate were combined with luciferase substrate  by automatic injection and light emission was measured for 10 seconds at room temperature in a luminometer .To determine viral infectivity, virus stocks were normalized for equal reverse transcriptase activity and used to infect LuSIV cells  for 10 min followed by two washes in PBS or fixed in FA buffer  for 20 min at room temperature followed by two washes in PBS. Coverslips were stored in PBS at 4\u00b0C until use. FA-fixed samples were permeabilized for 30 min at room temperature in permeabilization buffer  prior to incubation with antibodies. For antibody staining, cover slips were incubated in a humid chamber at 37\u00b0C for 30 min with primary antibodies at appropriate dilutions in 1% BSA in PBS. Cover slips were washed once in PBS  and incubated with Texas-Red- or Cy2-conjugated secondary antibodies (diluted in 1% BSA in PBS) for 30 min at 37\u00b0C in a humid chamber. Cover slips were then washed twice with PBS and mounted onto microscope slides with glycerol gelatin  containing 0.1M N-propyl gallate  to prevent photo bleaching and were stored at 4\u00b0C in the dark until analyzed by confocal microscopy.For confocal microscopy, a Zeiss LSM410 inverted laser scanning microscope was employed. The microscope was equipped with a krypton/argon mixed-gas laser and was operated by the Microcosm Renaissance 410 (v2.3.4) software package. Images were acquired with a Plan-Apochromat 63x/1.4 oil immersion objective . Additional optical magnification (up to 5-fold) was achieved using the zoom feature of the image acquisition software. For two-color analysis, objects were excited using 488/568 nm laser lines. Green and red emissions were recorded through appropriate filters (515\u2013540 nm band pass filter for Cy2 and 590 nm long pass filter for Texas-Red) and stored in separate (red and green) image channels. At the same time, bright field images (Nomarski optics) were collected and stored in a third (blue) channel. Image quality was enhanced during data acquisition using the Renaissance 410 line average feature (8 or 16x).None declared.S. K. carried out immunoblot analyses, infectivity assays, and was involved in the construction of plasmids and the production of antibodies. E.M., M.A.K., H.T., S.O., and R.G. participated in immunoblot analyses, infectivity studies, sample preparations, data validation, and overall experimental design. K.S. conceived of the study, performed IFA analyses, and coordinated the study. S.K. and K.S. participated in the writing of the manuscript. All authors read and approved the final manuscript."}
+{"text": "APOBEC3G  has antiretroviral activity associated with the hypermutation of viral DNA through cytosine deamination. APOBEC3G has two cytosine deaminase (CDA) domains; the catalytically inactive amino-terminal domain of APOBEC3G (N-CDA) carries the Vif interaction domain. There is no 3-D structure of APOBEC3G solved by X-ray or nuclear magnetic resonance.We predicted the structure of human APOBEC3G based on the crystal structure of APOBEC2. To assess the model structure, we evaluated 48 mutants of APOBEC3G N-CDA that identify novel variants altering \u0394Vif HIV-1 infectivity and packaging of APOBEC3G. Results indicated that the key residue D128 is exposed at the surface of the model, with a negative local electrostatic potential. Mutation D128K changes the sign of that local potential. In addition, two novel functionally relevant residues that result in defective APOBEC3G encapsidation, R122 and W127, cluster at the surface.The structure model identifies a cluster of residues important for packaging of APOBEC3G into virions, and may serve to guide functional analysis of APOBEC3G. Primate APOBEC3G has antiretroviral activity associated with the hypermutation of viral DNA through cytosine deamination (for recent review see There is no 3-D structure solved by X-ray or NMR nor an accurate model of APOBEC3G available. APOBEC3G relates to APOBEC family and AID (activation-induced deaminase) at the sequence level. Recent comparative modeling work for APOBEC-1 and AID In the present work, we model human APOBEC3G, with particular emphasis on the N-terminal domain. Using mutant data of the N-CDA, we mapped critical residues for packaging of APOBEC3G into viral particles on the new structure model of the huAPOBEC3G N-CDA. We completed the analysis by mapping N-CDA residues that are under positive evolutionary pressure in primate APOBEC3G.While this work was concluded, Huthoff and Malim provided a detailed molecular genetic analysis of the N-CDA region spanning amino acids residues 119 to 146 Ref . This anhttp://www.ncbi.nih.gov/) was defined as target sequence. The newly crystallized Human APOBEC2 dimer http://www.rcsb.org/pdb/Welcome.do) served as template.The huAPOBEC3G sequence  http://bioweb.pasteur.fr/seqanal/interfaces/dssp-simple.html) http://bioinf.cs.ucl.ac.uk/psipred/) http://www.jalview.org/) We used the align2d function of MODELLER program (http://swissmodel.expasy.org/anolea/) The target-template alignment was used to build the model by satisfaction of spatial restraints. The ANOLEA program  Generation of 100 models allowed selection of the best model candidate based on the global ANOLEA score. Final refinement for alignment of gap regions was performed by generating 100 additional models, followed by selection of the best model on the basis of the ANOLEA score. The final model was energy-minimized using the CHARMM program (http://foldx.embl.de/) http://www.cgl.ucsf.edu/chimera/) The CHARMM program was used to calculate the solvent accessible surface area of the final model. Additionally, using the FoldX program (The plasmid expressing a hemagglutinin (HA)-tagged form of APOBEC3G was a kind gift from M. Malim. A series of APOBEC3G alanine and specific mutants was constructed with the QuickChange Mutagenesis kit (Stratagene). The collection was already constituted, and it was not defined on the basis of the new structural data. HIV-1 particles were produced by transient transfection of 293T cells with Fugene (Roche) of a wild-type or of a Vif-defective HIV-1 proviral clone. Viral titers were determined in single-round infectivity assays by applying filtered supernatant from producer cells on HeLa-CD4-LTRLacZ indicator cells. Virion infectivity was derived by dividing the infectious titer by the amount of physical particles.HIV-1 particles were produced by transient transfection of 293T cells with Fugene (Roche). 1 ml of virus was then spun in Eppendorf tubes at 13'000rpm in a microfuge at 4\u00b0 for 90 minutes, without sucrose cushion. Pellets were resuspended in PBS 1% Triton, and the virion amount was measured by a standard RT assay. Normalized amount of virions were then loaded on standard Laemmli protein gels to perform Western Blots. Cell extracts were obtained through a standard RIPA extraction procedure. The HA tag was detected with the mouse hrp-coupled anti-HA 3F10 antibody (Roche). PCNA (proliferating cell nuclear antigen) was detected with the mouse Ab-1 antibody (Oncogene Science). The HIV-1 capsid was detected with the murine anti-p24 antibody produced from the AIDS Research and Reference Reagent Program #183-H12-5C.2+2+ with E60, a residue not present at the corresponding position in huAPOBEC3G N-CDA. The corresponding region of huAPOBEC3G C-CDA was modeled ab initio. The target-template alignment generated by MODELLER agreed with the secondary structure alignment  residues of the huAPOBEC3G N-CDA Amino acids substitutions at positions Y13, F17, F21, R29, T32, Y37, K40, S45, L49, L62, E85, Y86, W90, I92, S93, P96, M104, F107, L108, L116, T117, I118, L123, Y125, F126, D130, Y131, E133, L135, L138, M152, C160 and F164 did not result in changes in anti-viral activity. Mutations S28A, F74L, W94L, Y124A, Y154A resulted in reduced inhibition of \u0394Vif HIV1, while mutations R122A and W127L completely abolished this activity Table 1.Prior evolutionary analysis of primate APOBEC3G identified a number of residues under diversifying (positive) selection \u03b14-\u03b25-\u03b15-\u03b16 configuration, while CDA structures such as human cytidine deaminase have the pattern \u03b11-\u03b21-\u03b22-\u03b12-\u03b23-\u03b13-\u03b24-\u03b25-\u03b14-\u03b15. In addition, the direction of \u03b24 and \u03b25 are different: parallel in APOBEC2 but anti-parallel in cytidine deaminase. We had previously used the yeast Cytosine deaminase  to address the position of the extra helix and the correct direction of \u03b24 and \u03b25.This work presents a model structure of huAPOBEC3G that captures information from the recently published APOBEC2 structure vif due to lack of packaging of APOBEC3G into viral particles. Interaction of APOBEC3G with the NC-domain of HIV-1 Gag and non-specific RNA binding leads to its encapsidation into progeny virions The model emerging from this analysis allows speculation on various functionally relevant structural details of interest for the understanding of the Vif-APOBEC3G interaction and the process of APOBEC3G encapsidation into HIV-1 virions. In contrast with previous secondary and tertiary structure models, the current model locates the distinctive extra alpha helix on the same planar surface as the pseudo-active site. We evaluated extensive mutation data on this surface, that characterized two functionally relevant residues R122 and W127 that resulted in failure to inhibit infection by HIV-1/\u0394Our results are consistent with the work of Huthoff and Malim Inspection of surface modifications conferred by various mutations highlights the structural and/or charge differences in this region as the molecular basis for disruption of the APOBEC3G packaging into HIV-1 virions, and modification in the interaction with Vif. The detailed model structure presented here could serve to advance rational drug design. The quality of a homology model is strongly related to the sequence identity with the structural template. The current model, based on a sequence identity of 27%, should be satisfactory in its global fold, as supported by the good ANOLEA energy score profile  monomer of APOBEC2, and the C-CDA to the middle (M) monomer. Panel B, ANOLEA scores. The three ANOLEA profiles and the secondary structure were aligned according to the target-template alignment. The ANOLEA profile excludes the uncharacterized linker (residues 195-214)(2.86 MB EPS)Click here for additional data file."}
+{"text": "APOBEC3G is an antiviral host factor capable of inhibiting the replication of both exogenous and endogenous retroviruses as well as hepatitis B, a DNA virus that replicates through an RNA intermediate. To gain insight into the mechanism whereby APOBEC3G restricts retroviral replication, we investigated the subcellular localization of the protein. Herein, we report that APOBEC3G localizes to mRNA processing (P) bodies, cytoplasmic compartments involved in the degradation and storage of nontranslating mRNAs. Biochemical analysis revealed that APOBEC3G localizes to a ribonucleoprotein complex with other P-body proteins which have established roles in cap-dependent translation (eIF4E and eIF4E-T), translation suppression (RCK/p54), RNA interference\u2013mediated post-transcriptional gene silencing (AGO2), and decapping of mRNA (DCP2). Similar analysis with other APOBEC3 family members revealed a potential link between the localization of APOBEC3G and APOBEC3F to a common ribonucleoprotein complex and P-bodies with potent anti\u2013HIV-1 activity. In addition, we present evidence suggesting that an important role for HIV-1 Vif, which subverts both APOBEC3G and APOBEC3F antiviral function by inducing their degradation, could be to selectively remove these proteins from and/or restrict their localization to P-bodies. Taken together, the results of this study reveal a novel link between innate immunity against retroviruses and P-bodies suggesting that APOBEC3G and APOBEC3F could function in the context of P-bodies to restrict HIV-1 replication. Successful replication of viruses and other intracellular pathogens in their respective host cells requires that they overcome a series of replication restrictions or \u201croadblocks\u201d established by the cell. In the case of HIV-1, the ability of the virus to replicate in human cells is dependent on its ability to neutralize APOBEC3G, a DNA editing enzyme that incorporates into virions and renders them noninfectious. Although a potentially devastating inhibitor of HIV-1 replication, the virus evades APOBEC3G by inducing its degradation during virus assembly. APOBEC3G is also capable of inhibiting the replication of other retroviruses as well as the hepadnavirus hepatitis B, a DNA virus that replicates through an RNA intermediate, suggesting that APOBEC3G may function in cellular defense against a broad range of viral pathogens. Here, Rana and colleagues present their findings that APOBEC3G localizes to specialized compartments in the cytoplasm of mammalian cells known as mRNA processing (P) bodies, which function in the degradation and storage of cellular mRNA. Furthermore, they show that APOBEC3G assembles into a ribonucleoprotein complex with P-body proteins involved in translation, translation suppression, RNA interference, and mRNA decapping. These novel and exciting findings have broad-scale implications for APOBEC3G function and for the role of P-bodies in both cellular defense against viruses and retroviral assembly. On virions ,12\u201315. T virions ; however virions , suggestough Vif \u201320, whicough Vif \u201325. ThesDespite these significant advances in our understanding of APOBEC3G biology, there remained a considerable lack of detail concerning the subcellular context in which APOBEC3G functions. APOBEC3G has been shown to localize throughout the cytoplasm and to concentrate within punctate cytoplasmic bodies . Howevervif-deficient HIV-1 replication (unpublished data). Similar analysis of HeLa cells that stably express APOBEC3G with a C-terminal c-Myc epitope tag (APO3G-Myc), which also renders these cells nonpermissive to vif-deficient HIV-1 replication [+ T cells isolated from peripheral blood mononuclear cells following in vitro activation . This finding indicated that cytoplasmic bodies were not static structures but rather were both dynamic and intimately linked to mRNA translation. This dependence on active translation was a strikingly similar feature of proteins that localize to mRNA processing (P) bodies ,30,42 whNext, we investigated whether APOBEC3G simply colocalized with these P-body proteins or if they coexisted within a complex in the cell. Using the YFP-tagged versions of the P-body proteins, we found that YFP-AGO2, YFP-eIF4E, YFP-eIF4E-T, YFP-RCK/p54, and YFP-DCP2 all coimmunoprecipitated with APO3G-HA A or endoSimilar to APOBEC3G, APOBEC3F is a potent inhibitor of HIV-1 replication and is targeted by Vif \u201351, whilSimilar to APO3G-HA, APO3F-HA localized throughout the cytoplasm and to RCK/p54-labeled P-bodies B, arrowsWhile APOBEC3G and APOBEC3F coassembled into a common RNP complex and both localized to P-bodies, APO3B-HA was largely restricted to the nucleus of 293T cells A, c and C114S, that continued to interact with APOBEC3G readily colocalized with APOBEC3G at P-bodies [C114S did not colocalize with Myc-AGO2 at P-bodies . In conP-bodies . These rP-bodies B, d. Takvif-deficient HIV-1, the potent antiviral activity of APOBEC3G is successfully neutralized by wild-type HIV-1 through Vif [According to our current understanding of APOBEC3G function, this host restriction factor limits the spread of HIV-1 infection, and other retroviruses , by packough Vif \u201320, whicough Vif ,24,58,59vif-deficient HIV-1 replication can be rendered nonpermissive through either the transient or stable expression of recombinant APOBEC3G. Previously, we investigated the subcellular localization of recombinant APOBEC3G in these cells lines and reported that the protein localized throughout the cytoplasm and also to punctate cytoplasmic foci [vif-deficient HIV-1 infection suggested to us that these structures could be relevant to the antiviral properties of APOBEC3G.Despite these significant advances in our understanding of APOBEC3G biology, there remained a considerable lack of detail concerning the subcellular context in which APOBEC3G functions. Cell lines that are permissive  to mic foci , which wmic foci . The fac+ T cells, establishing that this was a bona fide property of APOBEC3G in cells that serve as a natural target for HIV-1 infection in vivo and leading us to investigate their identity. Our initial studies revealed that APOBEC3G cytoplasmic bodies were distinct from TRIM5\u03b1 cytoplasmic bodies and further that these bodies did not overlap with endocytic vesicles including early endosomes, late endosomes, or lysosomes . Using translation inhibitors to monitor the kinetics of cytoplasmic body assembly and disassembly, we observed that they disappeared from the cytoplasm within 60 min of cyclohexamide treatment. This dependence on active translation was a remarkably similar property of proteins that localize to mRNA processing (P) bodies, specialized compartments within the cytoplasm of both yeast and mammalian cells where nontranslating mRNAs accumulate and are subject to degradation or storage [We began this study by confirming that endogenous APOBEC3G also localizes to cytoplasmic bodies in primary peripheral blood CD4 storage \u201329,61. T storage ), transl storage ,32), RNA storage ,38,39,62 storage ,28,40).  storage  reportedOur studies on APOBEC3F and APOBEC3B revealed a possible link between the localization of APOBEC3G and APOBEC3F to P-bodies with potent anti\u2013HIV-1 activity. APOBEC3F, which shares approximately 50% sequence identity with APOBEC3G, exhibits potent anti\u2013HIV-1 activity, and is targeted by Vif for proteasome-mediated degradation \u201351, and C114S mutant, a nonfunctional Vif mutant that continues to interact with APOBEC3G [Considering a primary function of HIV-1 Vif is to restrict the incorporation of APOBEC3G into virions, we also determined whether Vif localized to P-bodies. Although the coexpression of Vif and APOBEC3G leads to a significant reduction in APOBEC3G levels, it is possible to detect cells where APOBEC3G and Vif are visible in the same cell . In thesAPOBEC3G , led to One of the more intriguing aspects of APOBEC3G biology is that it targets a broad range of both exogenous ,3,4 and C114S mutant, and pNL-A1\u0394vif were generous gifts of Dr. Klaus Strebel [APOBEC3G expression vectors pAPO3G-CFP, pAPO3G-YFP, and pAPO3G-HA were described previously . Express Strebel . Antibod+ T cells were isolated  from PHA/IL-2 activated human peripheral blood mononuclear cells cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 \u03bcg/ml streptomycin. The H9 lymphoid T cell line  was cultured in RPMI 1640 medium modified as above. The human 293T embryonic kidney and HeLa cervical carcinoma cell lines were maintained in a humidified incubator (5% CO2) at 37 \u00b0C in Dulbecco's modified Eagle's medium (Invitrogen) also modified as above. The HeLa-APOBEC3G cell line (referred to here as HeLa-APO3G), which stably expresses APOBEC3G with C-terminal c-Myc epitope tag (APO3G-Myc), was obtained through the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, from Drs. Klaus Strebel and Eri Miyagi [Primary CD4i Miyagi . 293T ani Miyagi .c protein assay . HA and CFP tagged proteins were precipitated by overnight incubation with either \u03b1-HA or \u03b1-GFP rabbit polyclonal antibodies directly conjugated to agarose beads (Santa Cruz Biotechnology). Samples were washed four times (15 min for each wash) in lysis buffer and eluted by boiling for 2 min at 100 \u00b0C in SDS-PAGE sample buffer . SDS-PAGE separation and immunoblot analysis of protein were performed as previously described [For immunoprecipitation, total cell extracts were prepared using Mammalian Protein Extraction Reagent  supplemented with 0.5% (v/v) Triton-X 100 (Pierce), 150 mM NaCl, 5 mM EDTA, and a 1:100 (v/v) dilution of a protease inhibitor cocktail for mammalian tissue. Extracts were clarified by centrifugation and protein concentration was determined by Described .d-lysine . Primary peripheral blood CD4+ T cells and H9 T lymphocytes were attached to cover slips using Cell-Tak cell and tissue adhesive (BD Biosciences) according to the manufacturer's instructions. Cells were fixed for 20 min with 3.7% (v/v) paraformaldehyde  in PBS for 20 min followed by permeabilization with 0.25% (v/v) TX-100 (Pierce) for 5 min. Samples were next washed three times (5 min for each wash) in PBS containing 0.1% (v/v) TX-100 (PBST). Samples were blocked for 30 min in PBST containing 2% (w/v) bovine serum albumin (PBST/BSA). All primary and secondary antibodies were diluted in PBST/BSA and secondary antibodies were directly conjugated to Alexa Fluor dyes . In certain cases, samples were counterstained with Hoechst 33258 to visualize the nuclei. Samples were visualized with a Leica confocal imaging spectrophotometer system (TCS-SP2)  attached to a Leica DMIRE inverted fluorescence microscope and equipped with an argon laser , two HeNe lasers , an acousto-optic tunable filter (AOTF) to attenuate individual visible laser lines, and a tunable acousto-optical beam splitter (AOBS). All images were acquired from a \u00d763, 1.32 NA oil immersion objective with variable digital magnification ranging from \u00d74 to \u00d78, and image analysis was performed using Leica Confocal Software (LCS) and Adobe Photoshop 7.0 .For immunolocalization, 293T and HeLa cells were seeded onto glass bottom micro-well dishes coated with poly-http://www.ncbi.nlm.nih.gov/Genbank) for the genes and gene products mentioned in this paper are YFP-tagged versions of LSM1 (NM_014462), AGO2 (NM_012154), eIF4E (NM_001968), eIF4E-T (NM_019843), RCK/p54 (NM_004397), and DCP2 (NM_152624).The GenBank accession numbers ("}
+{"text": "Inhibition of HIV-1 replication is thought to be the result of APOBEC3G-induced hypermutation of the viral genome that occurs early during reverse transcription. Against this backdrop is a new report from the Uchiyama laboratory that proposes deaminase-independent restriction of HTLV-1 by APOBEC3G . These findings combined with recent reports of deaminase-independent inhibition of Hepatitis B virus as well as HIV-1 suggest that cytidine deaminase activity and antiviral activity may be separable functional properties of APOBEC3G.APOBEC3G is a cellular cytidine deaminase that was recently identified as the Vif-sensitive antiviral host factor responsible for the restriction of  The identification of APOBEC3G (APO3G) in 2002 as the long elusive cellular target of the HIV viral infectivity factor (Vif)  has trigvif gene whose function is to prevent the packaging of APO3G into virus particles  [in vivo remains to be determined given the fact that expression of APO3G in human hepatocytes \u2013 the primary target for HBV \u2013 is very low.Primate lentiviruses such as HIV and SIV have adapted to APO3G with the help of the virus-encoded us (HBV)  and was us (HBV) . Inhibitus (HBV)  and APO3us (HBV) . Howeverus (HBV) . The pre2:32). HTLV-1 differs from HIV-1 in that it produces only very low levels of cell-free infectious virions suggesting a mode of virus transmission that is dependent on close cell-to-cell contacts [Against this backdrop appeared a study by the Uchiyama laboratory investigating the potential antiviral activity of APO3G towards HTLV-1 (Sasada et al. Retrovirology 2005, contacts ,8. Intercontacts . These rcontacts . In the contacts . Surpriscontacts . On the contacts . Thus, wcontacts . Such a contacts ."}
+{"text": "Single residue changes in the Vif protein sequence are sufficient to cause the loss of Vif-induced APOBEC3 neutralization. Interestingly, not all the detected defects lead to a complete inactivation of Vif function since some mutants retained selective neutralizing activity against APOBEC3F but not APOBEC3G or vice versa. Concordantly, independently hypermutated proviruses with distinguishable patterns of G-to-A substitution attributable to cytidine deamination induced by APOBEC3G, APOBEC3F, or both enzymes were present in individuals carrying proviruses with completely or partly defective Vif variants. Natural variation in Vif function may result in selective and partial neutralization of cytidine deaminases and thereby promote viral sequence diversification within HIV-1 infected individuals.The HIV-1 Vif protein counteracts the antiviral activity exhibited by the host cytidine deaminases APOBEC3G and APOBEC3F. Here, we show that defective  vif counters the antiretroviral activity of APOBEC3G and APOBEC3F by inducing their degradation.Host cells express DNA-editing enzymes, termed APOBEC3G and APOBEC3F, that are capable of profoundly attenuating the replication of retroviruses. However, human immunodeficiency viruses use an efficient approach to \u201cneutralize\u201d these cellular enzymes: specifically, the viral gene This study systematically analyzes natural variation in the ability of Vif to neutralize APOBEC-mediated HIV-1 editing and demonstrates that some Vif mutants selectively fail to efficiently \u201csilence\u201d APOBEC3 enzymes. These findings extend the conventional view that errors of reverse transcription and recombination are largely responsible for the rapid evolution of HIV-1 in vivo. Indeed, variation in Vif function may profoundly impact the extent and direction of viral sequence evolution within HIV-1-infected individuals. Thus, innate cellular defenses that likely evolved to restrict retroviral replication might have been usurped by HIV to accelerate viral sequence diversification and escape from immune control and inhibition by antiretroviral drugs. A variety of intrinsic mechanisms that protect organisms from retroviral infection and corresponding viral escape strategies have evolved as a consequence of the coexistence of retroviruses and vertebrates \u20134. LentiWhile both APOBEC3G and APOBEC3F  exhibit anti-HIV-1 activity \u201312,16, AAPOBEC3G- or APOBEC3F-catalyzed deamination is not highly sequence-specific, and many cytidines on the minus strand of nascent retroviral genomes or reporter genes can be deaminated ,14,18\u201320vif alleles remains largely unexplored, we examined the extent of variation in APOBEC3G and APOBEC3F neutralizing activity among naturally occurring HIV-1 Vif variants. Surprisingly, we found that apparently intact but inactive Vif variants are frequently detected in viral sequences from very different sources. Importantly, we also found Vif variants that selectively fail to neutralize APOBEC3G and/or APOBEC3F activity, as well as proviruses in LTNPs that appeared to be independently hypermutated as a result of APOBEC3G or APOBEC3F action. These studies indicate that sporadic inactivation of Vif likely occurs rather frequently in vivo, and that natural variation in Vif function is likely to profoundly impact the extent and direction of viral sequence evolution within HIV-1-infected individuals.Thus far, analyses of natural variation in HIV-1 Vif function have been restricted to prediction of defects resulting from gross mutations . Indeed, some reports suggest that long-term non-progressors (LTNPs) harbor grossly mutated Vif more frequently than do those with progressive HIV disease . Howevervif alleles, because replication-defective HIV-1 variants should not be obscured by superimposed replicating virus. Moreover, some viral DNA sequences in these individuals contained numerous G-to-A changes in the 5\u2032LTR (P2) [gag (P3) [Two distinct sources of Vif alleles were selected in order to either maximize or minimize the occurrence of inactive variants. The first group was derived from uncultured DNA isolated from peripheral blood mononuclear cells (PBMCs) of three extensively studied individuals  with non-progressive HIV-1 infection for more than 20 y , 30\u201335. LTR (P2)  and in ggag (P3) . Amplifivif alleles while minimizing the introduction of new mutations. V1, V3, and V4 were obtained from recently infected individuals, and V2 was obtained from a chronically infected patient. A second set of Vif variants was derived from four short-term HIV-1 isolates  . Vif varvif sequences obtained from these sources is depicted in vif sequences from each individual or isolate diverged by 3\u20139% from a prototype vif allele (from HIV-1 NL4\u20133), a value typical of North American subtype-B vif sequences , six sequences  contained one or more premature stop codons. Interestingly, each of these resulted from a G-to-A mutation in the context of a Trp codon (TGG to TAG). In addition, a subpopulation of obviously defective Vif sequences from P3  that contained premature stop codons were assumed to be inactive. Therefore, of the 79 sampled vif alleles, comprising 61 distinct proteins, at least 15 were unable or poorly able to counteract APOBEC3G antiviral activity  that occurred at a position that is invariant in the HIV-1 sequence database.In the presence of APOBEC3G, NL4\u20133 Vif harboring the mutations K22E, S32P, Y40H, E45G, F115S, G138R, or L150P failed to restore HIV-1 infectivity A. ConverGag-Pol sequences from patients and viral isolates harboring defective Vif alleles  ,33, we nGag-Pol sequences from the LTNPs contained about 3-fold fewer changes relative to an NL4\u20133 HIV-1 reference sequence than did the viral isolate sequences. This is perhaps due to the fact that the three LTNPs became infected more than 20 y ago, quite early in the North-American HIV-1 epidemic and around the time the viral isolate NY5, which comprises the 5\u2032 portion of the reference sequence NL4\u20133 [In general, the ce NL4\u20133 , was obtGag-Pol clones  in LTNP DNA and viral isolates, it seems probable that Vif is sporadically inactivated at some frequency by reverse transcriptase errors or cytidine deamination in all HIV-1-infected individuals. We also found multiple, independent hypermutated proviruses in two of the three LTNPs studied. It is likely that the relative ease of detection of extensively hypermutated proviruses is an unusual property of LTNPs [While defective of LTNPs  and hypeA recent report provides independent evidence for the occurrence of sporadic Vif inactivation in vivo: indeed, more than 9% of proviral sequences derived from resting CD4+ cells of HAART-treated patients with plasma viremia below the level of detection were found to be hypermutated .Site-directed mutagenesis studies revealed a number of residues and domains located throughout Vif that are essential for infectivity and viral replication in non-permissive cells \u201344. We iThe high adenosine content of lentiviral genomes  and the vif genes were amplified by nested PCR using high-fidelity polymerase and cloned into the expression vector pCRV1, as previously described [6 PBMCs [DNA was extracted from either patient's PBMCs , or from PBMCs used for viral propagation  using DNeasy DNA isolation kit . Samples were obtained in 1995 and 2000 for P1, in 2000 and 2001 for P2, and 2003 for P3. Because clinical materials from the mid-1990s were not available for P3, we reconstructed the two P3\u2032s vif alleles that were previously described and contained amino acid substitutions relative to a P3 sequence from 2003 (Y40H and F115S ). Full-lescribed . In orde[6 PBMCs ), multip[6 PBMCs . Wild-tyvif variants. PCR fragments were cloned into TOPO XL vector  and sequenced bidirectionally. To ensure appropriate sampling in the setting of low proviral load, we performed multiple PCR reactions in parallel and cloned the fragments from five to seven PCR reactions separately. Phylogenetic relationships were assessed by the Neighbor Joining method (PAUP software). Analysis of the G-to-A substitutions was performed using the HYPERMUT program [A region spanning the Gag, protease (PR), and half of the reverse transcriptase gene  was amplified from all three LTNPs and the four viral isolates using nested PCR and the same DNA samples used as a source of  program .HIV-1 vector particles were generated by transfecting 293T cells with plasmids expressing HIV-1 gag-pol (pCRV1/Gag-pol), a packaHIV-1 vector particles were generated by transfection of 293T cells, as for the analysis of Vif function with two differences: First, APOBEC3G or APOBEC3F but no Vif expression plasmid was included in the transfection mixture. Second, to minimize the possibility that transfected DNA rather than de novo synthesized viral DNA would be amplified and sequenced, the pV1/hrGFP plasmid vector was omitted from the transfection mixture and a 293T-derived cell line carrying an integrated V1/hrGFP vector genome was used to generate vector particles. Vector particles generated by this method were used to infect MT4 cells and DNA was extracted 8 to 10 h later using a DNeasy DNA isolation kit. hrGFP sequences were amplified by PCR, cloned into TOPO vector, and sequenced bidirectionally. Sequences (450-nucleotide fragment) from 10 to 15 clones were aligned using the DNAStar software package.Figure S1(24 KB PDF)Click here for additional data file.Table S1(16 KB PDF)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the vif and gag-pol sequences are DQ097739\u2013DQ097768.The GenBank ("}
+{"text": "The APOBEC3G protein represents a novel innate defense mechanism against retroviral infection. It facilitates the deamination of the cytosine residues in the single stranded cDNA intermediate during early steps of retroviral infection. Most poxvirus genomes are relatively A/T-rich, which may indicate APOBEC3G-induced mutational pressure. In addition, poxviruses replicate exclusively in the cytoplasm where APOBEC3G is located. It was therefore tempting to analyze whether vaccinia virus replication is affected by APOBEC3G.The replication of vaccinia virus, a prototype poxvirus, was not, however, inhibited in APOBEC3G-expressing cells, nor did other members of the APOBEC3 family alter vaccinia virus replication. HIV counteracts APOBEC3G by inducing its degradation. However, Western blot analysis showed that the levels of APOBEC3G protein were not affected by vaccinia virus infection.The data indicate that APOBEC3G is not a restriction factor for vaccinia virus replication nor is vaccinia virus able to degrade APOBEC3G. Poxviridae in particular, evolved several mechanisms for immune evasion [During evolution, eukaryotic cells had to cope with a large amount of pathogens. The interaction of host and pathogen required defense responses in the host, which resulted in the development of the innate immune system. Due to this high selection pressure, pathogens developed strategies to escape or manipulate the host immune defense.  evasion ,2. The b evasion .APOBEC3G is a recently discovered defense mechanism against retroviral infection . The proIn addition to its anti-retroviral function ,6, APOBETo assess the impact of APOBEC3G on the VACV life cycle, we used HeLa-APOBEC3G cells which were stably transfected with an APOBEC3GmycHis expression plasmid encoding a Myc- and 6-His-tagged protein . IntraceIn addition, we sought to assess whether other members of the APOBEC3 gene family are able to constrain VACV replication. We tested the influence of APOBEC3G, -F and -H, and mouse APOBEC3 on VACV replication by transient transfection of expression plasmids into BHK cells, followed by infection with VACV-WR at an MOI of 0.05. Viral titers were measured 0, 24 and 48 h after infection by titration on RK13 cells. Although the transfection rate was usually around 50%, expression of the APOBEC3 proteins in BHK cells had no influence on VACV replication Figure .During wild-type human immunodeficiency virus (HIV) infection, APOBEC3G is inactivated by the HIV accessory protein Vif, which targets it for degradation by the ubiquitin-dependent proteasomal pathway. Therefore, APOBEC3G only restricts Vif-deleted HIV ,15,10. CPoxviruses are large cytoplasmic DNA viruses and infection of a cell initiates drastic responses that aim to eliminate virus-infected cells. This innate immune response is able to restrict a multitude of viruses by various strategies. The APOBEC3 gene family contains recently discovered factors that are able to restrict retroviruses, hepadna viruses and parvoviruses ,7,8. TheAPOBEC3G, -F and -H, and mouse APOBEC3 are located in the cytoplasm of the cell, the location of poxviral replication. Most poxvirus genomes are relatively A/T-rich which could be a consequence of APOBEC3-induced mutational pressure . It was,in vitro and is able to infect virtually all cell types [VACV has a very broad host range ll types . Surprisll types . Howeverll types .Poxviruses have developed several strategies to evade the innate immune system . Like HIUsing transient transfections, we could show that APOBEC3G, -F or -H, or mouse APOBEC3 expression has no effect on VACV replication and VACV infection does not lead to a degradation of the APOBEC3G protein.The following plasmids were used for transfections: pcDNA-APOBEC3G-MycHis encoding a C-terminally Myc-tagged human APOBEC3G , human A293T and NIH 3T3 cells were grown in Dulbecco's Modified Eagle's Medium  supplemented with 10% fetal calf serum (FCS); . HeLa, HeLa-APOBEC3G and BHK cells were grown in Roswell Park Memorial Institute-1640 Medium  supplemented with 10% FCS. RK-13 cells were grown in Eagle's Minimum Essential Medium  supplemented with 10% FCS and 1% Non-Essential Amino Acids .6 cells in a 10 cm tissue culture plate. The cells were transfected with 2 \u03bcg Gag/Pol expression plasmid (pHIT60) [5 NIH 3T3 cells. 48\u201372 hours after transduction, the numbers of GFP-expressing cells were detected by FACS analysis. The titers are given in relative infectious units and are representative data of three independent experiments.To generate MLV-based vector particles, a day before transfection, cells were seeded at a density of 2 \u00d7 10(pHIT60) , 1 \u03bcg ec(pHIT60) ) and 3 \u03bc(pHIT60)  using th6 cells in a 10 cm tissue culture plate one day before transfection. The cells were transfected with the following expression plasmids [Lentiviral vectors were generated by seeding 293T cells at a density of 2 \u00d7 10plasmids  using thFor the analysis of VACV replication, cells (BHK or 293T) were transfected with 8 \u03bcg plasmid DNA, encoding APOBEC3 proteins and infected with vaccinia virus WR 48 h after the transfection. Cells were harvested at the indicated time points and vaccinia virus was titrated on RK-13 cells.Transfection of APOBEC3G expression plasmid was analyzed by intracellular immunofluorescence staining with a mouse anti-Myc antibody  and a FITC-conjugated anti-mouse antibody after permeabilization of the cells with BD Perm/Wash Buffer . The numbers of FITC-stained cells were detected by FACS analysis.Cell lysates were obtained and Western blot analysis was performed as described previously . WesternThe author(s) declare that they have no competing interests.MK, YS and YM-F performed the experiments. MK, YS, CM, GS and BS participated in the design of experiments, oversight of the conduction of the experiments, and in the interpretation of the results."}
+{"text": "Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cellular protein with a broad antiviral activity. It inhibits infectivitiy of a wide variety of retroviruses by deaminating deoxycytidine (dC) into deoxyuridine (dU) in newly synthesized minus strand DNA, resulting in G-to-A hypermutation of the viral plus strand DNA. To clarify the mechanism of its function, we have examined the antiviral activity of APOBEC3G on human T-cell leukemia virus type 1 (HTLV-1), the first identified human retrovirus.In this study, we have demonstrated that overexpressed as well as endogenous APOBEC3G were incorporated into HTLV-1 virions and that APOBEC3G inhibited the infection of HTLV-1. Interestingly, several inactive mutants of APOBEC3G also inhibited HTLV-1 and no G-to-A hypermutation was induced by APOBEC3G in HTLV-1 genome. Furthermore, we introduced the human immunodeficiency virus type 1 (HIV-1) vif gene into HTLV-1 producing cell line, MT-2, to antagonize APOBEC3G by reducing its intracellular expression and virion incorporation, which resulted in upregulation of the infectivity of produced viruses.APOBEC3G is incorporated into HTLV-1 virions and inhibits the infection of HTLV-1 without exerting its cytidine deaminase activity. These results suggest that APOBEC3G might act on HTLV-1 through different mechanisms from that on HIV-1 and contribute to the unique features of HTLV-1 infection and transmission. APOBEC3G, also known as CEM15 -4. The p5to 106 is infectious [in vivo [gag, pol, and env genes, HTLV-1 genome has four open reading frame (ORF) regions at its 3' end, which encode regulatory proteins including Rex and Tax. Although the functions of other encoded proteins such as p12, p13, and p30 have been under investigation [HTLV-1 is a member of retroviruses which is the etiologic agent of adult T-cell leukemia(ATL)  and HTLVfectious . The fac[in vivo ,19. Furt[in vivo . In additigation ,22, any In this report, we have investigated the antiviral activity of APOBEC3G on HTLV-1. We examined the packaging of APOBEC3G into HTLV-1 virions, induction of mutations in the viral genome, and regulation of the viral infectivity. Our finding would be a clue to understand the unique infectious mechanism of HTLV-1.We first examined the incorporation of APOBEC3G into HTLV-1 virions. We transfected HEK293T cells with an infectious molecular clone of HTLV-1 (K30) and infectious molecular clones of HIV-1 with or without vif  with or without an expression vector for HA-APOBEC3G and performed Western blotting to detect APOBEC3G in producer cells and produced virions. Incorporation of APOBEC3G was clearly detected in HTLV-1 virions produced from cells cotransfected with HTLV-1 K30 and APOBEC3G expression vector Fig. , lane 2.We next examined whether APOBEC3G packaged into HTLV-1 virions deteriorated the infectivity of the virus. For this purpose, we employed the PCR-based infectivity assay as previously described  with modGpG sequence which is the preferred substrate for APOBEC3G, suggesting that these mutations were induced by APOBEC3G, although the frequency is very low as seen with HBV [To confirm the above hypothesis, we examined whether APOBEC3G induces G-to-A hypermutation in HTLV-1 DNA. p12 region was amplified from target cell DNA and sequenced. We detected a few G-to-A mutations in HTLV-1 K30 genome integrated into target cell DNA in the presence of APOBEC3G Fig. , but notwith HBV . In contwith HBV -8. AccorFinally, we examined the antiviral activity of endogenous APOBEC3G. First, we confirmed the function of endogenous APOBEC3G in MT-2 cells by infection with HIV-1 wild type (WT) and \u0394Vif virions. WT virus could replicate in MT-2 cells, but \u0394Vif virus not (data not shown), indicating that endogenous APOBEC3G in MT-2 cells may be able to function as an anti-HIV-1 factor or that there may exist other APOBEC3 protein members sensitive to Vif. Based on this result, we performed an infectivity assay using HTLV-1 virions produced from MT-2 cells. Since we found that endogenous APOBEC3G was incorporated into HTLV-1 virions produced from MT-2 cells Fig. , we intrin vivo and in vitro [In this study, we have demonstrated that APOBEC3G has an antiviral activity on HTLV-1. APOBEC3G was efficiently incorporated into HTLV-1 virions and inhibited the infectivity of HTLV-1 without inducing G-to-A hypermutation. First, we showed that APOBEC3G, overexpressed or endogenous, was efficiently incorporated into HTLV-1 virions. Our finding suggests that HTLV-1 cannot exclude this protein from visions unlike HIV-1 -4,6-8. Pin vitro ,25. We cSecond, we also showed that APOBEC3G inhibited the infection of HTLV-1. Because of low infectivity of cell-free HTLV-1 virions, we could not detect p19 production in the supernatant of infection culture (data not shown). Instead, we performed an infectivity assay as described previously with modification , in whicTo confirm the notion above, we prepared MT-2/Vif cells to block incorporation of endogenous APOBEC3G into HTLV-1 virions. Expression of Vif in MT-2 cells reduced the expression of APOBEC3G and its incorporation into virions. In the presence of Vif, APOBEC3G in MT-2 cells seemed to be ubiquitinated and degraded by the proteasome, because we detected two bands of APOBEC3G in MT-2/Vif cells by immunoblotting, of which the upper band might indicate mono-ubiquitinated APOBEC3G, while the faded lower band indicate the intact APOBEC3G remained Fig.  and 3F. During the preparation of this manuscript, Navarro et al. reported that HTLV-1 is relatively resistant to the antiviral effect of encapsidated APOBEC3G . In thatFinally, our findings have also broadened the spectrum of antiviral activity of APOBEC3G and further studies on the mechanisms of the antiviral activity of APOBEC3G on HTLV-1 will provide us with new insights into the function of this molecule as an antiviral innate immunity.APOBEC3G is incorporated into HTLV-1 virions and inhibits the infection of HTLV-1 without exerting its cytidine deaminase activity. This suggests that APOBEC3G might act on HTLV-1 through different mechanisms from that on HIV-1 and contribute to the unique features of HTLV-1 infection and transmission.Expression vectors for hemagglutinin (HA)-tagged human APOBEC3G (APOBEC3G), its point mutants , and murine APOBEC3G (muAPOBEC3G) were described previously ,7. pNL43HEK293T cells were maintained in Dulbecco's modified Eagle's medium  containing 10% fetal calf serum, penicillin, streptomycin, and glutamine (Invitrogen). SupT1 cells and MT-2 cells were maintained in RPMI 1640  containing 10% fetal calf serum, penicillin, streptomycin, and glutamine. MT-2/Mock and MT-2/Vif cells were established by transduction of retrovirus vectors  and selection with Neomycin .Western blotting was performed to detect expression of APOBEC3G, its mutants, and muAPOBEC3G in producer cells, and their incorporation into virions as described previously . In brieTo confirm the incorporation of APOBEC3G into virion, HTLV-1 K30 virions were purified by sucrose density equilibrium gradients as previously reported with slight modifications [Infectivity of HTLV-1 was detected as previously reported with slight modifications . In brieMutations in HTLV-1 DNA were detected by sequencing p12 region of HTLV-1 integrated into target cells . PreparaThe author(s) declare that they have no competing interests.AS designed research, performed research, contributed vital new reagents, analyzed data, and wrote the paper. AT-K designed research, performed research, contributed vital new reagents, analyzed data, wrote the paper, and organized research. KS performed a part of research. MK performed a part of research. AA performed a part of research. MH performed a part of research and contributed vital new analytical tools. KI contributed vital new analytical tools and analyzed data. YT contributed vital new reagents. TU analyzed data, drafted the paper, and organized research."}
+{"text": "Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4G107S.pik1 is essential. In cells with pik1 deleted, ectopic expression of a loss-of-function allele, created by fusion to a temperature-sensitive dihydrofolate reductase, allowed normal cell proliferation at 25\u00b0C. At 36\u00b0C, cells arrested with abnormally thick, misplaced or supernumerary septa, indicating a defect late in septation. In addition to being Golgi associated, ectopically expressed GFP-tagged Pik1 was observed at the medial cell plane late in cytokinesis. New alleles, created by site-directed mutagenesis, were expressed ectopically. Lipid kinase and Cdc4-binding activity assays were performed. Pik1D709A was kinase-dead, but bound Cdc4. Pik1R838A did not bind Cdc4, but was an active kinase. Genomic integration of these substitutions in S. pombe and complementation studies in Saccharomyces cerevisiae pik1-101 cells revealed that D709 is essential in both cases while R838 is dispensable. In S. pombe, ectopic expression of pik1 was dominantly lethal; while, D709A,R838Apik1 was innocuous, R838Apik1 was almost innocuous, and D709Apik1 produced partial lethality and septation defects. The pik1 ectopic expression lethal phenotype was suppressed in G107Scdc4. Thus, D709 is essential for kinase activity and septation.Gene deletion revealed that Pik1 kinase activity is required for septation. The Pik1 R838 residue is required for important protein-protein interactions, possibly with Cdc4. Saccharomyces cerevisiaeDrosophila melanogasterS. pombe is a multistep process involving division site selection, contractile ring formation and contraction, membrane expansion/ingression and formation/dissolution of primary and secondary septa Phosphoinositides are involved in signal transduction, regulation of actin cytoskeletal organization, cytokinesis, and membrane traffic in eukaryotic cells, including in yeasts S. cerevisiae and S. pombe each appear to have 3 distinct PtdIns 4-kinases. In the budding yeast these are; Lsb6p, a type II enzyme; Stt4p, a type III\u03b1 enzyme; and Pik1, a type III\u03b2 enzyme pik1-101, arrests without completing cytokinesis LSB6 is non-essential STT4 and is required for actin nucleation and endosome motility S. pombe, locus SPAC343.19 appears to encode a type II enzyme, locus SPBC577.06c appears to encode a type III\u03b1 enzyme, and pik1 (SPAC22E12.16c) is a putative type III\u03b2 enzyme. Sequence comparisons suggest that S. cerevisiae PIK1 and S. pombe pik1 are orthologs; although, sequence similarities outside the lipid kinase domains are weak . The S. cerevisiae enzyme is in the nucleus and associated with the Golgi S. pombe protein appears associated with the Golgi S. pombe has not been reported. The localization of S. pombe Pik1 during the cell cycle and the importance of this enzyme for cell division have yet to be determined.In S. cerevisiae Pik1 interacts with a number of proteins, including Frq1 S. pombe Pik1 and the corresponding homologs of the S. cerevisiae proteins, Ncs1, Rad25 and Rad24, have not been reported.S. pombe Pik1 has been reported to interact with Cdc4 cdc4 (G107Scdc4) rng2 gene encodes a protein related to the human IQGAP1 protein, which binds actin and calmodulin and is a potential effector for the Rho family of GTPases. Cdc4 presumably interacts with Rng2 at one or several of its 6 IQ-motifs cdc4 mutations have been identified that cause temperature-dependent failure of cytokinesis G19Ecdc4 or G107Scdc4) were very stable, even at the restrictive temperature, suggesting that failure of cytokinesis is due to subtle changes in structure that may impair protein interactions R33Kcdc4) was shown to affect actin-myosin function in sliding filament motility assays at the non-permissive temperature cdc4 alleles  and a modified myosin that lacked both IQ domains and thereby unable to bind Cdc4, still showed lethality at the non-permissive temperature Cdc4 also interacts with Rng2, another contractile ring component, which is required for its assembly S. pombe pik1 and S. cerevisiae PIK1 are conserved. We hypothesized that in S. pombe, Pik1 lipid kinase activity and possibly its Cdc4-binding activity are involved in a specific aspect of cytokinesis. To test this hypothesis, we created and characterized a number of new pik1 alleles; including, a chromosomal pik1 deletion allele, a conditional loss-of-function allele, a fluorescently tagged allele, and 3 point mutation alleles. The latter 3 alleles were designed to affect Pik1 lipid kinase and Cdc4-binding activities. Alleles were assessed after genomic integration or ectopic expression, and for complementation of the conditionally lethal S. cerevisiae pik1-101 allele.It is not known if the essential functions of S. pombe strains  for screening . The double mutant D709A, R838Apik1 was generated by replacing the BamHI-AgeI fragment of D709Apik1 with the corresponding fragment from R838Apik1  (Genbank accession number FJ918574). Primer sequences are available upon request.A pik1, producing substitution R838A, or D709A, or both. Allele replacement constructs consisted of the last 380 codons of pik1 (pik1472\u2013851), the native stop codon, the nmt1 terminator region, ura4 gene cassette and 700 bp of genomic DNA downstream of the pik1 coding region. As a result, the pik1 locus was modified by the presence of nmt1 transcription termination sequences and by the presence of a downstream ura4 cassette. To control for these changes, the wild-type sequence was integrated into the pik1 gene by the same method. Diploid cells (N250 X N253) were transformed, plated at 30\u00b0C on EMM \u2013adenine \u2013uracil. Colonies were tested for integration by colony PCR and growth on EMM \u2013uracil. Tetrad analysis was used to establish the essentiality of each haploid pik1 allele.Homologous recombination in diploid cells ts). Long-lived at 25\u00b0C, at 36\u00b0C it is rapidly degraded by the N-end rule pathway ts coding region from pPW66R pik1 cDNA coding region in pREP41X ts-pik1 and the disrupted chromosomal pik1 allele , was selected by random spore analysis. In N1366, the sole source of Pik1 is episomal.The N-degron approach fuses a protein of interest to one that is conditionally unstable. The latter consists of monoubiquitin, an arginine residue, and a thermolabile dihydrofolate reductase  were resuspended in 50 ml medium at 25\u00b0C and incubated for 220 min. Cells were sampled every 20 min. and examined immediately by microscopy to evaluate the intracellular localization of 2XeGFP-Pik1 or fixed with formaldehyde, incubated for 30 min. on a rotating wheel, washed 3 times in phosphate-buffered saline and kept at 4\u00b0C for microscopic examination To examine cell cycle dependent changes in 2XeGFP-Pik1 localization, a Cell morphology and mitotic index were examined by bright-field and fluorescence microscopy after formaldehyde fixation and Calcofluor White  and 4\u20326,-diamidino-2-phenylindole (DAPI) staining An Olympus 1X70 inverted microscope with 60\u00d7 1.4NA Plan-apo objective, appropriate filter sets and a RT-Slider (SPOT) CCD camera  was used for bright-field and fluorescence microscopy. Images were cropped and processed for brightness and contrast with Spot32 Advanced software.pik1-td cells were examined by transmission electron microscopy as described Potassium permanganate-fixed pik1 alleles were introduced into cdc4 or G107Scdc4 strains. Starter cultures in EMM \u2013leucine +thiamine were incubated overnight at 30\u00b0C or 25\u00b0C (for temperature-sensitive strains). Cells were collected by centrifugation , washed 3 times in sterile distilled water, placed in 50\u2013100 ml cultures at 105 cells/ml, and incubated for 24 h. at 30\u00b0C or 25\u00b0C, + or \u2212 thiamine. Cell numbers were estimated with a hemocytometer. Cells were collected by centrifugation, and either washed and fixed for microscopic examination, or lysed with a \u2018mini\u2019 French pressure cell at 900 p.s.i. for protein estimation, immunoblotting, lipid kinase assays and ELISA.Recombinant pREP vectors Starter cultures in EMM \u2013leucine +thiamine were incubated to saturation (24\u201336 hours) at 30\u00b0C or 25\u00b0C. Cells were collected by centrifugation and washed 3 times in sterile distilled water. Aliquots (5 \u00b5l) from each of 4, 10-fold serial dilutions were spotted onto EMM \u2013leucine, + or \u2212 thiamine, + phloxin B plates and incubated at 30\u00b0C for 5\u20136 days.pik1 expression vectors were cultured for 24 h., + or \u2212 thiamine, at 30\u00b0C or 25\u00b0C. Cells were harvested by centrifugation (700\u00d7g for 5 min.), resuspended in 25 mM HEPES, pH 7.4, 10 mM MgCl2, and passed through a \u2018mini\u2019 French pressure cell 3 times at 900 p.s.i. Cell lysate protein content was estimated 32P] ATP for 15 min. at room temperature and the reaction stopped by addition of 6 M HCl to a final concentration of 1.7 M. Lipids were extracted with three volumes of chloroform:methanol , vortexed for 10 s. and centrifuged in an Eppendorf microcentrifuge at maximum speed for 5 min. 2 gas. The lipids were resuspended in 4 \u00b5l of chloroform:methanol  and spotted onto a Silica gel 60 thin layer chromatography (TLC) plate previously baked for 30 min. at 100\u00b0C. The TLC plate was placed in a chromatography chamber pre-equilibrated for two hours with freshly made developing solution (1-propanol\u22362 M acetic acid (13.7\u22367 vol\u2236vol)). Lipid separation was for 6\u20138 h. The TLC plate was dried overnight and either exposed to Kodak BioMax XAR film for 2\u20133 days or scanned under 10% methane in argon using a BIOSCAN AR-2000 imaging scanner for radio-TLC. WinSCAN 2D software version 1.05 was used to visualize the distribution of radioactivity on the plate. The silica carrying the radiolabelled lipid was added to Aquasol (Perkin-Elmer) for liquid scintillation counting. Data were corrected for counting efficiency and decay and expressed as disintegrations per minute (DPM). Lipid standards purchased from Avanti Polar Lipids P, PtdInsP2 and PtdInsP3) were run in parallel and visualized using iodine vapour.Cells carrying GAL4-DB (DNA binding domain) fused in-frame to a cdc4 cDNA sequence, and the tryptophan selectable pBI771 vector carrying sequences encoding the GAL4-TA (trans-activating domain) fused in-frame to residues 507 through 851 of wild-type or mutant pik1 alleles. Both vectors were introduced simultaneously into S. cerevisiae YPB2 cells using a lithium acetate procedure. Cells were selected for the presence of both plasmids by colony formation on SD \u2013leucine \u2013 tryptophan plates at 30\u00b0C for 5 days. A positive protein interaction was then identified by colony formation at 30\u00b0C for 7\u20139 days on SD \u2013leucine \u2013tryptophan \u2013histidine +3-amino-1\u2032,2\u2032,4\u2032-triazole (3-AT). X-gal colony filter assays were also performed to confirm positive yeast two-hybrid interactions Assays were performed as described S. pombe cultures, initially at 1\u00d7105 cells/ml, carrying pREP1 plasmids expressing wild-type or mutant pik1 alleles, were incubated + or \u2212 thiamine for 24 h. at 30\u00b0C. Cell extracts were prepared using a French press \u2018mini\u2019 cell (3 passages at 900 p.s.i.) in phosphate-buffered saline (PBS) with protease inhibitors  at 4\u00b0C. Multiwell plates were coated by incubating with purified Cdc4 protein S. pombe pik1 wild-type and mutant cDNA coding regions were cloned (NdeI \u2013 BamHI) in S. pombe expression vectors pREP1, pREP41, and pREP81 pik1 coding regions flanked by the nmt1 promoter and terminator sequences were then cloned (PstI \u2013 SstI) in S. cerevisiae expression vector, YEplac181 S. cerevisiae cells carrying PIK1 or pik1-101 were transformed with each of the resulting vectors using lithium acetate 5 cells/ml at 25\u00b0C. After 18\u201320 h., cells were recovered by centrifugation, washed 3-times in sterile water, and resuspended at 2\u00d7107 cells/ml. Aliquots (5 \u00b5l) of serial 10-fold dilutions were spotted onto SD \u2013Leu plates and incubated at a permissive (25\u00b0C) or restrictive (37\u00b0C) temperature for 5 days.pik1/\u0394pik1::ura4. Analysis of the germination and colony formation potential of spores from azygotic asci confirmed the essential nature of pik1 at 30\u00b0C. Of the four spores from pik1/\u0394pik1::ura4 diploid cells, only two formed colonies at 30\u00b0C . Most of the spores that did not form colonies at 19\u00b0C or 25\u00b0C germinated and divided a few times, in contrast to most of the spores at 36\u00b0C which did not germinate or germinated, but did not divide. Thus, pik1 is essential for vegetative cell division. pik1 also appears to be required for spore germination at the higher temperatures (30\u00b0C\u201336\u00b0C).Tetrad analysis was performed on the hemizygous diploid strain,  at 30\u00b0C . To evalR\u0394pik1:: Kan strain that carried an expressed episomal pik1 cDNA sequence. To achieve this, the pik1 coding region was replaced with a RKan gene cassette in haploid cells that carried an episomal pik1 cDNA sequence under the control of a highly attenuated (pREP81) thiamine-repressible nmt1 promoter . The nmt1 promoter is leaky and some level of expression is observed, even in the presence of thiamine \u0394pik1::RKan chromosomal allele, the level of pik1 expression from a thiamine-repressed, highly attenuated nmt1 promoter is sufficient for cell growth and division.Plasmid loss studies were carried out on a haploid pik1 cDNA coding region was fused to sequences encoding a ubiquitin, an arginine residue, and a temperature-sensitive dihydrofolate reductase fusion protein (Ub-R-DHFRts) pik1-td (pik1-temperature dependent). Ub-R-DHFRts is a thermolabile protein that unfolds at elevated temperatures to expose a destabilizing N-end residue, making the protein susceptible to degradation by ubiquitin-dependent proteolysis. At 25\u00b0C, the Ub-R-DHFRts-Pik1 fusion protein should be stable. At 36\u00b0C, the Ub-R-DHFRts moiety should unfold and cause degradation of Pik1. The effects of loss of Pik1 function were assessed by ectopic expression of pik1-td under the control of an attenuated nmt1 promoter in cells lacking the chromosomal pik1 coding region (\u0394pik1::ura4). The pik1-td cells were cultured at 25\u00b0C in the presence of thiamine. These cells had a normal proliferation rate . The fusion protein was below the detection limit in cells cultured under repressed conditions. Cells incubated in the presence or absence of thiamine had similar growth rates, morphology and septum and actin distributions (not shown). Thus, the fusion of 2XeGFP to Pik1 had no apparent effect on Pik1 functions. In the absence of thiamine, 2XeGFP-Pik1 fluorescence was observed as a punctate pattern throughout the cytoplasm and around the cell periphery. In about 8% of the cells, a fluorescent medial band was observed in addition to the dots (Sequences encoding two tandem eGFP (enhanced green fluorescent protein) proteins were fused to the 5\u2032end of a the dots . No medithe dots .pik1 was expressed in cdc25-22 cells synchronized by temperature block and release. At the restrictive temperature (36\u00b0C), cdc25-22 cells arrest in G2/M cdc25-22 block and release method. The punctate fluorescence throughout the cytoplasm was still observed at all time points although it was quite faint. Thus, there is a cell cycle dependent recruitment of Pik1 to the medial region of dividing cells, at about the same time as deposition of septum material occurs.To evaluate the cell cycle dependence of recruitment of Pik1 to the medial region, 2XeGFP-http://pfam.sanger.ac.uk/) http://ca.expasy.org/prosite/). To evaluate the importance of Pik1 lipid kinase and Cdc4-binding activities for cytokinesis, we created alleles that were impaired for each of these two functions.Previously, the 345 C-terminal amino acids of Pik1 (a.a. 507\u2013851) were shown to interact with Cdc4 D709Apik1  is present in the C-terminal region of Pik1 (pik1 homologs (R838Apik1 (Cdc4 is known to bind to IQ domains of type II myosins  of Pik1 . The fir of Pik1 . Furtherhomologs . The cenik1R838A .S. cerevisiae cells were co-transformed with the \u2018bait\u2019 vector carrying the S. pombe cdc4 cDNA sequence fused to the GAL4-BD (DNA binding domain) and with the \u2018prey\u2019 vectors carrying the C-terminal region of S. pombe wild-type and mutant pik1 sequences fused to the GAL4-TA  domain. As observed previously pik1 sequence indicating that the GAL4-TA domain by itself did not result in a positive interaction phenotype . Thus, derepression of the ectopic expression of each of the pik1 alleles results in elevated levels of the corresponding protein in the cells.As Cdc4 interaction with Pik1 in the yeast two-hybrid assay involved only the C-terminal end of Pik1, we wished to perform immunosorbent assays to assess the binding of Cdc4 to the full-length protein. Western blot analysis was performed to determine if the wild-type and mutant forms of Pik1 accumulated to detectable levels upon ectopic expression . These col cells , -ve. Thxpressed . The sigpik1 alleles cultured in the presence or absence of thiamine. Binding of Pik1 to Cdc4-coated wells was monitored with a primary antiserum against Pik1 and a secondary antibody-HRP conjugate P3 reference lipid-standards (data not shown). PtdIns(4)P and PtdIns(3)P migrate with the same Rf value and are not resolved with this assay system pik1 allele had higher incorporation of 32P label into PtdInsP when expression was derepressed . Cells ectopically expressing the D709Apik1 allele had cell lengths at septation, and septum and contractile ring formation comparable to cells cultured under repressed conditions or carrying the empty vector  that caused dominant lethality . These diploid strains were incubated on ME plates at 25\u00b0C for 2 days to obtain azygotic asci which were transferred to YES plates. Spores from each of 8\u201310 asci were separated with a micromanipulator and incubated at 30\u00b0C for 5 days. As a control, strain N1550 was constructed in which one copy of the pik1 locus was fully wild-type, while the other copy was wild-type with respect to the coding sequence, but had the same modifications to the 3\u2032-untranslated region. In 8 of 10 tetrads, each of the 4 spores formed a colony, although 2 colonies were much larger (D709Apik1/pik1 strain (N1565) revealed that the D709 residue is essential, presumably reflecting a requirement for Pik1 lipid kinase activity at 30\u00b0C. In 6 of 8 azygotic asci dissected, only 2 of the 4 spores formed colonies . In contrast, four colonies were observed from each tetrad in 8 of 10 azygotic asci dissected from the R838Apik1/pik1 strain (N1582) , only 2 of the 4 spores formed colonies and these cells were wild-type for pik1  ; R838A, for pik1 .S. cerevisiae PIK1, which are lost in pik1-101 cells at the restrictive temperature, can be provided by expression of the S. pombe pik1 gene. However, S. pombe cells are highly sensitive to changes in pik1 expression , or an attenuated (Pnmt41) or a highly attenuated (Pnmt81) version of the promoter nmt1 terminator sequence was included in these expression cassettes. We performed all of the experiments described below both in the presence and absence of thiamine. Since the results were indistinguishable only those experiments in the absence of thiamine are shown . This effect was not observed when the attenuated promoters were used. We observed reproducible, but partial complementation of the lethal phenotype of pik1-101 at 37\u00b0C with Pnmt1, reduced partial complementation with Pnmt41, and no complementation with Pnmt81 (not shown). These results indicate that S. pombe pik1 can provide essential functions of Pik1 in an S. cerevisiae loss-of-function mutant and that S. pombe nmt1 promoter sequences are useful in S. cerevisiae. Immunoblot analysis, using a polyclonal anti-S. pombe Pik1 serum that detected this protein in S. pombe cell extracts (S. cerevisiae pik1-101 cells (not shown).We wished to perform complementation studies to determine if the essential functions of pression . If S. cre shown . In initextracts , failed S. pombe Pik1 in S. cerevisiae, we expressed an eGFP-pik1 fusion allele under the control of Pnmt41 in S. cerevisiae PIK1 and pik1-101 cells. Unfortunately, the fluorescent signal from the eGFP-S. pombe Pik1 fusion protein was insufficient for the purpose of determining its subcellular distribution. Immunoblot analysis again failed to detect this protein in extracts from transformed S. cerevisiae PIK1 cells (not shown). Colony formation assays revealed that expression of eGFP-pik1 in PIK1 cells was innocuous at both 25\u00b0C and 37\u00b0C . The pik1-101 allele appears to be either kinase-dead or greatly reduced for kinase activity S. cerevisiaepik1-101 strain possesses sufficient residual kinase activity in the nucleus at the restrictive temperature.pik1-td strain is associated with failure of processes in late cytokinesis . The interaction between Pik1 and Cdc4 may be functionally important; however, whether it affects the activity or localization of Pik1 is unknown.Control experiments revealed that ectopic expression of wild-type le rings . In star protein  and of l protein ; yet, th protein . Examinannocuous . Since e7S cells . We had Overall, these results suggest that Pik1 is a lipid kinase that is recruited to the medial cell plane late in cytokinesis and is required for septation. These results also suggest that protein-protein interactions involving the R838 residue of Pik1, possibly with Cdc4, are functionally significant, although perhaps not essential."}
+{"text": "Although APOBEC3G protein is a potent and innate anti-HIV-1 cellular factor, HIV-1 Vif counteracts the effect of APOBEC3G by promoting its degradation through proteasome-mediated proteolysis. Thus, any means that could prevent APOBEC3G degradation could potentially enhance its anti-viral effect. The UBA2 domain has been identified as an intrinsic stabilization signal that protects protein from proteasomal degradation. In this pilot study, we tested whether APOBEC3G, when it is fused with UBA2, can resist Vif-mediated proteasomal degradation and further inhibit HIV-1 infection.APOBEC3G-UBA2 fusion protein is indeed more resistant to Vif-mediated degradation than APOBEC3G. The ability of UBA2 domain to stabilize APOBEC3G was diminished when polyubiquitin was over-expressed and the APOBEC3G-UBA2 fusion protein was found to bind less polyubiquitin than APOBEC3G, suggesting that UBA2 stabilizes APOBEC3G by preventing ubiquitin chain elongation and proteasome-mediated proteolysis. Consistently, treatment of cells with a proteasome inhibitor MG132 alleviated protein degradation of APOBEC3G and APOBEC3G-UBA2 fusion proteins. Analysis of the effect of APOBEC3G-UBA2 fusion protein on viral infectivity indicated that infection of virus packaged from HEK293 cells expressing APOBEC3G-UBA2 fusion protein is significantly lower than those packaged from HEK293 cells over-producing APOBEC3G or APOBEC3G-UBA2 mutant fusion proteins.Fusion of UBA2 to APOBEC3G can make it more difficult to be degraded by proteasome. Thus, UBA2 could potentially be used to antagonize Vif-mediated APOBEC3G degradation by preventing polyubiquitination. The stabilized APOBEC3G-UBA2 fusion protein gives stronger inhibitory effect on viral infectivity than APOBEC3G without UBA2. There is an active and antagonistic host-pathogen interaction during HIV-1 infection. Upon infection by HIV-1, host cells react with various innate, cellular and humoral immune responses to counteract the viral invasion. Limited and transient restriction of viral infection is normally achieved. However, HIV-1 overcomes these antiviral responses through various counteracting actions. For example, APOBEC3G , a host innate antiviral protein ; whereasAPOBEC3G is a member of cellular cytidine deaminase family. At the late phase of viral life cycle, APOBEC3G is encapsided into the virus particles through interaction with viral Gag protein -8. SpeciUbiquitin-associated domain 2 (UBA2) is typically 45 amino acids long that specifically bind to both mono- and polyubiquitins . HomonucSince Vif promotes APOBEC3G degradation through proteasome-mediated proteolysis of ubiquitinated proteins, and because UBA2 decreases protein degradation through this pathway, we hypothesize that UBA2, if fused with APOBEC3G, should be able to act as a \"stabilization signal\" and to protect APOBEC3G from Vif-mediated degradation. Here we tested this hypothesis by comparing protein stability of normal APOBEC3G protein with the APOBEC3G-UBA2 fusion proteins in the presence of Vif. To gain additional functional insights into the molecular mechanism underlying the ability of UBA2 to prevent protein degradation, the effects of UBA2 on APOBEC3G protein degradation under the conditions of excessive polyubiquitination or the lack of proteasome activity were examined. The effect of UBA2 on APOBEC3G stability and its impact on viral infectivity was also investigated.To test whether UBA2 can stabilize APOBEC3G protein, UBA2 was fused at the C-terminal end of APOBEC3G Fig. . The APOvif gene expression . Together, these data suggested that the wild type UBA2, when it is fused with APOBEC3G, is indeed able to stabilize APOBEC3G and renders it more resistant to Vif than the untagged APOBEC3G.Approximately equal amount of protein was produced in each of these plasmid constructs without ion Fig. . When vills Fig. , lane 5.lls Fig. , lane 6.One possibility for the observed resistance of APOBEC3G-UBA2 to Vif could be explained by the reduced binding of APOBEC3G-UBA2 to Vif. To test this possibility, Myc-tagged Vif was pull-down by immunoprecipitation in the APOBEC3G-producing HEK293 cells. Western blot analyses were carried out to measure the bindings of different APOBEC3G constructs to Vif. As shown in Fig. via Cullin5-EloB/C E3 ligase to induce polyubiquitination of APOBEC3G . Cellular lysates were prepared and the protein concentration was determined using the Pierce protein assay kit. For immunoblotting, an aliquot of total lysate (50 \u03bcg of proteins) in 2 \u00d7 SDS-PAGE sample buffer (1:1 v/v) was electrophoresed and transferred to a nitrocellulose filter. Filters were incubated with appropriate primary antibody in Tris-buffered saline  and 5% skim milk or 5% BSA overnight. The primary antibodies include anti-APOBEC3G antibody at a 1:500 dilution (NIH reagents program), anti-Vif antibody at a 1:200 dilution (NIH reagents program), anti-HA antibody at a 1:1000 dilution, and anti-\u03b2-actin (Sigma) antibody at a 1:3,000 dilution. After washing, the filter was further incubated with secondary antibody in TBS-Tween-20 (TBS-T) buffer for 1 h. Protein bands were visualized by an ECL detection system. Goat anti-mouse or anti-mouse IgG-HRP conjugate  were used as secondary antibodies according to the corresponding primary antibodies.p.t., HIV-1 viral particles were harvested from the supernatants of HEK293 cells by centrifugation at 1,000 rpm/min for 5 min. The isolated viral particles were split into 2 ml aliquots and stored in -80\u00b0C. To ensure equal levels of viral infection, the viral stocks were normalized by determining levels of p24 antigens in each viral stock. The level of p24 antigen was determined by using a commercial p24 antigen kit from ZeptoMetrix Co.  following the manufacture's instructions.Plasmid DNA was transfected into HEK293 or CEM-SS cells by using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. To create stable APOBEC3G-expressing cell lines, the plasmid DNA of pcDNA3.1(-)-Apo-E/Hygromycin, pcDNA3.1(-)-Apo-U/Hygromycin, pcDNA3.1(-)-Apo-M/Hygromycin was transfected into HEK293 cells. HEK293 cells that stably produce a high level of APOBEC3G, APOBEC3G-UBA2 or APOBEC3G-UBA2* were first established by selection of hygromycin resistant cells (300 \u03bcg/ml) for 2 weeks and verified by the Western blot analyses Fig. . To gene6 CEM-SS cells were either mock infected or infected with 3000 TCID50 of HIV-1NL4-3 and HIV-1NL4-3\u0394Vif. The viral replication was measured by p24 antigenemia.To evaluate the suppressive effect of APOBEC3G, APOBEC3G-UBA2 and APOBEC3G-UBA2* on viral replication in proliferating CD4+ T-lymphocytes, CEM-SS cells (APOBEC3G negative CD4+ T-lymphocytes) that stably express a plasmid control, APOBEC3G, APOBEC3G-UBA2 and APOBEC3G-UBA2* were established. 1 \u00d7 102 incubator. After 2 hours incubation, 1.5 ml complete DMEM medium was added to each well. The cells were further incubated under the same condition for 48 hrs after incubation. The media were removed and 2 ml fixing solution  was added to each well. Cells were washed twice with PBS and then fixed for 5 min. with 600 \u03bcl of staining solution . Cells were then incubated for 2 hrs at 37\u00b0C in a non-CO2 incubator. Staining was stopped by removing the staining solution and washed twice with PBS. Blue dots were counted as infected cells as described previously [t-test was used to determine potential significant difference among different treatment groups.MAGI assay was used to determine the viral infectivity as previously described . Brieflyeviously  under thp.i.-post-infection; p.t.-post-transfection.HIV-1-human immunodeficiency virus type 1; Vif-viral infectivity factor; APOBEC3G- apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G; UBA2-ubiquitin-associated domain 2; UBL-ubiquitin-like domain; The authors declare that they have no competing interests.LL and DL carried out all of the experiments. JYL provided supervision of the study and co-mentored LL's Ph.D. dissertation. RYZ supervised and directed the designed studies and co-mentored LL's Ph.D. dissertation. All authors read and approved the final manuscript."}
+{"text": "Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells.Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid \u201ceat-me\u201d signal, phosphatidylserine (PS), makes them recognizable  Professional phagocytes, particularly macrophages, are very attractive targets for selective drug delivery because these cells: i) host a variety of pathogens with significant public health impact, ii) play a critical role as orchestrators of inflammation as they regulate the production and release of pro- and anti-inflammatory mediators, reactive oxygen (ROS) and nitrogen species (RNS), particularly after exposure to particles Macrophage recognition and uptake of apoptotic cells  is an important type of cell/cell communications regulating inflammation sn-Glycero-3-Phosphocholine (DOPC), 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine]  (DOPS), 16\u22360-6\u22360 NBD PS, 1-Palmitoyl-2-[6-[amino]dodecanoyl]-sn-Glycero-3-Phospho-L-Serine  and 16\u22360-6\u22360 NBD PC, 1-Palmitoyl-2-[6-[amino]dodecanoyl]-sn-Glycero-3-Phospho-choline  were from Avanti Polar Lipids Inc. . HEPES, MgCl2, KCl, NaCl, phenylmethylsulfonyl fluoride, glutaraldehyde, osmium tetroxide, potassium ferricyanide, diethylenetriaminepentaacetic acid (DTPA), zymosan and Hoechst 33342 were from Sigma-Aldrich . RPMI, DMEM medium, Ca2++Mg2+-free PBS were purchased from Invitrogen Corporation .1,2-Dioleoyl-5 as the iron-containing catalyst precursor, and purified by acid treatment to remove metal contaminates 2/g. Surface area was determined by Brunauer, Emmett, and Teller (BET) analysis, and diameter and length was measured by TEM.SWCNT  produced by the high pressure CO disproportionation process (HiPco) technique 2SO4 and 35% aqueous H2O2 and sonicated in ultrasonic bath  for 24 hrs at 0\u00b0C. The dispersion was then heated to 70\u00b0C for 10 min for \u201cpolishing\u201d the nanotubes. This solution was then diluted 10-fold by deionized water and filtered through PTFE membrane (100 \u00b5m pore size). The collected sample was thoroughly washed with deionized water and vacuum dried at 110\u00b0C for 30 min. Thus obtained short SWCNT were dispersed in 25 mM HEPES buffer  by sonication to final concentration 0.5 mg SWCNT/ml.The chemical cutting of SWCNT was performed as reported previously Transmission electron microscopy was conducted in a FEI-Morgani TEM operated at 80 KV equipped with a soft imaging system charge-coupled device (CCD) camera. TEM samples were prepared by drop casting the solution on copper grid and the excess drawn off with filter paper. The grid was negatively stained with 2% uranyl acetate solution for a minute.Zeta potential and particle size were determined on the Malvern Zetasizer Nano . The analysis was conducted according to standard operating procedure  for this instrumentation type.2, clean quartz filter punch and placed in Petri dish. The samples were allowed to dry overnight in a dessicator located in the balance room. The quartz punch was then analyzed with the Sunset thermal-optical carbon analyzer .Elemental carbon was analyzed using modified method NIOSH 5040 (NMAM 5040) After sonication, 20 \u00b5L of SWCNT in 25 mM HEPES buffer (pH 7.4) was placed on a freshly cleaved mica which was subsequently rinsed with DI water while being spun at 950 rpm to remove the excess of the buffer solution. The samples were air-dried prior to imaging. AFM images were collected in a tapping mode with a Multimode Nanoscope IIIa microscope  in air.2SO4 plus H2O2 have been utilized. The morphology of thus obtained SWCNT preparations was assessed by TEM, SEM as well as AFM. Following the chemical cutting of SWCNT, the nanotubes were used for coating with cargoes . Therefore, neither phospholipids nor cyt c were exposed to aggressive environments employed for the SWCNT cutting protocol. SWCNT were sonicated with either 2.5 mM DOPC or 5 mM DOPC: DOPS at the ratio of 1\u22361 (3 cycles 30 s), then washed 4 times with 25 mM HEPES, pH 7.4. After each washing, samples were centrifuged at 50,000 g for 30 min at 4\u00b0C. To prepare fluorescently labeled nanotubes, SWCNT were sonicated with either PC or PC/PS liposomes containing NBD-PC or NBD-PS , respectively. For the PS-coated Annexin V treated SWCNT, PS-coated SWCNT were incubated with Annexin V (25 mg/mg SWCNT) in Annexin V binding buffer for 5 min at room temperature and then washed twice with 25 mM HEPES, pH 7.4 to remove non-bound Annexin V. After each washing, PS-coated/Annexin V treated SWCNT were centrifuged at 50,000 g for 15 min. To prepare PS/cyt c/SWCNT, nanoparticles (0.3 mg/ml) were incubated in 25 mM HEPES buffer, pH 7.4 with 50 \u00b5M cyt c for 30 min at room temperature. To remove non-bound cyt c, SWCNT were washed twice with 25 mM HEPES buffer pH 7.4 and centrifuged at 50,000 g for 30 min at 4\u00b0C. After that, cyt c/SWCNT were sonicated in the presence of 3 mM PC\u2236PS liposomes (at the ratio of 1\u22361) 3 cycles for 30 s and then washed 4 times with 25 mM HEPES. After each washing, samples were centrifuged at 50,000 g for 30 min at 4\u00b0C. Coated SWCNT were finally suspended in 25 mM HEPES pH 7.4  to prevent oxidative damage to lipids and protein) using the same volume as the original suspension. Endotoxin content in SWCNT suspensions and its amounts present in the medium during incubations were approximately 300\u2013500 times lower than those causing stimulation of macrophages.In all presented experiments, the SWCNT subjected to chemical cutting by HPhospholipids from coated SWCNT were extracted using Folch procedure Specific-pathogen-free adult female C57BL/6 mice (7\u20138 wk) were supplied by Jackson Lab  and weighed 20.3\u00b10.2 g at time of use. Animals were individually housed in AAALAC-approved NIOSH animal facilities in microisolator cages for one week prior to use. Autoclaved Beta Chip bedding  was changed weekly. Animals were supplied with water and Harlan Teklad, 7913, NIH-31 Modified Mouse/Rat Diet, Irradiated  and housed under controlled light, temperature and humidity conditions. Experiments were conducted under a protocol approved by the Animal Care and Use Committee of the NIOSH. Mice were randomized into three experimental groups treated either with non-coated SWCNT, PC-coated SWCNT or PS-coated SWCNT on day 0. Animals were sacrificed on day 1 following exposures.Pharyngeal aspiration was used for particulate administration to C57BL/6 mice. Briefly, after anesthization with ketamine and xylazine anesthesia , the mouse was placed on a board in a near vertical position. The animal's tongue was extended with lined forceps and a suspension of particulates  was placed in the posterior of pharynx. The tongue was held until the suspension was aspirated into the lungs. All mice in particle and PBS groups survived this exposure procedure. This technique provides good distribution of particles widely disseminated in a peri-bronchiolar pattern within the alveolar region 2++Mg 2+-free PBS at a volume of 0.9 ml for first lavage (kept separate) and 1.0 ml for subsequent lavages. Approximately 5 ml of BAL fluid per mouse was collected and pooled in sterile centrifuge tubes. Pooled BAL cells were washed in Ca+2+Mg+2-free PBS by alternate centrifugation (800\u00d7g for 10 min at 4\u00b0C) and resuspension.Mice were weighed and euthanized with intraperitoneal injection of sodium pentobarbital  (>100 mg/kg). The trachea was cannulated with a blunted 22 gauge needle, and BAL was performed using cold sterile CaPrimary microglia was isolated from brains of postnatal day 5 rats as described 6 cells/ml in RPMI-1640 medium. Monocytes were separated by adhesion to tissue culture plastic for 1 h at 37\u00b0C with a 5% CO2 atmosphere and non-adherent cells were removed by several washes with PBS. Human monocyte-derived macrophages (HMDMs) were cultured for 3\u20134 days in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin and 100 \u00b5g/ml streptomycin , and 50 ng/ml human recombinant M-CSF (50 ng/ml\u200a=\u200a7500 IU/ml) . For isolation of dendritic cells, peripheral blood mononuclear cells were harvested as described above, and washed three times with PBS, followed by resuspension in MACS-buffer (80 \u00b5l/107 cells) containing 0.5% BSA, 2 mM EDTA in PBS. Anti-CD14 microbeads  were added according to the manufacturer's instructions. After 30 min at 4\u00b0C, the CD14-positive cells were separated from the solution by autoMACS (Miltenyi Biotec), and analyzed by flow cytometry  to check for CD14+ cell purity. Monocyte-derived dendritic cells (MDDC) were generated essentially as described before 5 cells/ml, at 37\u00b0C in a 5% CO2 atmosphere. Both cytokines were from Biosource International . After 6\u20137 days, the cell surface molecules CD1a, CD11c, CD14 and CD83 were analyzed by flow cytometry as described below, to confirm an immature, phagocytosis-competent phenotype with low CD83 expression.Mononuclear cells were prepared from buffy coats  obtained from healthy adult blood donors by density gradient centrifugation using Lymphoprep  or Ficoll Paque , as described previously HeLa cells and RAW264.7 macrophages  were grown in DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 units/ml penicillin and 100 \u00b5g/ml streptomycin.SH-SY5Y neuroblastoma cells (ATCC) were kept in DMEM\u2236F12 (1\u22361) supplemented with 2 mM glutamine, 1% non essential amino acids and 15% fetal bovine serum (FBS).6/ml for RAW 264.7 macrophages and HeLa cells, 0.5\u00d7106/ml for HMDM and MDDC, 3\u00d7105/ml for microglia and SH-SY5Y neuroblastoma cells) were exposed to non-coated SWCNT or PC-coated SWCNT, PS-coated SWCNT  for 2 h (and 24 h for some experiments using MDDC) in serum-free medium, except for primary human phagocytes which were maintained in cell culture medium supplemented with 10% heat-inactivated serum so that cell viability was not compromised. For the purpose of uniformity we present the condition of exposure to SWCNT using the ratios of SWCNT (\u00b5g)/106 cells. The ratios SWCNT to cells were 125\u2013150 \u00b5g/106 cells. In the experiments with cyt c/PS-coated SWCNT chloroquine (100 \u00b5M) was applied as endosome disruptor and cells were incubated for 15 min at 37\u00b0C. Cells were washed once with DMEM medium and then incubated in DMEM medium containing 10% FBS for additional 2 h. At the end pointed incubation, cells were washed with PBS, collected and used for assessment of caspase 3/7 activity and externalized of PS. Cytotoxicity was confirmed using Trypan blue exclusion as well as LDH release  kit).Cells  in normal culture medium for different time periods. At the end of incubation, the medium was collected and subjected to 50,000 g centrifugation at 4\u00b0C for 30 min to remove nanoparticles from the solution. The supernatant was then used to measure cytokines. R&D Quantikine \u00ae immunoassay kit  was used for measurements according to the manufacturer's manual.RAW macrophages were seeded at 2.5\u00d710Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assay kit .PS exposure was determined by fluorescently microscopic detection of annexin V as outlined in the annexin V-FITC apoptosis detection kit . Cells were analyzed under a Nikon ECLIPSE TE 200 fluorescence microscope  equipped with a digital Hamamatsu CCD camera (C4742-95-12NBR) using the MetaImaging Series\u2122 software version 4.6 . A minimum of 300 cells were analyzed per experimental condition.Fluorescence intensity of HMDM or MDDC incubated in the presence or absence of NBD-labeled PC/PS-coated SWCNT was measured with a FACScan flow cytometer  equipped with a 488 nm argon laser. Ten thousand events were gated for live cells based on forward and side scatter characteristics were collected for each sample and data were analyzed using CellQuestPro software (Becton Dickinson). For monitoring of DC phenotype, cells were labeled with fluorescent phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) specific for CD1a  and CD11c (Becton Dickinson); and with fluorescein isothiocyanate (FITC)-conjugated mouse mAbs specific for CD83 and CD14 (Becton Dickinson) according to the manufacturer's instructions. Control samples were labeled with isotype-matched antibodies conjugated with the same fluorochrome. Fluorescence was measured with a FACSCalibur flow cytometer (Becton Dickinson) and data were analyzed using CellQuestPro.Cells were seeded on cover slides 18 h before treatment, 20 \u00b5g/ml of various functionalized SWCNT were added and incubated in DMEM with no phenol red at 37\u00b0C for 2 h. At the end of incubation cells were gently washed 3 times with PBS, fixed by 2.5% paraformaldehyde at RT for 5 min and examined under a Nikon ECLIPSE TE 200 fluorescence microscope  equipped with a digital Hamamatsu charge-coupled device camera (C4742\u201395-12NBR) and analyzed using the MetaImaging Series\u2122 software version 4.6 .5 cells per well the previous day. The normal DMEM medium was replaced with phenol red free RPMI 1640 medium the next day and incubated with LysoTracker Red- DND99 (50 nM) for 1 h to label lysosomes. Prior to incubation with LysoTracker Red, RAW 264.7 macrophages (5\u00d7105 cells per well) were incubated with cocktail of endocytosis inhibitors containing a mixture of nystatin (25 \u00b5g/ml), genistein (200 \u00b5M), chlorpromazine (6 \u00b5g/ml) and brefeldin A (10 \u00b5g/ml) for 30 min as described 6 cells). The cells were incubated with particles for 5 minutes for the purpose of confocal microscopy. Cells were then washed three times with PBS and fixed using 2% paraformaldehyde. Nuclei were stained with Hoechst 33342. Cells were imaged using Olympus Fluoview 1000 confocal microscope.RAW 264.7 macrophages were seeded on LabTek chamber slides at a density of 5\u00d710Following incubation for 2 or 24 h with NBD-labeled SWCNT, MDDCs were fixed in 4% formaldehyde for 15 min. Staining of cell membranes was carried out using anti-HLA-DR mAb (Becton Dickinson). A secondary goat anti-mouse mAb labeled with Alexa Fluor 546  was used for detection. Slides were mounted with anti-fading Vectashield mounting medium . Fluorescent images were acquired on a confocal laser-scanning microscope  equipped with one argon and two HeNe lasers. NBD was excited with a 488-nm laser line detecting light in the wavelength region of 560\u2013700 nm. Alexa 546 was excited by a 543-nm laser line with detection of light in the region of 560\u2013700 nm.Macrophages fixed in cold 2.5% glutaraldehyde were rinsed in PBS, post-fixed in 1% Osmium Tetroxide  with 0.1% potassium ferricyanide  dehydrated through a graded series of ethanol, from 30 to 100%, and then critical point dried in a critical point dryer, Emscope CPD, . Following critical point drying, the samples were attached to aluminum SEM specimen mounting stubs (Electron Microscopy Sciences) and then sputter coated with a gold palladium alloy . Following processing, samples were analyzed using a JEM 6330F microscope .4 and 1% K3Fe(CN)6 for 1 hour. After 3 PBS washes, the pellet was dehydrated through a graded series of 30% to 100% ethanol then incubated in Polybed 812 epoxy resin  for 1 h. After several changes of 100% resin over 24 h, pellet was cured at 37\u00b0C overnight with additional hardening at 65\u00b0C for 48 h. Ultrathin (60 nm) sections were collected on 200 mesh grids and stained with 2% uranyl acetate in 50% methanol for 10 min followed by 1% lead citrate for 7 min. Sections were observed on a JEM 1210 electron microscope  at 80 kV.Macrophages were fixed in 2.5% glutaraldehyde for 1 h, pelleted, and re-suspended in 3% gelatin in PBS, olidified at 4\u00b0C, and then re-fixed for 15 min. Pellets were washed 3 times in PBS and then postfixed in 1% OsOp< 0.05.The results are presented as mean\u00b1s.d. values from at least three experiments, and statistical analyses were performed by either paired/unpaired Student's t-test or one-way ANOVA. The statistical significance of differences was set at We prepared SWCNT coated with DOPC, as a control (PC-coated SWCNT), or a mixture of DOPS, plus PC (PS-coated SWCNT) by incubating nanotubes in the presence of liposomes containing these lipids (PC or a mixture of PS plus PC at a molar ratio of 1\u22361). Successful integration and the content of PS and PC associated with SWCNT were confirmed by direct assessment of the phosphorus content after HPTLC separation of phospholipids . We founAnalysis performed by NMAN 5040 revealed that purified SWCNT were comprised of 99.7 wt% elemental carbon. Determinations of carbon content for non-coated, PS-coated, PC-coated and cyt c/PS-coated SWCNT showed that non-covalent functionalization of SWCNT with phospholipids and protein (cyt c) expectedly resulted in increased carbon content in SWCNT suspensions. Based on our HPTLC-based determinations of phospholipid content of PC- and PS-coated SWCNT, the expected content of elemental carbon in the suspensions of these coated SWCNT should be 81 and 61 ng/\u00b5l, respectively. Direct estimates of elemental carbon content were 75 and 59 ng/\u00b5l for SWCNT functionalized with PS and PC . These nThere was essentially no difference in the organization and structure of SWCNT between non-coated samples and PS- or PC-coated samples. Assessments of size distribution by dynamic light scattering (DLS) showed that SWCNT , PS-coatEvaluations of zeta potentials for SWCNT, PC-coated SWCNT, PS-coated SWCNT, PS-NBD-coated SWCNT, PC-NBD-coated SWCNT, and SWCNT containing cyt c showed that they ranged from \u221240 to \u221250 mV . CoatingTransmission electron microscopy (TEM) of negatively stained non-coated, PS-coated, and PC-coated SWCNT showed that they had typical fibrous morphology and were represented mostly by single nanotubes, as well as by ropes of nanotubes  and entaBoth Trypan Blue exclusion test and LDH release assay demonstrated that neither non-coated SWCNT nor phospholipids-coated SWCNT induced any cytotoxic effects in RAW 264.7 macrophages after 4 hrs of co-incubation . MoreoveTo investigate interactions of SWCNT with the surface of macrophages, we performed SEM imaging . We founTEM evaluations of the uptake of SWCNT showed that RAW264.7 macrophages readily phagocytozed PS-coated SWCNT  while a To more quantitatively characterize uptake of phospholipid-coated SWCNT by macrophages we utilized fluorescently labeled PS, NBD-PS, for coating SWCNT; NBD-PC was utilized in control experiments. After extensive washings, SWCNT incubated with NBD-PS or NBD-PC displayed characteristic fluorescence spectra confirming that the coating with phospholipids was successful . We studVisually, we observed accumulation of \u201cblack\u201d (SWCNT-containing) pellets after sedimentation of cells. To quantitatively characterize this effect, we performed measurements of optical density of cell suspensions incubated with PS-coated SWCNT and PC-coated SWCNT. A broad absorbance of SWCNT with characteristic maxima at 1050\u20131060 and 1260\u20131270 nm was detected. The intensity of absorbance at 1050\u20131060 nm was 1.3-fold higher in PS-coated SWCNT than in PC-coated SWCNT samples. We further used fluorescently-labeled phospholipids to characterize the presence of phospholipid-coated SWCNT in cells. We found that NBD-PS coated SWCNT co-incubated with RAW 264.7 macrophages yielded a higher fluorescence response from cell suspensions than NBD-PC coated SWCNT . Further5 cells/well) with either NBD-PS-coated SWCNT or NBD-PC-coated SWCNT (150 \u00b5g/106 cells). We found that NBD-PS-coated SWCNT were readily taken up by macrophages. As shown in Additionally, to prove co-localization of SWCNT with phagolysosomes we used confocal microscopy. To this end, we treated RAW 264.7 macrophages (5\u00d710## P<0.05) in the control. Notably, in PS-coated SWCNT, TNF-\u03b1 levels dropped to 368\u00b133 pg/ml. No significant changes in the content of TNF-\u03b1 was found after SWCNT coating with PC (445\u00b121 pg/ml). In contrast, the production of anti-inflammatory cytokines is known to be stimulated by PS-dependent pathways Apoptotic cells with externalized PS quench the production and release of pro-inflammatory cytokines by macrophages We further determined whether PS-coated SWCNT could be employed for the delivery of physiologically active agents to macrophages. Because cyt c released from mitochondria into the cytosol acts as an effective activator of caspases and a death-signal Importantly, cyt c-loaded PS-SWCNT caused a marked increase of caspase activity  and PS eTo determine whether PS-coated SWCNT can be recognized and taken up by other types of professional phagocytes, we performed experiments using microglia from rat brain. The experiments using SEM  and TEM in vitro uptake of NBD-PC- versus NBD-PS-coated nanotubes  and human monocyte-derived dendritic cells (MDDC) also preferentially recognized and engulfed PS-coated SWCNT compared to PC-coated SWCNT . The latanotubes , and theanotubes  and TEM While ingestion of apoptotic cells is not absolutely specific to phagocytes, the rates of uptake by professional phagocytes - macrophages and microglial cells \u2013 are significantly higher compared to non-professional phagocytes  in vivo. To this end, we used an established mouse model of SWCNT pulmonary exposure through pharyngeal aspiration Because PS is an important recognition signal for alveolar macrophages in the lung Specific interfacing of SWCNT with phagocytic cells of the immune system \u2013 macrophages, microglia, and dendritic cells - is important for several reasons. The first one is that SWCNT can be used for simultaneous targeted delivery of several different regulators/inhibitors with a potential to release them in temporally and spatially predetermined ways to control the bioactivity of a specific cell population during physiologically critical events. As macrophages can host a number of pathogens in vivo whereby alveolar macrophages display enhanced uptake of PS-coated SWCNT during an inflammatory pulmonary response induced by aspiration exposure. Together, our in vitro and in vivo studies demonstrate that PS-functionalization renders nanotubes appetizing to phagocytes. We believe that analysis of SWCNT uptake by phagocytes based on the employment of fluorescently-labeled phospholipids (NBD-PS and NBD-PC) provided more quantitatively reliable data. In this case, however, the limitations of quantitative assessments might be due to a possibility that fluorescently-labeled phospholipids could also modify (promote or decrease) the uptake of these fluorescent phospholipid coated SWCNT by macrophage. To minimize this interference, mole fraction of fluorescently-labeled phospholipids in the mixture with non-labeled phospholipids of the same type in all experiments did not exceed 10 mol%. Notably, the TEM-based evaluations of phagocytotic activity towards SWCNT were in good agreement with independent assessments performed by confocal imaging of NBD-labeled phospholipids-coated SWCNT.Understanding of major principles of particle recognition by macrophages has long been a controversial issue. Because non-functionalized nanoparticles are prone to aggregation, sonication of non-coated SWCNT was performed before adding them to cells. Under these conditions, no significant uptake of non-coated SWCNT by RAW 264.7 macrophages occurred during the 2 h incubation period. In contrast, Dumortier et al. in vitro and in vivoPS-induced responses initiate signaling cascades in macrophages, switching off their pro-inflammatory activation pattern, and turning on the production of anti-inflammatory cytokines and chemokines Mycobacterium tuberculosis and Listeria monocytogenes. Molloy et al. L. monocytogenes was reported to induce apoptosis in vitro and in vivo in a variety of cell types with the exception of macrophages which represent the predominant compartment of bacterial multiplication and die as a result of necrosis. Shifting the equilibrium from necrosis to apoptosis in L. monocytogenes infected macrophages is believed to constitute a promising therapeutic strategy Importantly, manipulating macrophage apoptosis can be a valuable therapeutic strategy in several diseases associated with the presence of intracellular pathogens in macrophages such as synthetic single-chain lipids around SWCNT to form supramolecular structures designed for the immobilization of histidine-tagged proteins; they did not investigate whether such nanotubes were ingested by cells. In addition, in a recent and elegant study, Liu et al. Dai et al Taken together, our findings highlight a novel strategy for the controlled delivery of relevant cargoes into specific cell populations. Further developments of the novel principle established in the current study, i.e. PS-coating of SWCNT for recognition and ingestion, may exploit cargoes covalently-conjugated with specific linkers that are hydrolysable extracellularly by activated macrophages (eg. superoxide-sensitive linkers) or intracellularly (eg. esterase-sensitive conjugates) for targeted delivery and release to regulate life-span and activity of phagocytes.Figure S1TEM images of bare SWCNT (A) and PS-coated SWCNT (B) deposited on mica substrates.(5.93 MB TIF)Click here for additional data file.Figure S2Viability of RAW264.7 macrophages exposed to SWCNT. RAW 264.7 macrophages were seeded at 1\u00d7104 cells/ well on 96 well-plate 12 hrs before treatment. Cells were incubated with non-coated SWCNT or SWCNT coated with either PS or PC in serum and phenol red free DMEM containing 100 \u00b5M DTPA for up to 4 hrs. At the end of incubation, the culture medium was collected, centrifuged to remove SWCNT and used for LDH assay with CytotoxOne kit. Data are mean\u00b1s.d., n\u200a=\u200a3.(0.50 MB TIF)Click here for additional data file.Figure S3PS-coated SWCNT effectively deliver cyt c into RAW264.7 macrophages, and activate apoptotic pathways (caspase 3/7). RAW264.7 macrophages were seeded at 0.5\u00d7105 cells/well 12 hrs prior to treatment. Cells were incubated with differently functionalized SWCNT in serum and phenol red free DMEM containing 100 \u00b5M DTPA for 1 hr at 37\u00b0C. At the end of incubation, cell were washed to remove SWCNT and treated with chloroquine (100 \u00b5M) in complete culture medium at 37 \u00b0C for 15 min. After that, cells were washed with complete DMEM and additionally incubated for 8 hrs. Cells were stained in binding buffer containing annexin V (0.5 \u00b5g/ml) for 5 min and counter-stained with Hoechst 33342 for nuclei at room temperature. The cells were examined under a Nikon ECLIPSE TE 200 fluorescence microscope  equipped with a digital Hamamatsu CCD camera (C4742-95-12NBR) and analyzed using the MetaImaging Series\u2122 software version 4.6 . A minimum of 300 macrophages were counted per experimental condition. Data are mean\u00b1s.d. (* p<0.01 vs untreated). Insert: Typical fluorescence image of RAW264.7 macrophages treated with cyt c/PS-coated SWCNT (staining with annexin V).(1.39 MB TIF)Click here for additional data file.Table S1(0.03 MB DOC)Click here for additional data file."}
+{"text": "The aims of this study were to develop a simple and rapid method of nanoparticle dispersion using a natural lung surfactant and to evaluate the effect of dispersion status of SWCNT on cytotoxicity and fibrogenicity \u00ae was used to disperse single-walled carbon nanotubes (SWCNT) in a biological medium. At physiologically relevant concentrations, Survanta\u00ae produced well dispersed SWCNT without causing a cytotoxic or fibrogenic effect. In vitro studies show that Survanta\u00ae-dispersed SWCNT (SD-SWCNT) stimulated proliferation of lung epithelial cells at low doses (0.04-0.12 \u03bcg/ml or 0.02-0.06 \u03bcg/cm2 exposed surface area) but had a suppressive effect at high doses. Non-dispersed SWCNT (ND-SWCNT) did not exhibit these effects, suggesting the importance of dispersion status of SWCNT on bioactivities. Studies using cultured human lung fibroblasts show that SD-SWCNT stimulated collagen production of the cells. This result is supported by a similar observation using Acetone/sonication dispersed SWCNT (AD-SWCNT), suggesting that Survanta\u00ae did not mask the bioactivity of SWCNT. Likewise, in vivo studies show that both SD-SWCNT and AD-SWCNT induced lung fibrosis in mice, whereas the dispersing agent Survanta\u00ae alone or Survanta\u00ae-dispersed control ultrafine carbon black had no effect.The natural lung surfactant Survanta\u00ae was effective in dispersing SWCNT in biological media without causing cytotoxic effects at the test concentrations used in this study. SD-SWCNT stimulated collagen production of lung fibroblasts in vitro and induced lung fibrosis in vivo. Similar results were observed with AD-SWCNT, supporting the conclusion that Survanta\u00ae did not mask the bioactivities of SWCNT and thus can be used as an effective dispersing agent. Since excessive collagen production is a hallmark of lung fibrosis, the results of this study suggest that the in vitro model using lung fibroblasts may be an effective and rapid screening tool for prediction of the fibrogenic potential of SWCNT in vivo.The results indicate that Survanta Advances in nanotechnology have made possible the fabrication of materials at the nanoscale level. Carbon nanotubes (CNT) are a major class of nanomaterials possessing unique mechanical, electrical, and thermal properties. As the use of CNT has become more widespread, there has been a great concern about their potential adverse effects on human health and the environment. Nanoparticles can come in contact with the human body through inhalation as well as ingestion and dermal deposition. Pulmonary exposure could occur due to aerosolization of nanomaterials including agglomerates of different size and shape. Individual CNT have a very high aspect ratio and can agglomerate into structures which are micrometers in diameter in the dry state or upon suspension in polar and non-polar solvents -4. Animain vitro and in vivo models have been developed. These studies often rely on the use of nanoparticle preparations suspended in physiological solutions. Since nanoparticles in solution tend to form coarse agglomerates in physiological media, development of methods to disperse nanoparticles is important in assessing their biological activities. Over the years, a variety of methods have been described to disperse nanoparticles, including the use of cell culture reagents [\u00ae for in vitro and in vivo studies. Survanta\u00ae is a surfactant replacement used by health care professionals for prevention and treatment of respiratory distress syndrome in premature infants. It is a sterile product consisting of phospholipids and surfactant-associated proteins SP-B and SP-C. Its commercial availability, biocompatibility and safety make this preparation an attractive dispersing agent for biological studies of nanoparticles. In the present study, we evaluated the ability of Survanta\u00ae to disperse SWCNT and investigated the bioactivity of dispersed SWCNT in vitro and in vivo. We also compared the effect of Survanta\u00ae-dispersed SWCNT to SWCNT dispersions prepared by previously published acetone/sonication and aerosolization methods [To aid the investigations of pulmonary responses to nanoparticle exposure, several reagents ,9, dimetreagents , acetonereagents , pluronireagents , Tween 8reagents , and pulreagents . Some of methods ,7,14.\u00ae-dispersed SWCNT (SD-SWCNT) are shown in Figure \u00ae used in this preparation is based on the content of lung surfactants found in rodent lung lavage fluids [\u00ae dispersed SWCNT into smaller size structures as shown by visual inspection  and Survantae fluids . Micromen Figure  and Tabln Figure . These rry SWCNT .vs. ND-SWCNT. The mean width of SD-SWCNT was 380 nm. About two-thirds of the SD-SWCNT were dispersed into structures with diameters less than the average diameter of 380 nm, and more than 8% of the dispersed particles were less than 100 nm in diameter  assay and by direct cell count. The results show that at the concentrations tested (0.036-36 \u03bcg/ml), Survanta\u00ae had no significant effects on the LDH release and cell number as compared to non-treated control  and suppressing effect at the highest dose tested (0.6 \u03bcg/cm2 or 1.2 \u03bcg/ml), whereas non-dispersed SWCNT had no effect on cell proliferation as compared to non-treated or Survanta\u00ae only treated control. These results suggest the importance of dispersion status of SWCNT on their bioactivity, which is supported by the observation that SWCNT dispersed by acetone/sonication (AD-SWCNT) also induced cell proliferation at the lowest dose tested . Western blot analysis of collagen I, which is the most abundant collagen in the lung [\u00ae alone and SD-UFCB had a minimal effect  SD-SWCNT or control treatments, and analyzed for lung collagen content at two weeks post-treatment. Figure \u00ae assay, whereas SD-UFCB or Survanta\u00ae only treatment had no significant effect as compared to non-treated control (No Tx). Similar results were observed with collagen I content as determined by Western blot assay , biocompatibility , and commercial availability particularly as a sterile preparation which greatly facilitates in vivo and in vitro studies that require sterile conditions. In addition, one would argue that inhaled SWCNT would initially interact with alveolar lining fluid, which is modeled by Survanta\u00ae suspension. However, the effectiveness of this preparation in dispersing nanoparticles and its possible interfering effect on the bioactivities of SWCNT are not known, and, therefore, are investigated in the present study. The data presented demonstrate that Survanta\u00ae when used at the indicated concentrations is effective in dispersing SWCNT, yielding nanoparticles with dimensions similar to those observed after aerosolization of dry SWCNT or acetone/sonication dispersion of SWCNT [\u00ae-dispersed SWCNT form much smaller structures with an average width of 0.38 \u03bcm and an average length of 1.42 \u03bcm. The majority of the dispersed SWCNT is in the form of small bundles with no or minimum detectable individual nanotubes. The reported average diameters of aerosolized dry SWCNT and acetone/sonication-dispersed SWCNT are 0.24 \u03bcm and 0.6 \u03bcm, respectively [The major goal of this study was to evaluate the suitability of utilizing Survantaof SWCNT ,14. Non-ectively ,14. Thesectively , structuectively , and CNTectively . The datectively .in vivo pulmonary exposure studies should be performed using an inhalation method, since it best mimics the human exposure condition. However, the need for specialized facilities and equipment, trained personnel, and large quantities of nanoparticles has limited the use of this technology. Pulmonary aspiration represents an alternative method that has proven useful in many pulmonary toxicity studies. This method is simple, economical, uses small amounts of nanoparticles, and provides deep lung deposition as well as high correlation to the administered dose [Ideally, red dose . Recent red dose ,6,14. Th\u00ae for nanoparticle dispersion provides an additional advantage over other methods of dispersion for pulmonary studies as it better mimics the natural lung condition. This is particularly important for in vitro studies which normally lack lung surfactants that could have an effect on cell interaction and bioactivity of nanoparticles. Previous studies have shown that lung surfactants aid in the displacement of particles from air to the aqueous phase and towards the lung epithelium [The use of Survantaithelium . In addiithelium . These s\u00ae is its possible adverse effects on cells and tissues or masking effect on the exposed particles [\u00ae, when used at the indicated concentrations, had no significant cytotoxic effect on lung cells in vitro and did not induce collagen production or mask the fibrogenic effect of SWCNT either in vitro or in vivo. The results of this study also indicate that dispersion status of SWCNT is a key determinant of its biological activities to induce cell proliferation and enhance collagen production.A key concern about the use of Survantaarticles ,22. Our in vitro and in vivo fibrogenic responses to SWCNT and control particles under different dispersion conditions. This finding suggests the potential utility of in vitro lung fibroblasts as a predictive model for in vivo fibrogenicity testing of CNT and other nanomaterials. Fibrogenicity testing of nanomaterials is usually performed using animals. However, this method of testing is time-consuming, laborious, and costly. This combined with the rapid growth in nanotechnology, which produces an uncountable number and variety of nanomaterials, makes it impractical to test all of these materials using animals. The in vitro model described here represents an alternative method that could serve as a rapid screening tool for fibrogenicity testing of a large number of nanomaterials. This model can also be used to conduct detailed mechanistic studies of the fibrogenic effect of nanoparticles, which may not be achievable in vivo.Another key finding of this study is the correlation between \u00ae. Survanta\u00ae was shown to be effective in dispersing SWCNT and caused no cytotoxic or fibrogenic effect to the test lung cells under the experimental conditions. SD-SWCNT and AD-SWCNT similarly stimulated collagen production of lung fibroblasts in vitro and both induced lung fibrosis in vivo, indicating the fibrogenicity of SWCNT and non-masking effect of Survanta\u00ae. The reported in vitro cell model system could potentially be used to aid the fibrogenicity testing of CNT of various size and functionalization as well as mechanistic studies of other nanoparticles.The present study describes a novel method of CNT dispersion using the natural lung surfactant Survanta5 as the iron-containing catalyst precursor. These SWCNT were then purified by acid treatment to remove metal contaminates for use in this study. Elemental analysis of the supplied SWCNT by nitric acid dissolution and inductively coupled plasma-atomic emission spectrometry  showed that the SWCNT were 99% elemental carbon and 0.23% iron. The specific surface area was measured at -196\u00b0C by the nitrogen absorption-desorption technique  using a SA3100 Surface Area and Pore Size Analyzer . The diameter and length distribution of poorly and well-dispersed preparations of SWCNT (without or with Survanta\u00ae) were measured by field emission scanning electron microscopy. The surface area of dry SWCNT was 400-1,000 m2/g, and the length and width of individual (dry) SWCNT were 0.1-1 \u03bcm and 0.8-1.2 nm, respectively. Characterization studies were performed at NIOSH research facilities as previously described [SWCNT  were produced by the high pressure CO disproportionation (HiPco) technique, employing CO in a continuous-flow gas phase as the carbon feedstock and Fe(CO)\u00ae  or by the acetone-sonication method as described previously [\u00ae-dispersed SWCNT (SD-SWCNT) were prepared by dispersing SWCNT (0.1 mg/ml) in PBS containing Survanta\u00ae (150 \u03bcg/ml) with light sonication  at a power of 130 W, frequency of 20 kHz, and amplitude settings of 60% for 5-10 seconds. Non-dispersed SWCNT (ND-SWCNT) were prepared similarly but in the absence of Survanta\u00ae. Acetone/sonication dispersed SWCNT (AD-SWCNT) were prepared according to the method previously described [SWCNT were dispersed by using Survantaeviously ). Survanrvanta\u00ae 10 \u03bcg/ml wImages of SWCNT suspensions were obtained by field emission scanning electron microscopy and nanoscale hyperspectral microscopy. To assess the size distribution of SWCNT samples, a sample of each was taken and filtered through a polycarbonate filter  to collect the particles. After washing with water and drying, the filter was cut into equal sections, mounted onto aluminum stubs with double-stick carbon tape, and sputter coated with gold/palladium. The deposited particles were viewed under a field emission scanning electron microscope  at 400 and 30,000 magnifications. The average length and width of the particles in each sample were determined by analysis of a minimum of 300 particles. The size and distribution values were determined from triplicate experiments. Representative micrographs of the SWCNT samples were taken using conventional and hyperspectral microscopy. The latter system  is capable of identifying specific material at a sub 100-nanometer resolution based on the material's unique spectral signature. Hyperspectral images of SWCNT were captured with the CytoViva spectrophotometer and an integrated CCD camera mounted on an Olympus BX-51 microscope at 400x.4 cells/well in Dulbecco's modified eagle medium containing 5% fetal bovine serum. The cells were treated with various concentrations of Survanta\u00ae, ND-SWCNT, SD-SWCNT, or AD-SWCNT at 37\u00b0C. At the indicated times after the treatment, cell supernatants were collected and analyzed for lactate dehydrogenase (LDH) as an indicator of cell toxicity. LDH activity was determined by LDH-catalyzed oxidation of pyruvate coupled with the reduction of NAD at 340 nm using a commercial assay kit and a Cobas Mira Plus transfer analyzer . Cell growth was determined by direct cell counting of the control and treated cells. The cells were trypsinized, suspended in 100 \u03bcl culture medium, and 10 \u03bcl samples of the suspension were mixed with trypan blue for cell number counting and determination of cell viability using a hemocytometer.Human lung epithelial BEAS-2B cells  were incubated in a 24-well plate at the density of 2 \u00d7 10\u00ae assay . Human lung fibroblast CRL-1490 cells  or mice were treated with Survanta\u00ae, SWCNT, or control particles as described below. Treated cells or mouse lung tissues were lysed and cell/tissue lysates were analyzed for protein content using a bicinchoninic acid protein assay kit . For Western blot analysis, equal amounts of protein per sample (25 \u03bcg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked with T-PBS (0.3% Tween-20 in PBS) containing 3% dry milk and incubated with primary antibodies specific for collagen type I and \u03b2-actin  at 4\u00b0C overnight. After three washes with T-PBS, the membrane was incubated with peroxidase-conjugated secondary antibody for 1 h and then washed with 0.05% Tween-20 in PBS. The immune complexes were detected by chemiluminescence  and quantified using analyst/PC densitometry software .Collagen content was determined by Western blotting and the Sircol\u00ae assay, cell/tissue lysates (50 \u03bcl) were incubated with Sirius red reagent (50 \u03bcl) for 30 min, after which the collagen-dye complex was precipitated by centrifugation at 16,000 g for 5 min. The precipitates were washed with ethanol and dissolved in 0.5 M NaOH. The samples were then introduced into a microplate reader and absorbance determined at 540 nm.For analysis of collagen content by the Sircol\u00ae assays.Pathogen-free male C57BL/6J mice  weighing 25-30 grams were used. The animals were individually housed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility and allowed to acclimate at least 1 week prior to use. All experimental procedures were conducted in accordance with a protocol approved by the NIOSH Institutional Animal Care and Use Committee. The animals were treated with the test materials by pharyngeal aspiration as described previously . BrieflyP < 0.05. When significant F-values were obtained, individual means were compared with control using a two-sided Dunnett's test. P < 0.05 was considered to be significant. Data are given as means \u00b1 SD. The size distributions of SWCNT particles in different media were determined using the procedures described in Hinds [Data were analyzed by ANOVA (STATGRAF). Bartlett's test was used to test for homogeneity of variances between groups. Statistical differences were determined by one-way ANOVA, with significance set at in Hinds .The authors declare that they have no competing interests.in vitro and in vivo experiments, and drafted the manuscript. VC participated in the design of the study, evaluation of results, and helped to draft the manuscript. AM participated in in vitro studies and Western blot analysis. BC participated in particle measurements and data analysis. RM participated in animal exposure studies. DS-B carried out the electron microscopy. YR participated in study design, particle dispersion, collagen assays, and manuscript preparation. All authors read and approved the final manuscript.LW conceived the study, carried out"}
+{"text": "Organisms adapt to infectious agents by developing protective responses, and conversely, infectious agents develop adaptive countermeasures to these responses. Host defenses against infectious agents include adaptive and innate immune responses . Recently, additional host defense systems against viruses have been identified. These include the TRIM A3 (hA3) genes, the feline four genes A3 (mA3) gene A3 genes in general show a high degree of polymorphic variation, suggesting that they are under strong selective pressure A3 proteins belong to a family of genes that encode DNA- and RNA-editing enzymes and confer innate immunity to HIV-1 and perhaps other viruses, such as hepatitis B virus (HBV) and human papilloma virus (HPV) vif gene. In vif-deficient HIV-1 producer cells, both hA3G and hA3F are packaged into progeny virions via interaction with the nucleocapsid (NC) protein and viral RNA. Once packaged, hA3 proteins inhibit infection in target cells by deaminating deoxycytidine residues on the DNA minus strand following reverse transcription, inducing G to A hypermutation in newly synthesized HIV-1 DNA. A3 proteins also inhibit replication by cytidine deaminase (CDA)-independent mechanisms vif+ HIV-1, Vif binds hA3G and hA3F and targets these proteins for ubiquitinylation and degradation in the proteosome, thereby overcoming the anti-viral activity hA3G, the first identified family member, was discovered because of its interaction with the HIV-1 virion infectivity factor (Vif) Several studies have examined packaging of A3 proteins from different species into retroviruses endemic to the species using transfected tissue culture cells and have suggested that these viruses are resistant to the A3 proteins of their natural hosts. For example, it has been shown that human T cell leukemia virus I (HTLVI), Mason Pfizer monkey virus (MPMV), and MLV do not efficiently package human, monkey, or mouse A3 proteins, respectively, because of weak interactions between the NC proteins and the host A3 In contrast to studies demonstrating that endogenous A3 proteins do not restrict retroviruses that infect the same species, there are many examples of cross-species restriction in cultured cells. Human A3B and A3C restrict SIV A3 gene and clearly demonstrate that the host A3 protein plays a role in virus infection and, more importantly, in virus-mediated pathogenesis.However, the studies showing that A3 proteins do not restrict viruses that infect the same species need to be re-evaluated in light of several recent studies with two mouse viruses, MMTV and MLV. These in vivo studies examined the role of A3 in the resistance and susceptibility to infection in different inbred strains of mice and in mice with targeted deletion of the mIn the first of these studies, our lab tested whether knockout mice that lack a functional A3 gene were susceptible to infection with MMTV Rfv3) locus was subsequently found to affect the ability of resistant mice, such as C57BL/6, to recover from virus infection, at least in part through the production of a high-level antibody response Rfv3 to a 0.83 centimorgan region of chromosome 15, close to the APOBEC3 locus Rfv3 and mA3 were one and the same, and their data suggest that this may be the case Two groups more recently demonstrated that mA3 also restricts Friend MLV (F-MLV) infection and virus-induced erythroproliferation The two groups differed in their conclusions regarding the mechanism of A3-mediated resistance. There are a number of differences between the A3 alleles in F-MLV-resistant and \u2013susceptible mice, leading to differences in mRNA expression levels, alternative splicing of the mRNA, and amino acid polymorphisms Rfv3 is mA3, the fact that mA3\u2212/\u2212 mice are more susceptible to infection by at least two murine retroviruses, MMTV and MLV, and that the loss of this gene in vivo leads to increased pathogenesis by these viruses, provides strong support that this host-encoded restriction factor does function against a natural pathogen.Importantly, these studies demonstrate that the tissue culture experiments that examine the role of host restriction factors can underestimate the role that these restriction factors play in vivo. The numerous studies regarding mA3's effect on MLV infection in cultured cells are conflicting, providing evidence for and against a role in restriction A3 genes, particularly A3G and A3H, are highly polymorphic, suggesting positive selection by viruses or retroelements A3B locus that leads to a fused hA3A/3B gene What does this mean for HIV-1 and other human pathogens and the role that hA3 proteins play in restricting HIV-1 infection? There is increasing genetic evidence that A3 proteins protect against infection by HIV-1 and other viruses. Indeed, one of the alleles that has been linked to individuals who have received multiple exposures to HIV but remained sero-negative maps to chromosome 22q12-13, which contains the human A3 family member genes A3 alleles and resistance to infection to HIV-1, HTLVI, HBV, HPV, and other viruses whose infection may be affected by A3 proteins in vivo.The mouse studies underscore the importance of using in vivo models to understand host restriction factors and their importance in limiting viral pathogenesis. Unfortunately, the lack of a good animal model means we can at present only infer that A3G or other A3 molecules are retained in the genome as anti-HIV-1 (or other viruses) restriction factors in humans and other species. In the absence of being able to test species-specific A3 molecules in vivo against species-endemic viruses, it is critical to look for genetic associations between polymorphic"}
+{"text": "Stacks is a software system that uses short-read sequence data to identify and genotype loci in a set of individuals either de novo or by comparison to a reference genome. From reduced representation Illumina sequence data, such as RAD-tags, Stacks can recover thousands of single nucleotide polymorphism (SNP) markers useful for the genetic analysis of crosses or populations. Stacks can generate markers for ultra-dense genetic linkage maps, facilitate the examination of population phylogeography, and help in reference genome assembly. We report here the algorithms implemented in Stacks and demonstrate their efficacy by constructing loci from simulated RAD-tags taken from the stickleback reference genome and by recapitulating and improving a genetic map of the zebrafish, Danio rerio.Advances in sequencing technology provide special opportunities for genotyping individuals with speed and thrift, but the lack of software to automate the calling of tens of thousands of genotypes over hundreds of individuals has hindered progress.  Recent work genotyping 100 stickleback fish at 45,000 loci  and economically valuable species such as cow  by extracting 45,547 reads each 60 bp long in both directions at each SbfI restriction enzyme cut site (CCTGCAvGG) . We re-diplodized the genome in silico by creating alleles  into which we uniformly introduced single nucleotide polymorphisms (SNP) at a rate of 0.5%. We \u201csequenced\u201d each allele to a depth determined by a draw from a Poisson distribution at three different mean sequencing depths  . For each \u201csequenced\u201d read, we simulated sequencing errors at a rate that increased linearly along the sequence to mimic Illumina reads . We investigated three mean error rates  to cover normal to high error rates. Each simulation run involved 10 replicates. For each dataset, ustacks was executed setting the within-individual distance parameter to two nucleotides and the stack-depth parameter to three identical reads.The Stacks. We executed the Stacks pipeline with a stack-depth parameter of three and a within-individual distance parameter of two and constructed a linkage map using JoinMap  and required a unique best hit to the reference genome or a top hit with a raw BLAST score at least an order of magnitude greater than the second best hit with 70% of the query sequence aligned. Genotypes for markers present in at least 36 of the 42 HS map cross individuals were exported into JoinMap 4.0  by BLASTn. These searches used an e-value cutoff of 1 \u00d7 10nMap 4.0 . LinkageStacks as a modular pipeline to efficiently curate and assemble large numbers of short-read sequences from multiple samples. Stacks identifies loci in a set of individuals, either de novo or aligned to a reference genome, and then genotypes each locus. Stacks incorporates a maximum likelihood statistical model to identify sequence polymorphisms and distinguish them from sequencing errors. Stacks employs a Catalog to record all loci identified in a population and matches individuals to that Catalog to determine which haplotype alleles are present at every locus in each individual. Stacks stores results in a MySQL database and displays them through a web interface that facilitates marker annotation. The database also allows linking markers to other sequence information, such as RNA-seq data  are disassembled, and the reads are set aside because these stacks are indistinguishable from stacks generated with sequencing error. Reads in a stack are primary reads, and reads that are set aside are secondary reads. The ustacks program calculates the average depth of coverage, then identifies stacks that are two standard deviations above the mean and excludes them, along with all stacks that are one nucleotide apart from these extremely deep (lumberjack) stacks, which usually represent repetitive elements.The ustacks (unique stacks) program reads cleaned sequences and distills data into unique, exactly matching stacks by loading reads into a hash table . Unique within-individual distance parameter). This configurable distance depends on the dataset\u2019s genetic properties, such as polymorphism rate and read length, and usually allows just a few nucleotide differences. To implement this comparison, ustacks breaks the sequence of each stack into a set of overlapping fragments of equal length k (k-mers)  and loads k-mers into the Dictionary    . The firctionary .The ustacks program queries the k-mer Dictionary with each k-mer from each stack to identify other stacks with matching k-mers. For pairs of stacks with sufficient numbers of matching k-mers, ustacks aligns the pair, naively matching nucleotide by nucleotide to verify that each pair of stacks is within the allowable nucleotide distance, and if they are, it records a match.The k-mer search algorithm transitively relates pairs of stacks. For example, if stacks 4 and 5 match and stacks 5 and 6 match with an allowable distance of one nucleotide, ustacks records two matching pairs . Then usIn a diploid genetic cross, homozygous and heterozygous loci should contain one and two stacks, respectively. Allowing for some error, if more than three unique stacks have been merged, or if the coverage of the merged stack is more than two standard deviations above the mean coverage, ustacks shunts the stack to the deleveraging algorithm to determine which subset of these large stacks is most likely to represent a locus see .within-individual distance parameter by default). Secondary reads that do not have a best match to a unique defined locus are discarded. At the end of this stage, Stacks has constructed a set of putative loci from high-confidence unique stacks and has buttressed locus depth by adding secondary reads.The process of merging stacks is iterative. With a user-specified distance of three nucleotides between stacks, ustacks first finds stacks that are a single nucleotide different and merges them, then continues at a distance of two, and finally at a distance of three. At the end of each round, ustacks excludes lumberjack stacks. Secondary reads , cstacks  initializes the Catalog. Each additional individual is then merged into the Catalog in turn. Individual loci are matched to those already in the Catalog using the same k-mer search algorithm used by ustacks, except that each locus is represented in the k-mer dictionary by the set of k-mers resulting from each haplotype at that locus. When two loci match, cstacks merges their SNPs in the Catalog. If, however, those SNPs have conflicting alleles , the merge fails and cstacks issues a warning. The cstacks program adjusts its haplotype calls based on the newly merged SNPs.between-individual distance parameter of cstacks allows for mismatches while merging loci into the Catalog. If each parent is fixed for a different allele at a particular locus, cstacks can detect the mismatch and properly merge the loci. This property is particularly useful when fixed differences occur, as in crosses between inbred populations or between divergent species.The To identify which locus/haplotype combinations are present in each individual in the population, sstacks (search stacks) matches every individual in the cross, including the parents and the progeny, against the Catalog . The sstStacks has identified haplotypes segregating in each individual in the population. Next Stacks identifies informative markers. The markers.pl program identifies mappable markers in the parents by downloading Catalog matches from the MySQL database and tallying up all the matching parental haplotypes. The markers.pl program characterizes parental loci into 10 classes of mappable markers, including loci that are segregating in the family due to variation in a single parent , loci homozygous within parents but heterozygous between parents (aa/bb), loci with two (ab/aa), three (ab/ac), or four (ab/cd) haplotypes, as well as other related types  and an export type (JoinMap or R/qtl). The genotypes.pl program maps haplotypes in the progeny to the marker types detected in the parents. First, genotypes.pl downloads from the database the set of loci containing mappable markers recorded by markers.pl. It then maps haplotypes: if the first parent has haplotypes GA and AC, and the second parent has the GA haplotype, Stacks declares an ab/aa marker for this locus. The genotypes.pl program maps GA to a, and AC to b in the parents and checks progeny to see which haplotypes each contains, recording the genotypes . Finally, genotypes.pl formats genotypes for use with the mapping program and outputs a properly formatted file. Users can specify the minimum number of matching progeny required for locus export.At this point, i.e., in the parents) is present in a progeny individual at a low frequency (less than 10% of reads in the stack), genotypes.pl corrects the genotype. Likewise, genotypes.pl removes a homozygous genotype call for a particular individual if the locus contains fewer than five reads supporting the genotype. Users can adjust these thresholds.Users can tell the genotypes.pl program to perform automated corrections for certain errors, including checking homozygous tags in the progeny to ensure that a SNP is not present. As described in Stacks would call the genotype as homozygous C, not being able to distinguish the single A from a sequencing error. But if a homozygous C call results in a double cross-over involving this single locus, the genotype is more likely to be heterozygous C/A with the A allele undersequenced. Users can make this correction through the web interface, and the corrected genotype will be included on the next execution of genotypes.pl.The genotypes.pl program can optionally output a file formatted for loading into the database. The web interface allows users to manually correct genotypes. For example, a stack for Locus 1 in one of the progeny  might haStacks can identify loci not only de novo as described above but also using a reference genome. The two processes differ: instead of building stacks and loci from similar sequence reads, Stacks first aligns sequence reads to the reference genome using Bowtie  for each sequence in a FASTA file, is a Catalog locus ID, and will store that sequence in the MySQL database linked to the Catalog locus. Therefore, in addition to mini-contigs, if ESTs are available or were constructed Stacks importing and exporting capabilities can associate Stacks markers with additional sequences, including mini-contigs and ESTs. These sequence sets can associate mappable loci in protein coding genes to orthologs in other species by BLAST searches, or to genomic contigs in an emerging reference genome.In summary, the Stacks provides a web-based interface for viewing, annotating and correcting loci in a population  known loci (Stacks identified most loci (81% correctly assembled), and at 40\u00d7 coverage, the error rate had little effect on the number of identified loci  . In contSbfI RAD loci in the stickleback genome into a smaller number of loci with very high depths of coverage, as indicated by the long right tail of the distribution that stretches far beyond the truncated display in the figure. The introduction of SNPs into the simulated reads at a rate of 0.5% caused a shoulder to appear on the distribution at 20\u00d7, half the depth of the main peak . In sum, these simulations demonstrate remarkable fidelity of locus identification, even in the face of mounting errors, when the sequencing depth is between 20\u00d7 and 40\u00d7.To explore the effects of error rate on locus quality, we studied, at three levels of mean coverage, the effects of a typical low error rate of 0.5% and an unusually high error rate of 3.0% . At 10\u00d7 Stacks works well, it should reconstruct a known genome map. To test this prediction, we constructed for Danio rerio a genetic map (RADmap) by using RAD-seq and Stacks to re-genotype a previously published doubled haploid mapping panel  that consists of 42 progeny , each with a length nearly identical to the original (vs. 3160 cM in the RADmap). With 7861 markers, our RADmap has nearly twice as many markers as the original HSmap (4073 markers), but it required less than 1% of the cost and took less than 1% of the time to genotype and construct. The RADmap and HSmap had nearly identical marker order ; differences could represent errors in either map.If A comparison of the zebrafish RADmap to the sequenced reference genome showed alignment of 5787 RADmap markers and revealed that marker order for the RADmap and the physical assembly generally agreed . An addivs. the reference genome identified several regions of low recombination rate per physical distance. One region in LG20 showed recombination suppression in the RADmap over a region of about 10 Mb , PCR errors, or sequencing errors. Loci that appear in a large number of individuals in a population or in a large number of map cross progeny are the most reliable. Once nterface  or by spStacks is its convenient web interface, which supports manual corrections. Iterative corrections can make significant improvements in a genetic map based on the principle that double recombinants in a short genetic distance are unlikely events. Manual examination of markers that expand the map can identify, correct, or remove troublesome genotypes, followed by re-exporting data and reconstructing the map. Reiteration can provide a genetic map with strong statistical support on all linkage groups.One of the key attributes of Stacks might erroneously confuse paralogs that have nearly identical sequences with alleles of the same locus. Fortunately, Stacks can detect \u201covermerging\u201d of paralogous stacks because all individuals homozygous for a specific sequence at one paralog and homozygous for a slightly different sequence in the other paralog would appear to be heterozygotes for the relevant SNP. In contrast, a meiotic mapping population that is segregating a SNP at one locus or a population in Hardy-Weinberg equilibrium would have, on average, only about half of the individuals being heterozygotes. In addition, a diploid individual will never have more than two alleles of a single locus, so if individuals are discovered with three or more alleles, paralogs are likely to blame. Stacks can detect markers in which observed heterozygosity is significantly different than expected and flag them. The problem of confusing paralogs with allelic variants is evolutionarily transitory. Identical stacks  are uninformative and don\u2019t cause a problem; furthermore, a few neutral mutations are sufficient for Stacks to identify paralogous loci, particularly if the user sets the within-distance parameter to a small value. In a recent study of trout populations, Stacks flagged loci that differed from Hardy-Weinberg expectations, thereby successfully removing the effects of the recent (25\u2013100 million years ago) salmonid genome duplication  and the experimental goals . In some cases, however, the indiscriminate filtering of loci that do not appear to meet Hardy-Weinberg expectations can lead to erroneous conclusions. For example, in a recent moss linkage map, 45% of the loci exhibited segregation distortion, likely due to lethal interactions between distant loci  more tractable in nonmodel species because the enormous linkage map provides a framework for the analysis of population genomic data. Stacks is available for download, along with a set of example data, tutorials, and other documentation at http://creskolab.uoregon.edu/stacks/.Nearly a century after the first genetic maps , Stacks,"}
+{"text": "Prior studies of appetite regulatory networks, primarily in rodents, have established that targeted electrical stimulation of ventromedial hypothalamus (VMH) can alter food intake patterns and metabolic homeostasis. Consideration of this method for weight modulation in humans with severe overeating disorders and morbid obesity can be further advanced by modeling procedures and assessing endpoints that can provide preclinical data on efficacy and safety. In this study we adapted human deep brain stimulation (DBS) stereotactic methods and instrumentation to demonstrate in a large animal model the modulation of weight gain with VMH-DBS. Female G\u00f6ttingen minipigs were used because of their dietary habits, physiologic characteristics, and brain structures that resemble those of primates. Further, these animals become obese on extra-feeding regimens. DBS electrodes were first bilaterally implanted into the VMH of the animals (n\u200a=\u200a8) which were then maintained on a restricted food regimen for 1 mo following the surgery. The daily amount of food was then doubled for the next 2 mo in all animals to produce obesity associated with extra calorie intake, with half of the animals (n\u200a=\u200a4) concurrently receiving continuous low frequency (50 Hz) VMH-DBS. Adverse motoric or behavioral effects were not observed subsequent to the surgical procedure or during the DBS period. Throughout this 2 mo DBS period, all animals consumed the doubled amount of daily food. However, the animals that had received VMH-DBS showed a cumulative weight gain  that was lower than the nonstimulated VMH-DBS animals , suggestive of a DBS-associated increase in metabolic rate. These results in a porcine obesity model demonstrate the efficacy and behavioral safety of a low frequency VMH-DBS application as a potential clinical strategy for modulation of body weight. For some individuals, obesity factors result in a condition of morbid obesity  which affects 5.7% of the population in the U.S. according to national survey data According to the U.S. Census Bureau statistics (data for 2006), 34.3% of the adult population in the US is considered obese, as defined by a Body Mass Index (BMI) over 30 kg/m2) and super obese patients (BMI>50 kg/m2) provide a rationale for exploring other effective modality options Present treatments to control morbid obesity include a wide variety of drugs and nutritional/dietetic counseling but increasingly only gastrointestinal tract surgical procedures, particularly the Roux-en-Y gastric bypass, have provided successful therapeutic approaches The potential for hypothalamic DBS clinical applications related to control of food intake, fat distribution, and body weight has not been fully explored. Collectively, data from prior animal studies that demonstrated modulation of food intake with electrical stimulation have provided the rationale for a small number of human DBS studies. However, in 2008 and 2010, two case reports described mixed results on the use of hypothalamic DBS to produce weight loss Miniature pigs (minipigs) have gained increasing importance as an alternative non-rodent species to the dog or monkey for basic and applied biomedical research. The pig genome relative to that of the rodent is more closely related to the human genome Rodent models of obesity have provided a wealth of information on basic mechanisms modulating hunger and weight regulation. However, there are several areas where studies in pigs may offer advantages as a model for aspects of human obesity. G\u00f6ttingen minipigs are similar to humans in digestive physiology, dietary habits, and fat deposition ad libitum, analogous to the overeating-craving food observed in obese humans. This increase in food intake results in significant weight gain ad libitum, but the females gain significantly more body weight.Minipigs will exhibit hyperphagic behavior when food is provided, Under conditions of food presentation in excess of the \u2018restricted\u2019 diet, the G\u00f6ttingen minipig can be used to model human pathologies linked to obesity ad libitum\u2019) in all animals for a 2 mo period to establish a controlled condition facilitating obesity, as similarly used and validated in prior studies In our study, we used female animals and administered a normative \u2018restricted diet\u2019 regimen that corresponded to 400\u2013450 g/day of feed for promoting weight maintenance throughout the pre-DBS period. Subsequently, the daily food was doubled to 900 g/day  VMH-electrical stimulation negatively modulated weight and food intake in rodent studies, we hypothesized that similar effects would be achieved by low frequency VMH-DBS in a G\u00f6ttingen minipig model of obesity. In this study, we have shown that stereotactic targeting methods and instrumentation that have been well-established in human DBS applications can be adapted to studies in the G\u00f6ttingen minipig to demonstrate efficacy of low frequency VMH-DBS for modulation of body weight gain.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and all procedures were approved by the Office of Animal Research Oversight/Chancellor's Animal Research Committee at the University of California, Los Angeles (ARC# 2010-089-01B). All efforts were made to minimize animal suffering. Surgeries were performed under general anesthesia with analgesic and antibiotic administration.Sexually mature female G\u00f6ttingen minipigs  aged 9\u201311 months and weighing 20\u201327 kg were entered into the study.ad libitum. At one mo after full recovery following the surgery, all animals (n\u200a=\u200a8) received for 2 mo a daily doubled-food feeding, 900 g/day, previously referred to as a \u2018Near ad libitum\u2019 diet regimen The animals were singly-housed within enclosures (1.1\u00d71.8 m) in a dedicated room maintained at 22\u00b0C, relative humidity 40%. The enclosures allowed for visual contact among animals. For the complete monitoring of food intake, pre- and post-surgery, the animals were fed once daily between 0730 and 0800 on a \u2018Restricted\u2019 diet regimen of 450 g/day , to promote normal weight maintenance without the development of obesity The feeding and behavioral observations were conducted by veterinary and lab personnel blinded to the experimental conditions. The animals in this study (n\u200a=\u200a8) were assessed individually at specified times pre-surgery, post-surgery, and during the DBS stimulation period. The motoric and affective behaviors of all animals were observed for \u223c4 wks pre-surgery, and subsequently for 4 wks post-surgery. Observations then continued for 2 mo, either on-DBS (n\u200a=\u200a4) or off-DBS . During 3 d/wk of behavioral testing, the animals were allowed to run along a 15 m long corridor. Five-point Likert scales were used to assess motoric behavior  and affective behavior . Affective behavior categories consisted of acceptance of petting, willingness to accept and eat small food treats, absence of any guarding response at the site of lead or IPG placement, and resting in a species-typical lateral recumbent position.2, and cardiac rate were monitored continuously throughout the procedures. Following the surgery, the animals were injected with analgesic Carprofen  for 3 d and antibiotic Baytril  for 5 d, respectively.The animals were initially anesthetized with Telazol  followed by endotracheal intubation. General anesthesia was maintained by inhalation anesthesia (isoflurane 0.5\u20132.0%). during the MR imaging and surgical procedures. A catheter was placed in the femoral vein for fluid administration. Body temperature, oxygen saturation, pCOA stereotactic frame designed for a porcine head The VMH target was planned according to regional landmarks identified in a published stereotactic brain atlas of the Gottingen minipig species Following the stereotactic target planning and target verification on the phantom, the CRW apparatus with target coordinates dialed-in, was attached to the frame affixed to animal's head for the subsequent stereotactic lead implants. After a midline skin incision, the dorsal skull surface was exposed and 1.5 mm diameter holes were drilled bilaterally for the approach to the hypothalamic targets The accuracy of electrode placement in relation to the set stereotactic targets was ascertained by intraoperative x-ray imaging with cross-hair reticles inserted into the CRW frame. The center of the crosshairs visualized in the image coincided with the center-of-arc point, i.e., the calculated and dialed-in stereotactic target for each VMH. Fluoroscopy allowed for intraoperative corrections of the electrode placement in the ventral-dorsal direction (Z-axis) and also for verification of the spacing between electrodes (2\u20133 mm) inserted parallel to the midline . This entire lead placement method was used for targeting both left and right VMH. The leads were then secured to the skull with titanium screws and mini-plates  as was recently suggested in a similar procedure on G\u00f6ttingen minipigs The proximal ends of the bilaterally implanted leads were connected to the extension , Plano, TX; a St. Jude Medical Company) which was tunneled subcutaneously to the dorsal part of the neck lateral to the right ear of the animal and connected to the implantable pulse generator (IPG)  which was subcutaneously implanted. The extension and IPG were secured in place with non-absorbable sutures. This IPG placement method allowed for unrestricted and free movement of the animal as well as for transdermal access for IPG programming and activation without the need to anesthetize or restrain the animal.2 that are significantly smaller than the clinical DBS electrode with a contact area of 0.06 cm2. When such small surface area electrodes are used, there is a concern that excess electric charge can be generated at the electrode surface that may potentially damage brain tissue. The relationship between electric charge and electrode surface area is expressed as charge density (microcoulombs/cm2/phase), i.e., the quantity of delivered electric charge divided by the surface area.In our study, we used custom-made DBS leads 2/phase is used as a recommended conservative upper limit for clinical DBS studies In prior electrical stimulation and DBS studies in brain, histologically-detected tissue damage or its absence was determined following applications of different combinations of stimulation parameters, 2/phase is considered not to result in tissue damage. In 2 electrode area, a curve that represents combinations of pulse width and current resulting in 30 microcoulombs/cm2/phase. Accordingly, any combinations to the left of that safety-threshold curve could have been selected. Since we wanted to maintain a low current amplitude for delivering DBS to a relatively small area of the hypothalamus, we initially used a low current (0.5 mA) that was matched with a long PW (507 \u00b5s) to achieve a maximal charge density that still remained <30 microcoulombs/cm2/phase.Thus, any combination of pulse width, current, and electrode area that yields less than 30 microcoulombs/cmFollowing the animal's full recovery from surgery (1 mo), the IPG was programmed with DBS parameter settings  and then activated in a monopolar mode. Contacts 0 and 4, i.e., the most ventral contacts in the left and right VMH leads, were programmed as cathode and the IPG case as anode. Those settings were used for Wks 1, 2, followed by subsequent ramping up of the amplitude: Wks 3\u20134 1.0 mA; Wks 5\u20138 1.5 mA. DBS was continuously active throughout the entire 8 wk stimulation period.In preliminary studies, we observed that the minipigs showed small daily weight fluctuations that were likely due to variable degrees of water and stool retention, as in humans. By design, we delivered DBS to effect a moderate and progressive weight change to achieve a statistically significant \u2018long term\u2019 result after a 2 mo DBS stimulation period. The weight measure after that time could then be reliably used to establish the efficacy of the DBS.After the 2 mo DBS stimulation period was completed, preprandial blood samples were obtained  for measurements of blood glucose levels using a glucose oxidase/electrochemical kit .Our primary outcome measure was cumulative weight change in the 2 mo period under the condition of either on- or off-DBS. Since a directional hypothesis was explicitly stipulated in advance of the study (as shown in After 2 mo of either on- or off-DBS (controls), all animals were euthanized with pentobarbital . The brains were removed within 20 min; 5 mm coronal blocks were obtained and were frozen for \u223c30 s in isopentane maintained at \u221235\u00b0C on dry ice, and then stored at \u221280\u00b0C. For each brain, the block containing the hypothalamus was cut into 20 \u00b5m sections on a cryostat (Leica CM 3050) maintained at \u221220\u00b0C  and thaw-mounted onto gelatin-subbed slides.2O2 solution in PBS for 10 min at room temperature to block endogenous peroxidase activity, rinsed and preincubated for 1 h in 3% normal horse serum (NHS) in PBS. Sections were then incubated with mouse monoclonal anti-GFAP , 1\u2236200, in PBS with 3% NHS for 1 h at room temperature. Following rinses, the sections were incubated with biotinylated horse anti-mouse IgG (1\u2236200)  for 1 h and then with ABC (Vector Labs), 1\u2236200, for 1 h at room temperature. GFAP immunoreactivity was visualized with DAB with nickel ammonium sulfate enhancement. Sections were counterstained with Pyronin Y.Selected sections were post-fixed for 10 min in acetone at \u221220\u00b0C or in 4% paraformaldehyde solution in 0.1 M PBS, pH 7.4, at room temperature for 15 min and then stained with hematoxylin and eosin, and cresyl violet to identify the terminal region of the implanted electrodes. Adjacent sections were then immunostained for GFAP. Briefly, slides with acetone fixed sections were incubated with 0.3% HThe motoric and affective behavior of all animals remained unchanged throughout the pre-surgery, and the post-surgery off-DBS and on-DBS periods. The on-DBS animals showed no adverse reaction to the initial activation of the DBS parameter setting at  or, subsequently, throughout the DBS stimulation when the amplitude was increased and maintained at 1.5 mA. These conclusions were based on the animals' overt behavior in different circumstances that included being frequently in a recumbent position during the day when observed through a viewing window on the door outside of their cage area, their rolling on their side to accept stroking/petting of their belly area, and maintaining that position for the duration of the interaction.A schematic drawing provides an estimate of the actual target area occupied by the custom-designed electrode . The resThe iPlan 2.6 software  was used to derive the bilateral VMH targets, with confirmation assessed either with a post-operative MR scan after the leads were implanted  or with The surgery and MRI procedures were unremarkable. The animals well-tolerated the general anesthesia (\u223c8 h) and they regained consciousness within 2 h after its termination at the end of the surgery. Normal appetite was observed the following day. Prior to initiation of VMH-DBS, we continued the pre-surgery standardized food regimen of 450 g/day for 1 mo post-surgery in order to observe maintenance of pre-surgery weight, affective and motor behaviors, and to allow complete recovery from the surgical procedure-DBS lead implants.2/phase threshold-safety curve, used for clinical DBS applications with commercial electrodes  yielded a charge density below the 30 \u00b5C/cmectrodes . Those sH&E histology and GFAP immunostaining did not show detectable signs of inflammation or reactive gliosis in the VMH brain region (data not shown).t-test, p<.05  that was significantly lower than the 9.4\u00b11.3 kg, measured for the nonstimulated VMH-DBS animals; one-tailed t, p<.05 .After the 2 mo DBS stimulation period, preprandial measurements of morning blood glucose levels (after overnight fasting) in on-DBS animals were 3.0\u00b10.3 mM (mean \u00b1 SEM) which were not significantly different from the glucose levels of 3.2\u00b10.5 mM measured in control animals. These glucose levels are within the range of mean control values (3\u20134.1 mM) reported for the G\u00f6ttingen minipig in prior studies The rationale for a clinical application of VMH-DBS to treat morbid obesity and severe eating disorders is based on an extensive literature demonstrating the regulation of food intake and satiety by the hypothalamus Consideration of a low frequency VMH-DBS clinical trial can be advanced upon demonstration of its efficacy and safety in preclinical studies in which aspects of the clinical procedures have been modeled. The primary goals of this research were to adapt human neurosurgical methods and DBS instrumentation for use in a large animal obesity model and then evaluate the effects of VMH-DBS on weight modulation and behavior. Our results showed that in the G\u00f6ttingen minipig under conditions of extra calorie intake, the continuous delivery of low frequency (50 Hz) DBS in the ventral hypothalamus region was associated with a lower weight gain compared to that in animals not receiving DBS.In the early 1960's DBS represents the clinical counterpart of electrical stimulation as used in animal studies. Generally, DBS applications in humans are classified bimodally by frequency range - either high (100\u2013185 Hz) or low (<100 Hz). Within each mode, further options for amplitude (either voltage or current) and pulse width then allow for a wide range of parameter settings combinations. However, in the absence of a detailed theoretical framework for understanding how those variables affect neuronal and glial tissues, the clinician usually uses combinations that follow prior protocols that demonstrated positive clinical outcomes, and then they further modify selected settings empirically to maximize efficacy and minimize adverse effects for individual patients.Molecular mechanisms underlying the therapeutic effects of DBS remain not well-defined. Initially, experimental evidence supported the theory that high-frequency DBS paradoxically acts like a lesion, i.e., via a neuronal \u2018depolarization block.\u2019 However, more recent studies have shown that while high frequency DBS does inhibit somatic activity near the DBS electrode, it can also increase regional output by directly activating axons of local projection neurons. Additionally, surrounding neuropil can be stimulated to different extents as the DBS intensity fades radially from the electrode placement. These \u2018secondary\u2019 effects of the high frequency DBS intensity may be analogous to those primarily produced by low-frequency DBS which is hypothesized to activate neurons  by enhancing their firing and responsivity to other neural inputs Since the early 2000's, the posterior hypothalamus has been targeted with high frequency DBS as treatment of cluster headaches Here, we attempted to address several of those issues. Firstly, we needed to develop and validate in our lab a large animal model of morbid obesity in which DBS instrumentation and methods could be applied. We greatly benefited from a wide range of prior brain studies in the G\u00f6ttingen minipig conducted by the Danish group over the last 10 years. In particular, their delineation of the hypothalamus cellular topography clearly indicated its potential applicability for VMH-DBS Accurate targeting of DBS electrodes to deep structures of the brain, e.g., the hypothalamus, is challenging since small initial trajectory errors are magnified as the depth of insertion increases. Also, transiting across cerebral ventricle membranes enroute to hypothalamus can cause distortion in the trajectory. We significantly obviated those issues with the use of a guide tube to support the minielectrode Our prior experience with neurosurgical stereotactic methods, instrumentation and MR imaging for implementing DBS in the human 2/phase. Although that value was higher than the 30 \u00b5C/cm2/phase limit recommended for clinical applications with the commercial electrodes, it was within a \u2018no tissue damage-zone\u2019 derived from multiple non-DBS electrical stimulation and DBS clinical studies 2) yields a charge density within the recommended safety zone.However, the selection of the initial DBS parameter settings was not evidence-based since the effects of systematic variations of low frequency VMH-DBS stimulation and pulse widths over long periods of time  in any animal species had not been previously described. Accordingly, we selected the stimulation frequency of 50 Hz, based on experimental literature of VMH electrical stimulation showing that a majority of studies with 50 Hz (range: 10\u2013100 Hz) resulted in either a reduction of food intake or an increase in energy utilization. We activated the most distal electrodes with a monopolar configuration to presumably affect a larger VMH region. As our main read-outs on VMH-DBS efficacy were the amount of daily food consumption and weekly weighing, we needed to maintain the same DBS settings for at least 2 wks to determine any significant change. Since our initial DBS settings were without apparent effect for 1\u20132 wks, we increased the amplitude to 1 mA and then observed a reduced increase in weight (<1 kg) from the previous wk in 2 of 4 animals, suggestive of a DBS effect. Insofar as all animals appeared to well-tolerate the VMH-DBS, we increased the current to 1.5 mA in all animals which was then continued for the second mo of stimulation. This resulted in a charge density of 76 \u00b5C/cmThe present study design with the read-out of weight change did not allow for accurate assessments of alternating short periods of 1\u20132 wks with on- and off-DBS. However, it was not our intention to induce significant weight fluctuations acutely with DBS since such short term effects may not extrapolate to a safe, long term human application. The overall effect of the VMH-DBS treatment for a 2 mo period of continuous stimulation did result in a significantly lower weight gain of \u223c10% relative to weight-matched DBS-off \u2018controls\u2019 for the same period of extra-calorie intake. We tentatively attribute this lower weight gain to an increase in metabolic rate insofar as all animals ate their entire daily food ration within 30\u201340 min of presentation throughout the 2 mo DBS period. This study was not designed to measure changes in metabolic rate, but now having established an effective set of VMH-DBS parameters, future studies employing indirect calorimetry to measure increases in metabolic rate would provide confirmatory data. Further, the use of that method would be ideal for conducting parameter sweeps of DBS settings that can be readily evaluated for their efficacy and reversibility. A VMH DBS-induced increase in metabolic rate has some support from prior experimental and clinical studies. In rodents, electrical stimulation in the hypothalamus resulted in metabolic rate increases In conclusion, this study has demonstrated that clinical neurosurgical instrumentation and methods can be applied to preclinical studies in a large animal model. We also demonstrated that DBS leads and electrodes can be scaled down to appropriate size for use in the minipig brain. The DBS that was targeted to the ventral hypothalamus of the Gottingen minipig effected a reduction in weight gain under conditions of extra-calorie intake. These results show that DBS can provide CNS neuromodulation within the hypothalamus and provide preclinical evidence in support for this DBS application as a potential strategy for the treatment of humans with morbid obesity."}
+{"text": "The objective of this observational study was to examine the key contributors to health outcomes and to better understand the health disparities between Delta and non-Delta counties in 8 states in the Mississippi River Delta Region. We hypothesized that a unique set of contributors to health outcomes in the Delta counties could explain the disparities between Delta and non-Delta counties.Data were from the 2014 County Health Rankings for counties in 8 states . We used the Delta Regional Authority definition to identify the 252 Delta counties and 468 non-Delta counties or county equivalents. Information on health factors  and outcomes  were derived from 38 measures from the 2014 County Health Rankings. The contributions of health factors to health outcomes in Delta and non-Delta counties were examined using path analysis.We found similarities between Delta counties and non-Delta counties in the health factors  that significantly predicted the health outcomes of self-rated health and low birthweight. The most variation was seen in predictors of mortality; however, Delta counties shared 2 of the 3 significant predictors  of mortality with non-Delta counties. On average across all measures, values in the Delta were 16% worse than in the non-Delta and 22% worse than in the rest of the United States.The health status of Delta counties is poorer than that of non-Delta counties because the health factors that contribute to health outcomes in the entire region are worse in the Delta counties, not because of a unique set of health predictors. The Mississippi River Delta Region is among the most socioeconomically disadvantaged areas of the United States. The Delta is defined by the Delta Regional Authority as 252 counties or parishes in 8 states near the lower half of the Mississippi River . These cAlthough several studies described the poor health status of the Mississippi River Delta Region, few used empirical methods to explain health disparities between the Delta and other regions. Bloom and Bowser  examinedwww.countyhealthrankings.org) \u2014 a collaborative effort between the University of Wisconsin Population Health Institute and the Robert Wood Johnson Foundation \u2014 compiles health-related information from various sources to produce annual rankings  by using means and standard deviations derived from the 8 Delta states. Where data were missing, state-level mean values were imputed. We multiplied z scores for positive outcomes, such as high school graduation rate, by \u22121 so that all measures followed the same scheme, in which a higher number indicates poorer health. Composite scores were then calculated for 16 subcategories of health factors , and two absolute fit indices   and tobacco use  were significant predictors of self-rated health; however, rurality was also a strong and significant predictor . Percentage of the population that was African American was again the strongest significant predictor of low birth weight , but family and social support  proved to be significant and a stronger indicator than sexual activity  in this sample. The top 3 significant predictors of YPLL in the non-Delta counties were community safety , rurality , and income .We used county-level data for a variety of health factors to identify the key contributors to health outcomes in the Mississippi River Delta Region and to better understand health disparities between Delta and non-Delta counties in the region\u2019s states. Contrary to our hypothesis, our main finding was that the primary contributors to health outcomes in Delta and non-Delta counties were similar, especially for self-rated health and low birth weight. The predictors were most varied for mortality, but 2 of the 3 significant predictors in Delta counties were also the strongest significant predictors in non-Delta counties. Overall, the health status of Delta counties appeared to be poorer than that of non-Delta counties because the factors that affect health the most in the entire region were worse in the Delta counties, and not because there is an entirely different set of health predictors in Delta counties than in non-Delta counties.These predictors also proved to be analogous to what was found by other studies examining predictors of similar health outcomes in a variety of populations. For instance, it is well understood that health behaviors are associated with self-rated health status. Two recent studies have presented evidence supporting the relationship between self-rated health and smoking, physical activity, and proper nutrition ,8. ZarinSimilarly, the link between African American race and low birth weight is well established. Women identifying as black or African American are twice as likely to have a preterm birth and 3 to 4 times as likely to have a very early preterm birth as other racial and ethnic groups in the United States and the United Kingdom ,10. In tIncome is also a widely accepted predictor of mortality. In the United States, data from the National Longitudinal Mortality Study show that those in the highest income bracket  can expect to live 6 years longer than those in the lowest bracket  . Income These findings suggest that the health disparities between Delta counties and non-Delta counties are related to differences in health factors that both areas have in common, rather than differences between the 2 areas in what affects health. For instance, for predictors of self-rated health status, we found no significant differences between Delta counties and non-Delta counties in tobacco use, but Delta counties scored worse in all 4 measures that make up the diet and exercise subcategory . Also, the African American proportion of the population, which was the strongest significant predictor of low birth weight, is roughly 4 times larger in Delta counties than non-Delta counties. Last, in regard to the primary contributors to our mortality measure, median household income is approximately 11% lower in Delta counties, and violent crime (one of 2 measures that make up community safety) is 53% higher.Overall, Delta counties performed worse than non-Delta counties and the national average on all but 3 measures. On average across all measures, values in Delta counties were 16% worse than those in non-Delta counties and 22% worse than those in the rest of the United States. This trend is similar to what was previously reported in a small set of studies describing health in the Delta region  or the Centers for Disease Control and Prevention\u2019s Healthy Living , which provide evidence ratings and implementation strategies for policies, programs, and system changes that improve health factors, can provide information on such efforts. We hope the findings of this study will assist local health officials, leaders, and policy makers in deciding how to allocate limited resources to improve the health of the Mississippi River Delta Region.Despite these limitations, this study contributes to the health disparities literature by examining contributors to health outcomes in the Mississippi River Delta Region. By examining the influence of 16 health factors comprising 35 measures on the health outcomes of mortality and quality of life, we saw that Delta counties and non-Delta counties were similar in regard to what contributes to health. Efforts to improve the health of Delta communities should focus on reducing the disparity in modifiable factors identified as predictors of health outcomes. For instance, our findings suggest that efforts to improve access to exercise opportunities  and a healthy food environment , and subsequent reductions in obesity, could lead to improved health-related quality of life. In addition, taking action to reduce violent crime could help to address the disparity in premature mortality in the region. Resources, such as the County Health Rankings\u2019 \u201cWhat Works for Health\u201d ("}
+{"text": "H. djakonovi sp.n., was discovered in Rudnaya Bay in the Sea of Japan. This is a sympatric species of the well-known and common species Henricia pseudoleviusculaHenricia species inhabiting the Sea of Japan. Nevertheless, these species can be distinguished by their abactinal spines: in both species, they are short and barrel-like, but the new species is the only Henricia species in Russian waters of the Pacific that possesses such spines with a massive, smooth, bullet-like tip. The spines in H.\u00a0pseudoleviuscula are crowned with a variable number of well-developed thorns. About half (<50%) of the abactinal pseudopaxillae in the new species are oval, not crescent-shaped as in H. pseudoleviuscula.A new sea star species,  Henricia Gray, 1840 (blood stars) belonging to the family Echinasteridae  are a group of organisms with poorly developed systematics despite their wide distribution and abundance in the world\u2019s oceans, especially in the northern Pacific , a name that has been widely applied to Henricia in the North Pacific, to only a portion of the cool temperate coastline of the western North America.Studies on North Pacific assessed , and 13 Henricia in the Asian fauna were also published . A similar species has not been reported from outside the Russian waters in Japan and Korea  remain poorly studied in the Northwestern Pacific, as recent studies have demonstrated  and Vostok Bay , respectively. These animals were released within several hours into the natural environment at the same sites where they were collected. Sea star collection is not regulated by Russian law. They were not collected in protected waters. The images were taken using a Nikon D7000 camera and a Nikon Nikkor 60 f2.8 lens. Skeletal spines were cleaned from soft tissues, and skeletal plates were denuded with sodium hypochlorite. Scanning electron images of the spines were obtained by using a Zeiss Sigma electron microscope after carbon coating.Sea stars were collected by SCUBA-diving in Rudnaya, Kievka, and Vostok bays in the Sea of Japan during 2015\u20132016, and the animals were preserved in 96% ethanol. The specimens were deposited in the collection of the Museum of National Scientific Center of Marine Biology, Vladivostok, Russia (MIMB). Life coloration, abactinal skeletal reticulation, and spines shape were checked in 25 and 218 specimens of http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:632d0662-da5f-4f7e-a63a-f112de99a7d5. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix MIMB331294 4 Jun 2016, Senkina Shapka pinnacle, Rudnaya Bay, 44.36\u00b0N 135.83\u00b0E, 16 m, leg. A. Chichvarkhin. Paratype. MIMB331304 4 Jun 2016, Senkina Shapka pinnacle, Rudnaya Bay, 44.36\u00b0N 135.83\u00b0E, 16 m, leg. A. Chichvarkhin.Holotype. Senkina Shapka pinnacle, Rudnaya Bay, NW Sea of Japan.Henricia pseudoleviuscula: MIMB331271 specimen4 Jun 2016, Senkina Shapka pinnacle, Rudnaya Bay, 44.36\u00b0N 135.83\u00b0E, 16 m, leg. A. Chichvarkhin; 2 specimens 5 Oct 2015, Senkina Shapka pinnacle, Rudnaya Bay,44.36\u00b0N 135.83\u00b0E, 15\u201318 m, leg. A. Chichvarkhin; MIMB331261 specimen 27 Jun 2015, Kievka Bay, Skaly Is., 5 m, leg. A. Chichvarkhin; MIMB33128, MIMB3313111specimens 23\u201325 Aug 2015, Vostok Bay, leg.K. Dudka; ZIHU-2397 (Hokkaido University) 2 specimens H. reniossa syntypes (marked as a \u2018paratype\u2019) 30 Sep 1906, Albatross station 5031 Bomase\u2019ri Shima.R to 7\u00a0cm; disc relatively large, rays long, fairly rigid, marginal and ventrolateral plates are similar, pillow-shaped; about a half of abactinal plates cross-shaped, arranged as roof tiles: each plate\u2019s proximal outgrowths cover adjacent proximal plate; elevated sides of cross-shaped plates form crescent-shaped pseudopaxillae with 20\u201330 spines, the other triangular or irregular-shaped abactinal plates lacking proximal outgrowths form oval pseudopaxillae; abactinal plates on disk close-set but not very tightly leaving space for papular areas; more than one intermarginal row, the longest intermarginal row contains 20 plates; color in life dark/dirty red with almost black spots and wide transversal lines; aboral side of disk dark, brownish-red divided into five triangular sectors with lighter lines connecting anal pore and disk margin. Abactinal and marginal spines blunt with rounded droplet-like apex, some apices bear few very short thorns.r\u00a0=\u00a011mm; R\u00a0=\u00a038 mm, r\u00a0=\u00a07.5 mm in paratype. The rays slender not very slim, slightly swollen at base, tapering to blunt tips. Most abactinal plates (>50%) cross-shaped with two proximal outgrowths covering adjacent plates, their elevated proximal sides form crescent-shaped pseudopaxillae. The other plates triangular or irregular shaped, lacking proximal outgrowths, form round and oval pseudopaxillae. Plates convex without ridges or tubercles. Abactinal surface  and disk margin (armpits)  and 4. AThe species was found on solid rock at the depths of 14\u201318 m at water temperature of 2\u20136\u00b0C.Henricia.The name is dedicated to AM Djakonov, the famous Russian (Soviet) echinoderm taxonomist who described several species in the genus Henricia djakonovi sp. n.is superficially very similar to sympatric H. pseudoleviusculaH. pseudoleviuscula is solid brown  the abactinal spines of H. djakonovi are with massive, smooth droplet-shaped tips,\u00a0while H. pseudoleviuscula has spines with the apices bearing 3\u201310 well discernible thorns  thear areas  and 4 ; e thorns  and 3E.Henricia djakonovi sp. n. and H. pseudoleviuscula can be easily distinguished from the other Henricia species by their spotted live coloration. The ventrolateral and marginal plates of H. reniossaH. pseudoleviuscula and H. djakonovi, although the type specimens of H. reniossa differ from Hayashi\u2019s images: their ventrolaterals, infero- and superomarginals are cross-shaped. Also, the abactinal spines of H. reniossa type specimens are different being club-shaped and bearing numerous long thorns. Few additional intermarginal rows are reported for H. reniossa asiaticaH.\u00a0djakonovi and H. pseudoleviuscula are subquadratic, do not possess a ridge, while in H. reniossa these plates are transversally elongated possessing a very distinctive ridge.Henricia specimens examined in the field in Vostok and Rudnaya Bays were unequivocally identified as H. pseudoleviuscula by their solid abactinal disk coloration, thorny spines, lack of papulae in the center of the disk, and single short intermarginal row. At least 10 individuals of H. djakonovi were found in the wild and from underwater images (In total, 243 spotted r images  on Senki"}
+{"text": "Klebsiella pneumoniae on a neonatal intensive care unit in a university hospital in Germany. This transmission occurred even though intensified infection control measures were in place, which impressively shows the importance of surveillance, outbreak management, and awareness of contributing factors regarding outbreak situations.Isolation precautions required for neonatal intensive care units are part of a bundle with the aim to prevent transmission, colonization, and infection with multidrug-resistant gram-negative pathogens as neonates face an increased risk of mortality and morbidity in case of infection. The following short report describes a transmission of 3MDRGN  Enterobacteriaceae. German standards go a step further and additionally recommend the isolation of patients with extended spectrum beta-lactamases- (ESBL-) producing Enterobacteriaceae in the neonatal ICU. Recommendations of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) for the general patient population are stratified according to species. They advise for single-room allocation only for Klebsiella ssp. harbouring ESBL genes but not for Escherichia coli containing the genes. This difference is based on the presumed different transmission potential on the species level. However, evidence is only limited and indirect.Detection of multidrug-resistant gram-negative pathogens (MDRGN) in prematurely and maturely born infants with the need of intensive medical care causes major consequences. In case of infection, broad spectrum antibiotics are needed for treatment. Infections due to MDRGN are associated with worse outcomes compared to infections due to susceptible isolates. Isolation precautions required for neonatal intensive care units (NICU) are part of a bundle with the aim to prevent transmission, colonization, and infection with MDRGN , 2. Due In Germany, the following recommendations are given for neonatal ICUs .3MDRGN is defined as a gram-negative pathogen resistant to three of the following four different classes of bactericidal antibiotics in vitro: broad spectrum penicillins, third or fourth generation cephalosporines, carbapenems (in neonates meropenem), and fluoroquinolones. 4MDRGN is a pathogen with in vitro resistance to all of the abovementioned antibiotics. As fluoroquinolones are not empirically used in neonates, the definition of 2MDRGN, a gram-negative pathogen resistant to cephalosporines and broad-spectrum penicillins, is particularly important for neonatal ICUs.In this context, current recommendations of the German Commission on Hospital Hygiene and Infection Prevention (KRINKO) at the Robert Koch Institute (RKI), Berlin, focus on a weekly screening for MDRGN , 3. The The combined neonatal-pediatric 20-bed ICU has the resources to care for ten extremely premature infants and ten pediatric patients with cardiac diseases.Klebsiella pneumoniae and 3MDRGN Escherichia coli were detected exclusively in a rectal swab without clinical symptoms. Therefore, the infant remained in single-room isolation (2015-11-19 to 2016-01-09). Being in a difficult general condition, the colonization evolved to an infection, and the infant needed specific antibiotic treatment.An eight months old female refugee, who had recently arrived from Iran travelling via Turkey and Greece, was admitted to our combined neonatal-pediatric ICU in November 2015. The infant suffered from a severe cardiac malformation, but previous contact to the healthcare system had been denied several times. The patient was isolated and screened for resistant bacteria at admission in accordance with the internal UMG guidelines. 3MDRGN K. pneumoniae in another infant with an identical antibiotic susceptibility profile within the weekly neonatal screening, but no E. coli. The infant shared an adjacent room with three more patients , swabs from surroundings in every patient room , and surroundings in parent rooms, kitchen, and laboratory. Extensive contact precautions for all patients on the ward, consequent isolation and cohorting of the four colonized patients, and all direct contacts were determined. Common areas as for the nursing and parent rooms were shut down, and conversations with parents and relatives in detail were accomplished. Furthermore, interdisciplinary rounds on the ward were arranged daily and documented in detail. Members of the executive board also were involved. All procedures were maintained until the end of January 2016.The swabs of the environmental screening were cultured in Caso-Bouillon (37\u00b0C/24 hours) and subcultured on nutrient agar plates, followed by differentiation and identification regarding morphological and biochemical characteristics.The patient screening samples of the weekly screening for MDRGN were examined regarding the minimum inhibitory concentration (MIC) as part of determination of resistance via Vitek-MIC.Klebsiella pneumoniae isolates were recovered: patients number 2 and 3  [Two of five 3MDRGN Germany) .Klebsiella pneumoniae occurred after a period of four weeks.Despite preemptive isolation and infection control measures and according to advanced infection control strategies, a transmission of 3MDRGN Staphylococcus epidermidis and aerobic spores could be detected. Many samples even turned out to be sterile. Microbiological results concerning medical devices and equipment in other rooms  also turned out to be sterile or colonized with pathogens without relevance concerning infection control. The frequent exploration of inanimate surfaces detected Enterococcus faecalis on one thermometer. 3MDRGN Klebsiella pneumoniae or 3MDRGN Escherichia coli was not found, neither in the bedroom cohorting the four patients colonized nor in adjacent rooms. A point source could not be identified by investigating the samples. None of all patients developed an infection with 3MDRGN Klebsiella pneumoniae except the index patient.All environmental samples turned out to be negative for facultative pathogens except the swabs taken from the nearest surrounding in the index patient room. On devices like stethoscope, tape measure, several buttons, or xylocaine gel, only Klebsiella pneumoniae isolates  indicated the similarity of the 3MDRGN isolates .All five patient samples turned out to be positive for ESBL (Vitec-MIC). Development of gentamicin-resistance often appears quickly due to the fact that ampicillin/gentamicin is a first-line antibiotic in pediatric therapy.Klebsiella pneumoniae isolates available (Isolate 2 and 3, corresponding to patient 2 and 3, All 3MDRGN The retrospective analysis revealed an emergency situation that had happened on the NICU affecting the two patient rooms only a few days before detection of the 3MDRGN colonization\u2014the same nursing staff had been involved in both of the rooms. Our hypothesis was a singular transmission during this situation.Further consequences of the outbreak management were the closure of beds and the postponement of elective admissions on the combined neonatal-pediatric ICU as well as on the adjacent neonatal standard care unit.Advanced infection control strategies were kept until the end of January 2016. Altogether 199 bed days at the neonatal-pediatric ICU and 122 bed days at the neonatal standard care unit were lost.K. pneumoniae but not of 3MDRGN E. coli occurred. This is in accordance with recent findings that K. pneumoniae is more transmissible than E. coli [Despite the implementation of intensified comprehensive infection control measures, beyond official requirements, transmissions of MDRGN cannot be completely avoided in clinical settings. In this case, it is remarkable that a transmission of 3MDRGN  E. coli \u201311.It becomes clear that proactive hygiene interventions influence the daily routine care on the NICU permanently. These interventions include strict isolation regimes for patients with colonization or infection as well as patients with contact to the index patients. Nevertheless, it is essential to remember basic infection control measures and to observe compliance of hand hygiene, contact precautions, and take care of staff training and instruction .E. coli and K. pneumoniae than nonrefugee pediatric patients, as seen in the described case and in the data analyzed at our university hospital [Pediatric refugee patients have a considerably higher prevalence of colonization with MRSA as well as cefotaxime and/or ceftazidime-resistant hospital . Screenihospital , 13, 14."}
+{"text": "Editorial on the Research TopicSecond Line Treatment of Non-Small Cell Lung Cancer: Clinical, Pathological and Molecular Aspects of Novel Promising DrugsLazzari et al.; Sullivan and Planchard; Tran and Klempner; K\u00f6hler). In addition, the introduction of immunotherapy, with anti PD-1 Pembrolizumab, in first-line treatment of NSCLC represents the best choice for EGFR, ALK, and ROS1 wild-type patients expressing PD-L1 on \u226550% of neoplastic cells . Despite the survival improvement achieved with these new therapeutic options in first-line treatment, about 30% of patients do not obtain a tumor response . Moreover, those patients, initially sensitive to these treatments, acquire resistance and develop tumor progression. Approximately 60% of the patients progressing from first-line therapy receiving further systemic treatment in the second-line setting  Also in second line, the armamentarium for the treatment of patients with NSCLC, includes a pletora of new drugs, such as immune checkpoint inhibitors  , third generation tyrosine kinase inhibitors (Osimertinib) , and anti-angiogenic agents (Nintedanib and Ramucirumab) .The advent of precision medicine and predictive molecular pathology led to a revolution in clinical management of patients with non-small cell lung cancer. The discovery of oncogene addiction allowed the development of targeted therapies that represent newer therapeutic options reserved to those patients harboring specific gene alterations, such as EGFR mutations, ALK, and ROS1 translocations . Probably the right way is to give all the available opportunities to patients, but challenges and pitfalls should be carefully debated.This exciting therapeutic scenario for NSCLC patients still has unsolved questions and challenging issues, in particular regarding the optimal selection of the patient population through the individualization of the correct methodology and biological source of material (tissues vs liquid biopsy) for clinical relevant biomarkers assessment (Taken together, the papers published in Research Topic \u201cSecond Line Treatment of Non-Small Cell Lung Cancer: Clinical, Pathological and Molecular Aspects of Novel Promising Drugs\u201d represent a critical discussion focused on the older therapies and the historical development of second line, putting into perspective the new agents available in clinical practice, defining their importance from a clinical point of view, but also to consider and exploit the complex molecular mechanisms responsible of their efficacy or of the subsequently observed resistance phenomena, to support the oncologist to design the best therapeutic strategies for NSCLC patients.UM and PP contributed equally to this paper.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": "Systematic reviews and meta-analyses are methodologically rigorous studies that are said to form the reference standard for summarising evidence to guide health care. Reporting quality of reviews is of critical importance in order to judge the quality and risk of bias in a review to ensure sound healthcare decisions are made. This is particularly important in the field of dermatology due to the growing number of systematic reviews and their key role in informing healthcare decision within dermatology. A contemporary and comprehensive review of the compliance of dermatology systematic reviews and meta-analyses with the PRISMA checklist, in the highest impact factor dermatology journals, has not yet been assessed. To our knowledge, our review represents the most extensive study assessing reporting quality of systematic reviews and meta-analyses published within dermatology to date.Our protocol is reported in line with the Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 guidelines. MEDLINE will be searched to look for systematic reviews and meta-analysis in selected years within the top four highest impact factor dermatology journals in 2017. Records and full texts will be screened independently by five researchers. Data will be extracted onto a standard data extraction database. A training session will take place to ensure accurate data extraction and scoring of studies with the PRISMA checklist. The data will be analysed and outcomes will be determined. Primary outcome will be the compliance of reviews with the PRISMA checklist. Whilst reporting quality and study quality are not the same, a poorly reported study is of limited value since it is difficult to make a full and transparent judgment of its utility without all of the necessary information Given the increase in systematic reviews within dermatology it is important, perhaps now more than ever, to ensure reviews are adequately reported 2Systematic reviews and meta-analyses form a critical part of dermatological research since they form the reference standard for summarising evidence to guide decisions within clinical dermatology, whilst also minimising bias It has previously been reported that systematic reviews and meta-analyses within dermatology were less likely to evaluate publication bias The compliance of dermatology systematic reviews and meta-analyses across all items of the checklist and in the highest impact factor journals is yet to be assessed. To our knowledge, our review will represent the most extensive study assessing reporting quality of systematic reviews and meta-analyses published within dermatology to date.33.1This systematic review will assess the compliance of systematic reviews and meta-analyses in leading dermatology journal with the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) Statement.3.2To assess whether compliance of PRISMA guidelines improves over time and whether this correlates with mandatory enforcement of PRISMA reporting or appointment of a dedicated systematic review editor. To assess: the variety of sub-topics reviewed, if reviews are registered, if protocols exist and if there is a difference in PRISMA compliance between Cochrane vs non-Cochrane reviews, to rank in order of compliance the items of the checklist across all studies and to assess if certain items are consistently poorly reported. Lastly to assess if there is an assessment of publication bias across systematic reviews as well as within its primary studies.3.3It is hypothesised that systematic reviews in leading dermatology journals are fully compliant with the PRISMA checklist. In addition to this, it is hypothesised that systematic reviews published more recently have higher compliance with the checklist.4a priori with the international prospective register of systematic reviews, Research Registry (review number: reviewregistry597) This protocol is in line with the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols (PRISMA-P) 2015 statement A search technique similar to a systematic review assessing the PRISMA compliance in craniofacial surgery reviews will be used, in order to increase comparability with previous similar work 4.1Inclusion criteria will include: systematic reviews and meta-analyses within dermatology, reviews only published within the top four highest impact factor journals as of 2017, reviews published in the years 2016/17, 2011/12 and 2006/07 and English language studies. The selected dates were chosen since 2016/17 is the most contemporaneous two-year period and five-year periods proceeding these years were chosen to allow for comparators.Exclusion criteria will include: articles that are not systematic reviews and meta-analyses, articles outside of the dates and journals previously mentioned, historical reviews, narrative literature reviews, grey literature or unpublished reviews.4.2https://jcr.incites.thomsonreuters.com, Thomas Reuters, New York, US) In order to locate the top four highest impact factor dermatology journals as of 2017, the Thomson Reuters InCites Journal Citation Reports was utilised  are PubMed indexed.4.3The search strategy aims to collect all systematic reviews and meta-analyses within the selected journals during the years aforementioned. The strategy will be developed in line with an information search specialist. Search strategy will include the use of Boolean logical operators to improve sensitivity of the search. The search terms will be \u201csystematic review\u201d OR \u201cmeta-analysis\u201d OR \u201cmeta-analyses\u201d AND \u201cJOURNAL NAME\u201d.An example full search strategy conducted on 1/8/18 includes:  AND \u201csystematic review\u201d[All Fields] AND (\u201c2006/01/01\u201d[PDAT]: \u201c2006/12/31\u201d[PDAT]).4.4There will be two stages to the screening and identification of study records. Firstly, five independent researchers will screen the titles and abstracts against inclusion and exclusion criteria. In the second stage of screening, full text will be assessed for inclusion and exclusion criteria. Any discrepancies between articles will be resolved by discussion or senior author decision (BG). Reasons for exclusion will be noted. Articles that have passed both stages of screening will be included for data extraction. Data will then be extracted onto a standard extraction database created on Google Forms . Duplicates will be removed before extraction.A training session will take place before data extraction focussing on accurate marking of included studies against the PRISMA guidelines. This training session will be conducted by a senior researcher and involve \u201cpractice\u201d marking of dermatology systematic reviews with the PRISMA checklist with any discrepancies within researchers being fed back to the team and discussed in training.4.5The following data items will be extracted from the articles: compliance of each article with the twenty-seven item PRISMA checklist, study authors, date of publication, journal name, dermatology sub-topic assessed by the review, country where review took place as assessed by first author, commercial or non-commercial funding, if a protocol exists, if it is a Cochrane review, if the review is registered. Overall outcome of the review will also be assessed with regard to whether the review showed a positive, equivocal or negative outcome with respect to the intervention being studied. Dermatology sub-topics will be divided using the Cochrane Skin Group titles categorised by the British Association of Dermatologists (BAD) diagnostic index 4.6Articles will be scored for compliance with the twenty-seven item PRISMA checklist. A score of one will be given for an article that meets all the criteria for a particular item, a score of zero for those that do not meet or partially meet the item requirements and not-applicable (N/A) if there are further concerns or if the particular item is not relevant to the article. For some studies, not all twenty-seven items of the checklist will be relevant in which case the maximum PRISMA score of an article will be calculated by subtracting the number of irrelevant items by twenty-seven. A compliance score expressed as a percentage will be calculated for each article.4.7Primary outcome will be compliance with the PRISMA checklist. Secondary outcomes assessed will be to assess whether compliance of PRISMA guidelines improves over time and whether this correlates with mandatory enforcement of PRISMA reporting or appointment of a dedicated systematic review editor. Other outcomes will be to describe sub-topics reviewed, to assess if reviews are registered, if protocols exist and if there is a difference in PRISMA compliance between Cochrane vs non-Cochrane reviews, to rank in order of compliance the items of the checklist across all studies and to assess if certain items are consistently poorly reported. Lastly to assess if there is an assessment of publication bias across systematic reviews as well as within its primary studies.4.8This will be assessed as to whether a systematic review or meta-analysis is compliant with PRISMA checklist items pertaining to bias, which is as follows: assessment of risk of bias in individual studies, assessment of risk of bias across studies, presenting data on the risk of bias within studies, presenting data on the risk of bias across studies.4.9Data will be analysed and assessed using Microsoft Excel . Continuous variables will be presented by their mean and ranges and categorical variables will be presented as percentages. The PRISMA compliance will be expressed as a percentage for each study. A preliminary pilot search conducted on 1/8/18 has identified 233 reviews and meta-analyses for screening.4.10a priori registration status. There will also be analysis of the checklist items that are consistently most and least compliant with ranking. No meta-regression or sensitivity analyses will be planned.The PRISMA score across all articles will be compared based on various factors such as: journal impact factor, year published, sub-topics, presence of a protocol, 4.11The authors aim to disseminate the results as widely as possible. The systematic review will have published in a peer-reviewed journal and will be presented in a broad range of national and international conferences. We will also be carrying out this review in line with PRISMA-2015 statement. This review will be carried out in line with Cochrane Handbook for Systematic Reviews and Interventions and will be compliant with PRISMA guidelines.5By conducting this systematic review, we aim to clarify deficiencies in systematic review reporting, contribute to improvement of methodology and therefore transparency of reporting within dermatology systematic reviews and meta-analyses. As a result, we hope this will ultimately ensure good clinical practice and patient care.Not applicable.The authors declare no commercial or other sources of funding for this review.BG and RA conceived idea for the study. BG drafted the manuscript. BG, RA and AJF authors critically revised the manuscript for intellectual content and approved the final version for publication. BG is guarantor for the review.None declared. No funding or sponsorship received for this study.BG is guarantor for the review.reviewregistry597."}
+{"text": "Of note, epigenetic changes are increasingly being reported to play an outstanding role in carrying deleterious information that, together with susceptibility genes, boost the development of metabesity in subsequent generations. In this context, it is noteworthy to mention that the transition from the pre-industrial era to the current high-technology society and global economy, even after suffering two world wars, has been very fast. By contrast, evolution-driven processes, such as biological ones, are slow. In fact, there is a general consensus that at the metabolic level, adipogenic processes and thrifty pathways prevail over those promoting energy expenditure in a way that currently leads to metabolic diseases by excessive energy storage. In such an imbalanced social\u2013biological scenario, genes that were beneficial in the past have shifted to becoming detrimental, i.e., favouring metabesity, which is quickly growing to reach pandemic proportions. Metabesity includes but is not limited to obesity (currently not considered a disease in most countries), cardiovascular disease, diabetes and metabolic syndrome. From a pathophysiological point of view, its progression comprises inflammatory and oxidative damage, insensitivity to key regulators, e.g., hormones like insulin and leptin, cell death and overload of the natural regeneration capacity of tissues. While different approaches aiming at tackling metabesity are being explored, regenerative medicine remains a promising one, though facing important obstacles.This Special Issue contains a series of new reviews and original research articles providing advances in this exciting field, highlighting some genes and processes that show potential in contributing to the development of new approaches for tackling metabesity. One such process is epigenetic regulation which, in the context of pancreatic regeneration in diabetes, is concisely but at the same time accurately reviewed by Dr. Collombat and co-workers . CurrentRegarding this latter factor, HMG20A, emerging evidence shows its involvement in neuronal and beta cell mature function . It is nIn addition, reflecting even more the current abundance of epigenetic studies in metabesity, Dr. Tinahones and collaborators here describe a differential methylation pattern of complement factor C3 in adipocytes from class 3 obese patients . AccordiOther interesting and emerging targets in metabesity are glucose regulated protein 78kD (GRP78), a chaperone known to reduce ER stress upstream of unfolded protein response (UPR) by improving protein folding. Dr. Lopez and co-workers show in this Special Issue that GRP78 overexpression in the ventromedial hypothalamus (a key brain area regulating thermogenesis) of rats fed a very high-fat diet decreases body weight, insulin resistance and hepatic steatosis, while increasing thermogenesis in brown adipose tissue and browning in white adipose tissue [As can also be inferred from this Special Issue ,7, the h"}
+{"text": "Vmax and reducing its Km, probably due to the elimination of uremic toxins during dialysis. At least 75% of alkaline phosphatase activity in human plasma was found to depend on a levamisole-sensitive enzyme probably corresponding to tissue non-specific alkaline phosphatase (TNAP). Dialysis increased total plasma protein concentration by 14% and reduced TNAP enzyme by 20%, resulting in an underestimation of pyrophosphate hydrolysis in post-dialysis plasma. Levamisole inhibited TNAP activity , reducing pyrophosphate hydrolysis in plasma and increasing plasma pyrophosphate availability. Alkaline phosphatase is also found in many tissues and cells types; therefore, our results in plasma may be indicative of changes in phosphatase activity in other locations that collectively could contribute significantly to pyrophosphate hydrolysis in vivo. In conclusion, these findings demonstrate that dialysis increases pyrophosphate hydrolysis, which, taken together with previously reported increases in alkalization and calcium ion levels in post-dialysis plasma, causes VC and could be prevented by adding calcification inhibitors during dialysis.Vascular calcification (VC) is associated with significant morbidity and mortality of dialysis patients. Previous studies showed an association between loss of plasma pyrophosphate and VC. Moreover, loss of pyrophosphate occurs during dialysis in this population, suggesting that therapeutic approaches that prevent reduction of plasma pyrophosphate levels during dialysis could improve the quality of life of dialysis patients. This study found that pyrophosphate hydrolysis was 51% higher in post- than pre-dialysis plasma. Dialysis sessions modified the kinetic behavior of alkaline phosphatase, increasing its  Hyperphosphatemia is a typical clinical manifestation in these patients. Increases in plasma phosphate levels have been associated with the prevalence of calcification2 due to the spontaneous formation of calcium-phosphate crystals3. Moreover, calcification has been found to contribute to the substantial morbidity and mortality rates in this patient population5.Vascular calcification is a common complication in hemodialysis patients and is associated with cardiovascular events and all-cause mortality7. The first involves a profound transition to a bone-forming phenotype, that results in the loss of vascular smooth muscle cells markers and the expression of osteochondrogenic markers9. The second consequence invokes apoptosis-dependent matrix mineralization, which has been detected both in cultured humans vascular smooth muscle cells11 and in arteries from pediatric dialysis patients12.There are two major consequences regarding the fate of vascular smooth muscle cells in phosphate-induced calcificationin vitro15 and in vivo19. Reductions in plasma pyrophosphate concentrations have been associated with vascular calcification20. Pyrophosphate is generated enzymatically via the hydrolysis of extracellular ATP by the enzyme ectonucleotide pyrophosphatase/phosphodiesterase (eNPP)21, and pyrophosphate is degraded to inorganic phosphate (Pi) mainly by tissue non-specific alkaline phosphatase (TNAP)22. Overexpression of TNAP in vascular smooth muscle cells is sufficient to cause ex vivo calcification in aortic rings22, and murine models with increased expression and activity of TNAP have demonstrated excessive vascular calcification23.Pyrophosphate is the main endogenous inhibitor of calcium-phosphate crystal formation and growth 25 may be due to increases in phosphatase activity25. To expand on these findings, this study analyzed the kinetic behavior of alkaline phosphatase activity in plasma from hemodialysis patients, the effect of dialysis on pyrophosphate hydrolysis, and the effect of alkaline phosphatase inhibition on pyrophosphate availability.Reductions in plasma pyrophosphate levels after dialysismax S)/(Km\u2009+\u2009S), where V is the velocity of pNPP hydrolysis, Vmax is the maximal velocity or capacity of pNPP hydrolysis, S is the concentration of pNPP and Km is the affinity constant. Analysis of the enzyme kinetics of plasma alkaline phosphatase showed that its Vmax was \u223c40% higher  and its apparent Km was significantly lower  after than before dialysis  hydrolysis in 40 pairs of samples were fitted to a Michaelis-Menten equation, V\u2009=\u2009 of 611.9\u2009\u00b5mol/L pyrophosphate  released from the hydrolysis of 32-pyrophosphate (32PPi) in plasma. 32Pi and 32PPi were separated by chromatography on PEI-cellulose plates and counted by liquid scintillation. 32PPi hydrolysis in plasma was linear over 8\u2009hours  and 560 fmol * hour\u22121 * \u00b5L\u22121 .Pyrophosphate hydrolysis was quantified as 32-phosphate  that in presence of phosphate (1.53\u2009\u00b1\u20090.17 pmol * mg\u22121 * min\u22121).Aortas were obtained from euthanized rats and digested over 10\u2009min in order to remove the adventitia layer. Then, pyrophosphate hydrolysis assay in absence of phosphate was first performed. Then, after washing five times in MEM media without phosphate, the same aortic rings were used for pyrophosphate hydrolysis assay in presence of 1\u2009mmol/L phosphate. Pyrophosphate hydrolysis was found to be 3.7-fold higher Fig.\u00a0 in absen25, have been associated with vascular calcification20. Because vascular calcification is the main clinical adverse effect in dialysis patients, largely determining their morbidity and mortality rates, further exploration of these findings may improve patient quality of life. This study showed that the reduction in plasma pyrophosphate levels following dialysis could be probably due to an increase in pyrophosphate hydrolysis. The ~51% increase in pyrophosphate hydrolysis was due primarily to increases in plasma alkaline phosphatase activity following dialysis. We found that the Vmax of this enzyme increased by \u223c40%, while its Km decreased by \u223c40%, from before to after dialysis. These findings are compatible with the presence of both competitive and non-competitive inhibitors, which are removed from plasma during dialysis. For example, the elimination of phosphate from plasma during dialysis25 may explain, at least in part, the increase in levamisol-sensitive alkaline phosphatase activity. Moreover, since alkaline phosphatase is found in many tissues and cells types (anchored in the cell membrane), pyrophosphate hydrolysis in isolated plasma is much less than in vivo. However, our results in plasma may be indicative of changes in phosphatase activity in other locations that collectively could contribute significantly to pyrophosphate hydrolysis in vivo. This could also explain the associated up-regulation of TNAP enzyme in uremic aorta shown in previously studies16, as a compensatory mechanism to improve the loss of hydrolysis capacity due to the phosphatase inhibition with uremic toxins (mainly phosphate).Reductions in plasma pyrophosphate levels, which occur following hemodialysisInteresting, high levels of plasma alkaline phosphatase are also associated with mortality in all stages of chronic kidney diseases. Our study revealed an increase in alkaline phosphatase activity in post-dialysis plasma. Therefore, this hidden consequence of hemodialysis increases our knowledge of the factors contributing to mortality in this population.22, had an IC50 value of 7.2\u2009\u00b5mol/L, with a concentration of 100\u2009\u00b5mol/L completely inhibiting alkaline phosphatase activity in human plasma. Interestingly, 75% of alkaline phosphatase activity in plasma is provided by a levamisole-sensitive enzyme, probably TNAP22. Although levamisole has been used as an anthelmintic treatment agent in humans, it has been replaced by more effective treatments. Levamisole could be used to prevent excessive pyrophosphate hydrolysis during dialysis sessions while developing more effective TNAP inhibitors. We found that the addition of levamisole to post-dialysis plasma reduced the hydrolysis of pyrophosphate, thereby increasing its availability.We also found that levamisole, an inhibitor of TNAPAlthough we found that the increased pyrophosphate hydrolysis in post-dialysis plasma was associated with an increase in phosphatase activity, the plasma concentration of TNAP was \u223c20% lower after than before dialysis. These findings suggest that ~51% higher pyrophosphate hydrolysis in plasma after than before dialysis is an underestimate. During dialysis, low molecular weight proteins are lost (<60\u2009kDa). These may include TNAP, with a molecular weight of \u223c53\u2009kDa, suggesting that the lower TNAP concentration after than before dialysis session may be the consequence of diffusion during dialysis.20, the findings of this study indicate that a loss of ability to prevent calcification plays a predominant role during this pathological process26. Vascular calcification is therefore associated with reductions in plasma pyrophosphate levels25 and increases in alkalization27 and calcium concentrations following dialysis. Supplementation with exogenous anticalcifying agents, such as pyrophosphate and TNAP inhibitors, may therefore inhibit or prevent dialysis-associated calcification.In conclusion, this study showed that pyrophosphate hydrolysis in plasma is greater after than before dialysis, despite the reduction in the level of TNAP protein, the main phosphatase in human plasma. Moreover, reduction in pyrophosphate levels is also be influenced by increments in tissue/cell TNAP activity. Because reductions in plasma pyrophosphate levels are associated with vascular calcification2). The dialysate was composed of 1.5\u2009mmol/L calcium, 35\u2009mmol/L bicarbonate, 0.75\u2009mmol/L potassium, 0.5\u2009mmol/L magnesium, and 140\u2009mmol/L sodium.Each patient underwent a conventional, purely diffusive 4\u2009hour (mid-week) hemodialysis session without hemodiafiltration, using a high flux helixone dialyzer  at final concentrations of 5\u2009\u00b5mol/L and 10\u2009\u00b5Ci/mL, respectively. After 4\u2009hours, the samples were chromatographed on PEI-cellulose plates , which were developed with 650\u2009mmol/L K2HPO4  pH 3, as described22. After radiography, the spots containing phosphate and pyrophosphate were removed and added to liquid scintillation fluid . Radioactivity was measured using the liquid scintillation analyzer Tri-Carb 2810TR (Perkin Elmer).Plasma samples (5\u2009\u00b5L) were incubated in 15\u2009\u00b5L Molecular Biology Water  containing pyrophosphate  and , where Top refers to the velocity of pNPP hydrolysis in the absence of inhibitor (levamisole), and Bottom refers to the maximal inhibition.The mean inhibitory concentration  was then calculated indirectly using the following equation: Ki\u2009=\u2009IC50 /[1\u2009+\u2009(S/Km)], were S is the constant concentration of pNPP (0.250\u2009mmol/L) and Km is the affinity constant of pNPP (0.082\u2009mmol/L).The inhibition constant, KMale Sprague-Dawley rats (8\u201312 weeks) were obtained from Charles River Laboratories (France). The protocol was approved by ethics committees both the FIIS-FJD  and Madrid Community (PROEX 427/15); and conformed to directive 2010/63EU and recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under RD1201/2005.15Then, the pyrophosphate hydrolysis assay was performed.Rats were euthanized via carbon dioxide inhalation and thoracic aorta tissue was perfused with saline and removed according to previously published protocolsex vivo in Minimum Essential Medium Eagle . To remove adventitia layer, rat aortas were digested for 10\u2009min with collagenase, as previously described28. Then, medial layer of the aortic rings were incubated ex vivo in MEM media containing 5\u2009\u00b5mol/L pyrophosphate and 32-pyrophosphate (32PPi) as a radiotracer. After the indicated time of incubation, ortophosphate was separated from pyrophosphate, as previously described15. Briefly, 20\u2009\u03bcL of sample was mixed with 400\u2009\u03bcL of ammonium molybdate  and 0.75\u2009mol/L sulphuric acid . Samples were then extracted with 800\u2009\u03bcL of isobutanol/petroleum ether (4:1) to separate the phosphomolybdate from the pyrophosphate . Next, 400\u2009\u03bcL of the organic phase containing phosphomolybdate was removed and subjected to radioactivity counting.For pyrophosphate hydrolysis experiment Fig.\u00a0, aortic 2PO4/K2HPO4 pH 7.4). Finally, the aortic rings were dried and weighed.In experiments shown in Fig.\u00a0Results are presented as mean\u2009\u00b1\u2009standard error of the mean (SEM), and were compared by the Wilcoxon matched pairs test. Statistical significance was determined using GraphPad Prism 5 software.\u00a0Supplementary Information"}
+{"text": "In this work, molecular modeling studies combining molecular docking, 3D-QSAR, MESP, MD simulations and free energy calculations were performed on pyridine amides and 1,2,4-triazolopyridines as 11\u03b2-HSD1 inhibitors to explore structure-activity relationships and structural requirement for the inhibitory activity. 3D-QSAR models, including CoMFA and CoMSIA, were developed from the conformations obtained by docking strategy. The derived pharmacophoric features were further supported by MESP and Mulliken charge analyses using density functional theory. In addition, MD simulations and free energy calculations were employed to determine the detailed binding process and to compare the binding modes of inhibitors with different bioactivities. The binding free energies calculated by MM/PBSA showed a good correlation with the experimental biological activities. Free energy analyses and per-residue energy decomposition indicated the van der Waals interaction would be the major driving force for the interactions between an inhibitor and 11\u03b2-HSD1. These unified results may provide that hydrogen bond interactions with Ser170 and Tyr183 are favorable for enhancing activity. Thr124, Ser170, Tyr177, Tyr183, Val227, and Val231 are the key amino acid residues in the binding pocket. The obtained results are expected to be valuable for the rational design of novel potent 11\u03b2-HSD1 inhibitors. This isozyme is mainly located in classical mineralocorticoid target tissues, such as salivary glands, kidney and colon, and its function is to protect the mineralocorticoid receptor from activation by cortisol [Obesity, diabetes mellitus and other metabolic syndrome manifestations are primary causes of morbidity and mortality around the world . The metcortisol ,12. Althcortisol . These fcortisol . These ocortisol ,16.A number of small molecular inhibitors of 11\u03b2-HSD1 have been disclosed in the past few years ,18,19, aAs a useful technology and tool for drug design, computer-aided drug design methods have been applied to discover and design new 11\u03b2-HSD1 inhibitors ,25,26,2750 values were converted into the corresponding pIC50 values and were used as dependent variables for subsequent 3D-QSAR analyses. The whole data set was divided into a training set of 31 compounds for 3D-QSAR model generation and a test set of 9 compounds for model validation, respectively, by considering both distribution of biological data and structural diversity. As shown in A total of 40 11\u03b2-HSD1 inhibitors, including pyridine amides and 1,2,4-triazolopyridines analogs, were collected from literatures reported by the same research group ,29 to be1 was retrieved from RCSB Protein Data Bank (http://www.rcsb.org/pdb/). 3D structures of all other compounds in both training and test sets were generated by modifying corresponding functional groups of 1 with the SKETCH module of Sybyl-X1.3 molecular modeling software package [The co-crystal structure (PDB ID: 3CH6 ) of 11\u03b2- package . Subsequ1 engaged in the co-crystal structure was used as a starting position to define the potential binding site. Each compound was then docked into the idealized active site with a \u201cwhole\u201d molecular alignment algorism [To explore the binding conformation of the above compound in the active site of 11\u03b2-HSD1, flexible docking simulations were performed using the Surflex-Dock program in Sybyl-X1.3 . The prialgorism  and the algorism . Herein,algorism , the consp3 hybridized carbon atom probe with a charge of +1.0 and van der Waals radius of 1.53 \u00c5. The cut-off value was set to the default value of 30.0 kcal/mol. For CoMSIA analyses, by using a common probe with a charge of +1.0, five similarity indices consisting of steric (S), electrostatic (E), hydrophobic (H), H-bond donor (D), and H-bond acceptor (A) fields were calculated for each lattice with a grid of 2 \u00c5. A Gaussian method was used to evaluate the mutual distance between the probe atom and each molecule atom in CoMSIA model generation. The attenuation factor was set to the default value of 0.3.Both comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods were applied for the development of 3D-QSAR models using the QSAR module of Sybyl-X1.3. To generate CoMFA model, steric and electrostatic fields were calculated at each point of regularly spaced grids of 2.0 \u00c5 using an 50) served as target variables. A minimum column filter value (\u03c3) of 2.0 kcal/mol was set to reduce noise and speed up the analysis. Leave-one-out (LOO) algorithm was adopted in the cross-validation analysis to obtain an optimal number of components (ONC), the lowest standard error of prediction and cross-validated correlation coefficient (q2). Then, the obtained ONC was used to calculate the non-cross-validated correlation coefficient (r2) of the produced PLS model. In addition, the statistical significance of the generated models was described by the standard error of estimate (SEE) and F probability value . The predictive capability of the 3D-QSAR models was further evaluated with the test set of molecules. The predictive correlation (r2pred) based on the test set molecules was computed using Equation (1) below:Partial least squares (PLS) ,36 metho1, 14, 32, and 39 were calculated to explain bioactivities and molecular properties using ab initio method. The docking-predicted conformation of each compound was used as an input conformer for density functional theory (DFT) calculations using Gaussian 09 program package [3). The overall molecular size and the distribution of positive or negative electrostatic potential were indicated by color coded isosurface values. The most negative and positive electrostatic potentials are colored deep red and blue, respectively, and the intermediated shades colored with cyan, yellow, and green indicate the moderate range of reactivity.Molecular electrostatic potential (MESP) is a useful feature in understanding the molecular electronic structure, chemical reactivity of the molecule, and structure-activity relationship studies . In the  package . Complet package . In orde package . MESP an package . In deta1, 11\u03b2-HSD1-11, and 11\u03b2-HSD1-14 were used as initial structures for MD computations, respectively. The force field parameters for each inhibitor were generated by the general AMBER force field (GAFF) using ANTECHAMBER program [ff03.r1 force field was applied to obtain force field parameters for protein and water molecules [+ or Cl\u2212 ions). Each complex was solvated in a truncated octahedron box of TIP3P water molecules [2). Secondly, residue side chains of protein were relaxed while backbone heavy atoms were restrained with a constraint force of 5 kcal/(mol\u2219\u00c52). Finally, the whole system was optimized without any restraint. In each step, structural optimization was implemented using 2500 steps of steepest descent followed by 5000 steps of conjugate gradient method.The MD simulations were performed to investigate different interaction modes of ligands with various bioactivities binding to 11\u03b2-HSD1 using the AMBER12 software package . The doc program , and the program  were use program . The staolecules . All misolecules . Consideolecules  with a m2) on the protein-ligand complex. Subsequently, the whole system was subjected to seven rounds of equilibrations in each time period of 1 ns at 300 K with a carefully decreasing restraint weights of 5, 3, 1, 0.5, 0.3, 0.1, and 0 kcal/(mol\u00b7\u00c52), respectively, without restriction on the solvation environment. Finally, 50 ns MD productions were run for each system in the NPT ensemble at 300 K with 1.0 atm pressure. During MD simulations, periodic boundary conditions were applied to avoid edge effects in all calculations. The particle mesh Ewald (PME) method was utilized to deal with the long-rang Coulombic interactions [\u22121 throughout the equilibration runs and with a collision frequency of 2.0 ps\u22121 throughout the production runs.After energy minimizations, each system was gradually heated in the NVT ensemble from 0 to 300 K over a period of 100 ps with a force constant of 10 kcal/:For each complex, 800 snapshots were extracted from the last 4 ns MD trajectories at 5 ps intervals to calculate the binding free energy using the parallelized python script  AMBER12 . For eacEinternal is omitted in the MD calculation.Since there is no covalent bond formed between the ligand and receptor, \u0394Gsol is the solvation free energy, which was composed of the polar and the nonpolar contributions:Gpol could be obtained by solving the Poisson-Boltzmann (PB) equation for MM/PBSA method [Gnonpol was estimated by:\u0394A method  or the GA method . Whereas2) was computed with a probe radius of 1.4 \u00c5 using the linear combination of pairwise overlaps (LCPO) method [2) and \u22121.008000 kcal/mol for PB calculations and 0.0072 kcal/(mol\u2219\u00c52) and 0 kcal/mol for GB calculations, respectively [The solvent accessible surface area  method . In thisectively .T\u0394S) was obtained from changes in the translational, rotational and vibrational degrees of freedom. It can be determined from the following equation:rij) to mimic the impact of solvent. In consideration of the high computational demand of this approach, only 40 snapshots taken from last 4 ns MD trajectories at 100 ps intervals were utilized to estimate the entropic contribution.The entropy ( AMBER12 . Prior tEvdW), electrostatic contribution (\u0394Eele), polar solvation contribution (\u0394Gpol), and nonpolar solvation contribution (\u0394Gnonpol). The vdW and electrostatic interactions between inhibitor and per-residue of 11\u03b2-HSD1 were calculated using the SANDER program of AMBER12. The polar contributions of the solvation free energy were computed by the GB model. The nonpolar contributions of the desolvation free energy were determined with SASA dependent terms. All energy decomposition analyses were performed on the basis of the same snapshots which were used in previous calculations.In order to investigate detailed binding modes between the protein and its inhibitors and to find key residues affecting the inhibitory activity, binding free energies were decomposed to each residue\u2032s contribution to ligand using MM/GBSA method. There were only molecular mechanic energies and solvation energies but not for entropies taken into consideration in this decomposition. The binding interaction of per inhibitor-residue includes four terms, van der Waals contribution  of 0.39 \u00c5, indicating the high reliability of Surflex-Dock in reproducing the experimental binding mode of 11\u03b2-HSD1 inhibitors. In this study, flexible docking calculations were performed on inhibitors of 11\u03b2-HSD1 to predict their potential binding conformations using the Surflex-Dock procedure. As shown in 1 in 11\u03b2-HSD1 is depicted in 1 may have strong parallel \u03c0-\u03c0 interactions with the side chain phenyl ring of Tyr177. Moreover, the central pyridine core is also parallel to the backbone of Gly216 and Leu217 with van der Waals interaction. The 3,3-dimethylpiperidinyl moiety is surrounded by the hydrophobic side chains of Ile121, Thr124, Tyr183, Ala223, Ala226, and Val227.The detailed binding mode of 5, 11, 14, and 39 were selected for detailed analyses of their binding mechanism on 11\u03b2-HSD1. The binding modes of these four compounds combined with the main residues of 11\u03b2-HSD1 are displayed in 5, 11, 14 and N1 and N2 atoms of 39 form hydrogen bonds with the side chain hydroxyl groups of residues Ser170 and Tyr183. Thus, these two residues may provide crucial hydrophilic interactions with an inhibitor binding to 11\u03b2-HSD1. In addition, as compared to the highly active compound 1, compound 5 contains a less hydrophobic 2-chlorophenyl moiety and a 4-methylpiperidinyl group resulting in a smaller hydrophobic space-filling in the binding pocket (5 has lower bioactivity than 1. As for compound 11 or 14 (2) between the central pyridine core and the 2-chlorophenyl moiety. Moreover, compound 14 with a strong electron withdrawing linkage (SO2) is detrimental to 11\u03b2-HSD1 potency. On the other hand, inactive compound 39 has a different binding mode in comparison to 1. Typically, the 2-methyl-4-chlorophenyl substituent of 39 approaches to a solvent exposed area of the binding site and is close to the hydrophobic residue Tyr177 with a weak T-shaped \u03c0-\u03c0 interaction. The N-acetylpiperidin-4-yl moiety is oriented to another side of the binding pocket, reducing the hydrophobic interactions with the side chain of Ile121 , 26.4% (E), 27.1% (H), 18.0% (D), and 18.1% (A), respectively. The electrostatic and hydrophobic contributions shared large parts for the inhibitory activity for 11\u03b2-HSD1 in the generated CoMSIA model. It was noticed that steric contribution in the CoMSIA model was much lower than that in the CoMFA model. As we know, the CoMFA method is restricted to electrostatic fields with Lennard-Jones and Coulomb potentials. This may introduce errors in scaling, alignment sensitivity, and interpretation of contours. However, the CoMSIA method has been developed to make usage of hydrophobic fields in addition to the electrostatic fields. It can improve these inherent deficiencies arising from the CoMFA method [50 values and residuals defined as the experimental minus the predicted pIC50 values are summarized in 50 values without deviation of more than one logarithmic unit. The correlations between the experimental and model-predicted bioactivities of all compounds are shown in r2pred of the CoMFA and CoMSIA models were 0.938 and 0.923, respectively. The high r2pred values indicate a good predictive ability of our generated 3D-QSAR models.The CoMSIA model was generated based on five fields, steric field (S), electrostatic field (E), hydrophobic field (H), H-bond donor (D), and H-bond acceptor (A), giving a cross-validated A method . Accordi1 was labeled on the map as a template for visualization to explore the structure-activity relationships of these 11\u03b2-HSD1 inhibitors. The contour analyses have been performed by dividing the total molecular area into three subdivisions  and 12 (\u2212O\u2212), the longer linkers of 9 (\u2212SCH2\u2212) and 10 (\u2212OCH2\u2212) may extend their phenyl ring B to the 5\u2032-position of 1\u2032s B-ring, making 9 and 10 more similar to 1 in shape. In fact, the B-ring of 1 was positioned at the edge of the binding pocket of 11\u03b2-HSD1 and extended to the solvent in the docking-simulated structural model of complex 11\u03b2-HSD1-1 , and two low activity compounds (14 and 39) selected from the docking study were analyzed based on MESPs, HOMO-LUMO parameters and dipole moments. In addition, Mulliken population analyses of studied inhibitors were also calculated to get more detailed insights at the electronic level.To understand surface electronic properties of 11\u03b2-HSD1 inhibitors and to comprehend the pharmacophoric features required for binding, two highly active compounds , which is also not in accordance with the large blue contour in the CoMSIA model. Thus, MESPs plotted over the low active compounds 14 and 39 indicated that their molecular electronic properties do not fit well with the required electrostatic features predicted by above generated 3D-QSAR models. As shown in 1, 32, 14, and 39, respectively. The distributions of HOMO and LUMO sites are distinct in the active and low active compounds. In the active compound 1, both HOMO and LUMO orbitals are overlapped significantly at A-ring and B-ring, while the carbonyl oxygen atom of amide and some positions of C-ring possess HOMO orbitals. Such extensive overlapping frontier orbitals indicated the highly reactive nature of the active compound. The HOMO orbital of 14 is located only at the carbonyl oxygen atom and C-ring. By comparing active 32 and low active 39, the TZP core possesses both HOMO and LUMO orbitals, whereas B-ring and the linker between A-ring and B-ring have the obviously different electronic features. The computed quantum chemical descriptors HOMO and LUMO values of these four molecules in both gas and solvation (water) phases are summarized in 1 and 32) show higher dipole moments than another two low active inhibitors (14 and 39), thus compounds 1 and 32 have much stronger hydrophobic interactions with the binding pocket than those of 14 and 39.The frontier orbitals HOMO and LUMO, which are quantum chemical descriptors, were calculated for these four molecules. The HOMO energy is closely related to reactivity to electrophilic attack while LUMO energy is closely related to reactivity to nucleophilic attack. Thus, the eigenvalues of HOMO and LUMO and their energy gap reflect the biological activity of the molecule. Usually, the decrease in the HOMO and LUMO energy gap explains the eventual charge transfer interaction taking place within the molecule under the influence of an external electric field. As shown in 2\u2212 would be a suitable group. The B-ring was predicted to be surrounded by several hydrophobic residues, like Met179, Ile230, and Val231. It is consistent with the experimental activity that appropriate linker between A-ring and B-ring would also make the distal aromatic ring deep into this sub-region to interact with these hydrophobic residues. In addition, a moderately bulky group substituted on the 5\u2032/6\u2032-position of B-ring would increase the activity for the sake of the existence of Met233. Moreover, since the B-ring extends to a relatively capacious region at the surface of the protein, modification at specific position of B-ring may improve the physicochemical property of an inhibitor without sacrificing the potency. The bulky and hydrophobic group with positive charges potential of the C-ring are also predicted to be acceptable for the inhibitors. Thus, proper substitutions at these regions could directly contribute to improving the bioactivities of 11\u03b2-HSD1 inhibitors.Combining the results from above docking simulations, 3D-QSAR analyses, and quantum chemistry computations, structural requirements for designing novel 11\u03b2-HSD1 inhibitors are summarized in Although docking simulations provide a good starting for further calculations with the purpose of predicting the binding modes, the solvent effect on the whole system and the potential conformational changes are not fully taken into account. Therefore, molecular dynamics simulations were undertaken to investigate the stability of the enzyme-inhibitor complex in aqueous solution.1, 11, and 14 with 11\u03b2-HSD1, respectively, were applied as starting structures for MD simulations to analyze the structural requirements for the inhibitory activity and compare the binding modes between the inhibitors and 11\u03b2-HSD1. The root-mean-square deviations (RMSDs) for all heavy atoms of each ligand, backbone atoms of whole protein, and backbone atoms of residues in the binding pocket within 5 \u00c5 around the ligand were analyzed to explore the dynamic stabilities of all systems. The RMSDs of each system relative to their starting structures are plotted in 11 and 14 with lower biological activities spent longer time to reach equilibrium  of C\u03b1 atoms of protein in each complex. For comparison, the corresponding values of RMSF obtained from the co-crystal structure of 11\u03b2-HSD1-means algorithm [ptraj module of AMBER12. The representative conformations were then extracted from the cluster and compared with the above docking-simulated structures. In general, the conformation of the binding pocket and the inhibitors were found to be stable during the MD simulations, suggesting the rationality and validity of the docking models. However, some differences could still be observed between the MD simulated structures and the docked models. The program LIGPLOT [1 and 11\u03b2-HSD1. In order to further investigate the receptor-ligand interactions in the binding process, we compared the conformations of complexes during the MD simulations with the initial structures obtained from docking simulations. Clustering grouping was performed to analyze the structural variations of each complex during the MD simulations. Based on the pairwise similarity measured by RMSD with the lgorithm , five cl LIGPLOT  was used1 complex  distances. As shown in 1 and the main chains of Thr124 and Tyr183, respectively. D2 and D4 represent the distances between the 3-fluoro-4-methylphenyl moiety of 1 and the main chains of residues Tyr177 and Val231, respectively. The stability of the interatomic distances reveals that these hydrophobic interactions are also favorable to stabilize the binding of 1 to 11\u03b2-HSD1. 11 and 11\u03b2-HSD1. As shown in 11 at the beginning  estimated by the NMODE module of AMBER12. The results of estimated free energies and energy components of each complex in combination with their experimental data are listed in Gexp) determined from the corresponding experimental IC50 values, the ranking of predicted binding affinities (\u0394Gpred(PB)) of inhibitors 1 , 11 , and 14  correlates well with their experimental ones . Therefore, MM/PBSA calculated results would be reliable for the analyses of the interaction mode of inhibitors binding to 11\u03b2-HSD1. In order to deeply understand which energy term has more impact on the binding of 11\u03b2-HSD1-inhibitor complexes, we compared four individual energy components, \u0394Eele, \u0394EvdW, \u0394Gnonpol, and \u0394Gele(PB) of these three complexes. As listed in EvdW and the nonpolar solvation contribution \u0394Gnonpol are of vital importance to the binding free energy. On the contrary, the sum (\u0394Eele +\u0394Gele(PB)) of the electrostatic interaction and the polar solvation contribution is considerably unfavorable for binding free energy in all three complexes. Furthermore, it should be noted that \u0394EvdW is much stronger than \u0394Gnonpol, indicating that van der Waals interactions may contribute mostly to an inhibitor binding to 11\u03b2-HSD1 receptor. The binding affinities of above three enzyme-inhibitors complexes were further calculated using the MM/PBSA method with the vibrational entropy term  and 14  than 1 , another residue Tyr183, which only has an H-bond interaction with compound 1 during MD simulations, has more energy contributions to 1  than 11  and 14 . Among the resides in the binding pocket of 11\u03b2-HSD1, residues Thr124, Tyr183, Val227, and Val231 have stronger interactions with compound 1 in comparison with aforementioned residues, while residues Leu126, Met179, and Val180 interact with compound 14 differently from the other inhibitors. The residue Thr124 showed higher energy contributions to 1  binding to 11\u03b2-HSD1 in comparison with 11  and 14  since the side chain of Thr124 has interaction with the dimethylpiperidyl moiety of 1. Among the hydrophobic residues in the binding pocket of 11\u03b2-HSD1, the residue Val231 contributes more than twice to interact with 1  than 11  and 14 . This observation is consistent with the docking result that compound 1 with the 3-fluoro-4-methylphenyl moiety has strong hydrophobic interaction with the side chain of residue Val231. Therefore, we may safely conclude that Thr124, Tyr183, and Val 231 could be the key residues for inhibitors binding to 11\u03b2-HSD1. In order to gain insight into the energy contributions of the important residues mentioned above and to represent the results more intuitively, we divided each residue\u2032s contribution into polar and nonpolar components (14 from the other compounds. In general, Ser170 has significantly hydrophilic energy contributions for these three compounds, while Tyr183 has a favorable hydrophobic contribution to the binding of an inhibitor since its aromatic ring. In the meantime, Tyr183 also shows greater hydrophilic energy contributions for these three compounds. Therefore, Tyr183 plays a more critical role in forming H-bond interaction with an active inhibitor than Ser170. Taken together, we may conclude that the H-bond interactions with Ser170 and Tyr183, and the hydrophobic interactions with Thr124, Tyr177, Tyr183, Val227, and Val231 play important roles in improving the 11\u03b2-HSD1 inhibitory activity.Most of the key residues are hydrophobic enough to form strong van der Waals interactions with the inhibitors. Particular attention had been paid to those residues with relatively large differences in the contribution to binding free energies. Typically, the residues Ser170 and Tyr183, which have H-bond interactions with the inhibitor as discussed above, showed different interaction contributions. Ser170 has a little more energy contributions to mponents . As show1 and 32 were vital for the potency. Similarly, the molecular electrostatic profiles of the low active compounds 14 and 39 were also consistent with the 3D contour maps obtained from the CoMFA and CoMSIA models. Moreover, molecular dynamics simulations were performed to assess the contributions of residues to ligand binding by using three inhibitors 1, 11, and 14, with different activities and diverse structure features. The calculated binding free energies were in good accordance with the biochemical results. The decomposition of binding free energy into each interaction components indicated that the van der Waals interactions provided the major driving force for the binding process, while free energy decomposition to each residue suggested that the residues Thr124, Ser170, Tyr177, Tyr183, Val227, and Val231 contributed predominately to the binding free energies. The pivotal hydrogen bond interactions with Ser170 and Tyr183 could help to enhance the inhibitory activity for 11\u03b2-HSD1. Overall, these results obtained from the computational approaches not only provide several possible mechanism interpretations at the molecular levels, but also can be helpful for the rational design of novel 11\u03b2-HSD1 inhibitors.In the current study, a combined computational approach was applied to identify the structural determinants and specific binding modes between 11\u03b2-HSD1 and its inhibitors. The protein-ligand interactions have been characterized through receptor-based 3D-QSAR models, MESPs, MD simulations and free energy calculations. The higher statistic values for CoMFA and CoMSIA models demonstrated their reliability and predictive capability. From contour maps of the 3D-QSAR models, structure feature requirements were obtained for different substituents on the scaffold. It was worth noting that an important hydrogen bond rich region was identified by the contour maps. MESPs and Mulliken atomic charges analyses integrated with 3D-QSAR suggested that the electronegative amide carbonyl oxygen atom and nitrogen atoms of 1,2,4-triazolopyridine core in active compounds"}
+{"text": "Prolonged political instability may have exacerbated gender inequitable beliefs in the Democratic Republic of Congo (DRC). The aim of this study was to assess attitudes related to gender-equitable norms and its determinants among young, church-going women and men in Kinshasa, DRC.Data were collected through a cross-sectional survey with 291 church-going women and 289 men aged 18\u201324\u00a0years old, residing in three disadvantaged communes of Kinshasa. Variables included sociodemographic characteristics, attitudes towards gender equality, and responses to issues related to the gender-equitable men (GEM) scale. The GEM scale is a 24 item-questionnaire developed to measure attitudes towards gender equitable norms. Logistic regression was applied to discover the associations between the independent variables and the GEM outcome.Our study reflected the existence of attitudes hampering gender equality that were endorsed by both women and men. For example, 91.4% of women and 83% of men agreed with the statement \u201ca woman\u2019s most important role is to take care of her home and cook for her family\u201d. Similarly, 88.3% of women and 82.9% of men concurred with the idea that men need more sex than women. These findings coexisted with a few equitable norms, because 93.7% of women and 92.3% of men agreed that a man and a woman should decide together if they want to have children. A positive association was found in both women and men between being educated, being single and separated and having supportive attitudes towards gender equality and a higher GEM scale score. Residency in Camp Luka and Masina was also a significant social determinant associated with equitable gender norms among men whilst job status was only significant among women.While both women and men had high levels of gender inequitable norms, those with more education, single, and with supportive attitudes to gender equality had high GEM scale scores. The results highlight an urgent need for the church to challenge and change gender norms among church youths. The United Nations has prioritized the achievement of gender equality and women\u2019s empowerment as one of the main ways of ensuring the sustainable development goals . The latConversely, having unprotected sex with several partners and using violence against women might be common expectations for men, suggesting that men may drive HIV in sub-Saharan Africa , 10. DesDespite some ambivalence and resistance , researcChurches are an integral part of social life in many African societies where they have wide networks and provide nearly 70% of health services and mainly to the most marginalized people . ChurcheFor instance, a study conducted in Zambia showed the way some men exhibited harmful norms of masculinities of having sex with multiple partners, perpetrating violence against partners and drinking heavily . When thWhile DRC is a country with vast natural resources, most Congolese live in abject poverty attributable to political instability, resource mismanagement, and armed conflict. DRC ranked both at the bottom of the Gender Equality Index (144th out of 148 countries)  and Human Development Index (176th out of 188) . DRC didThe distribution of the population according to the religious affiliations in DRC is: Catholic (31%); Protestant 30%); other Christian Churches (34%); indigenous religions (3%); and Muslims (2%) . The Ecu%; other This cross-sectional survey was carried out in Bumbu, Camp Luka, and Masina, three deprived peri-urban communes located in Kinshasa, the capital of DRC. The study sites were purposively selected given the youth work that the \u00c9glise du Christ au Congo and Salvation Army was undertaking in these areas. In addition, the first author had collaborated with the leadership of these churches through his engagement with EHAIA .The first author acquired the French translated questionnaire IMAGES from Promundo, a nongovernmental organisation founded in Brazil, which promotes equitable masculinities and equitable gender relations locally and globally . We pret.The local parish pastors recruited eligible participants for this study based on the following criteria: to be young women and men (18\u201324\u00a0years old), belonging to the Salvation Army and the \u00c9glise du Christ au Congo, living in the three selected communes during the time data was collected, able to read and write in French, and volunteered to be involved in the study. We decided to interview this target group because young people aged 18\u00a0years do not need parental permission for participating in research and those aged 24\u00a0years are still considered as youths according to the World Health Organisation definition .The authorisation to carry out this study was provided by the leadership of both churches involved and the first author made contacts with the respective pastors who recruited the respondents at the congregational levels, mainly on Sundays. Once the church youths had gathered to take part in the study, the first author provided them with a self-administered questionnaire. The participants read and filled in the questionnaire autonomously within the relatively quiet church premises. In some instances, particularly for respondents who found certain statements difficult to grasp, the first author read the questions aloud to enable everyone to complete the survey. In general, it took approximately one hour for each participant to fill in the questionnaire.The study was carried out from March to April, 2016 and in total 750 church-going youths were invited to participate. However, 88 participants were excluded for not meeting the inclusion criteria : 67 did not turn up, and 15 declined the invitation for personal reasons. Finally, 580 were included, of whom 291 women and 289 men completed the questionnaires .sociodemographic characteristics, attitudes towards gender equality, and the Gender Equitable Men scale (GEM) [Our questionnaire is an adapted version of the International Men and Gender Equitable Survey (IMAGES) that was organised in three groups: le (GEM) . In addiAlthough gender was dichotomised into women and men , age was. Our survey included 16 statements and responses fell into four categories as participants were asked to ascertain if they (1) completely or (2) partly agreed or they (3) partly or (4) completely disagreed with the statements. An index, created by the sum of all of the items, was developed and divided into terciles representing low, medium, and high levels of attitudes towards gender equality. Responses by young, church-going women and men to the questions regarding attitudes are reported in This part of the original IMAGES questionnaire comprises several themes, notably men and women\u2019s practices, attitudes related to gender norms, gender equality, household dynamics and the men\u2019s involvement as fathers, intimate partner violence, health, and stress . After w. Initially, the GEM scale comprises 24 items related to gender and domestic chores, violence, sexual relationships, masculinities, and sexual and reproductive health. While our study included a list of 16 statements, we excluded the eight remaining GEM items after reflecting on the misunderstandings faced by young people involved in the pilot study [The GEM scale was originally developed in low income settings in Brazil and used as a tool to measure changes in gender-related interventions , 38, 39.ot study . A similot study , 31, 36.ot study . The Croot study .p\u2009<\u20090.05) in the crude model were included in the adjusted models. Once the logistic regressions were established, the goodness-of-fit of the models was calculated using the Hosmer Lemeshow\u2019s goodness-of-fit test to estimate that the presumed models were appropriately specified in the third step. Finally, given the relative small sample, predictable variables were organized in five groups. P-values were not significant, indicating a good model fit.As mentioned at the outset of this paper, the respondents\u2019 survey responses were written on the questionnaire format that the first author entered into an Excel sheet and transferred later on the whole data file to STATA 13.1 for statistical analysis. Because the study was looking for differences between women and men, all analyses were stratified by gender. First of all, the percentages of responses related to sociodemographic characteristics of young women and men, attitudes towards gender equality, and the GEM scale were calculated. Statistical gender differences for the various domains of the GEM scale were assessed using the chi-squared test. In the descriptive part, we used terciles as to do so is part of the GEM guidelines . But we This study was approved in 2010 by the institutional review board of the School of Public Health at the University of Kinshasa, DRC. The study\u2019s aim and purpose were explained to the women and men taking part, who were ensured anonymity. Written informed consent was obtained from all study participants.Table\u00a0The next section summarises the views expressed by the participants by domains of the GEM scale Table\u00a0. More woThe results of the logistic regression of factors associated with high GEM scale scores among young, church-going women and men is presented in Table\u00a0Our study provides evidence of attitudes and beliefs that may hamper gender equality and demonstrates that women and men in this study endorsed inequitable gender norms. Nonetheless, a positive association was found for both women and men between being educated, being single and separated, and having supportive attitudes towards gender equality and higher GEM scale scores.Overall, our findings revealed similarly poor gender-equity scores for both women and men for gender and domestic chores, masculinities, and sexual and reproductive health; these findings are in line with the DRC-IMAGES study . For insBy contrast, respondents\u2019 views about violence against women and sexual relationships were sharply different among women and men. In this regard, the DRC-IMAGES study  and our Several issues might explain some of the differences between the DRC-IMAGES study  and oursIn comparison to the DRC-IMAGES study, our church-going youths mainly scored gender inequitable statements for several reasons. The prevailing notions of gender in DRC are largely based on norms rooted in unequal power relations characterized by violence experienced by women and men\u2019s domination in decision making process . In addiWe expected that our study population might have been different from the general public in terms of attitudes towards equality because of the church influence. Compared with DRC-IMAGES, our findings suggest that their attitudes were not so divergent. Support for gender inequitable norms by church youths might also be due to biased interpretations of some narrow Christian views about patriarchy, which tend to give more power to men, including the control of women\u2019s bodies and sexuality , 46. In Our findings suggest that women endorsed most of the GEM inequitable statements, indicating that they may experience constant unequal power relationships and therefore internalized inequitable gender norms . The DRCA study conducted in Zambia suggested that women\u2019s attitudes and practices around gender norms might partly stem from interpretations of the biblical story of creation as evidence of women\u2019s inferiority in relation to men in relationships . Also, b. If educated youth can internalise equitable gender norms and act on them in their daily lives, then a shift in generational gender norms can gradually take place in Africa.The level of education attained by women and men, especially those with a secondary education or higher, emerged as a consistent predictor of more equitable attitudes and is similar to that of the Mali-IMAGES study . These tWe found that single women and men achieved high GEM scale scores, suggesting that those who may not have experienced a long term serious relationship might have not been exposed to unequal power relationships. And in turn, this might help explain why they have higher GEM scores. From this perspective, research has suggested that certain marriages might be seen as spaces surrounding harmful norms of masculinities, which may reinforce patriarchal attitudes that make it difficult to negotiate recent societal changes, such as gender equality , 58. OurOur study has some limitations that should be considered when interpreting the results. The relative small sample size could not have identified some relationships by using logistic regression, and a potential selection bias might have been operating because respondents were selected by parish pastors. Because the participants mainly came from the Salvation Army and from specific disadvantaged areas of Kinshasa, the findings might not be generalised to other urban or rural churches of the country. As with any survey carried out on sensitive issues regarding gender equity and sexuality and despite the promise of participant anonymity, some sensitive issues might have been under- and/or over-reported. Although the GEM scale was initially constructed for men, it was applied to both women and men participating in this study to make the findings comparable .Although most research on gender equity attitudes focuses on men, our study findings suggest that both young women and men had high levels of gender inequitable norms. Hence, our study may have acted as a spotlight, revealing many harmful gender norms that may prevail in the churches and in the society at large. For instance, more women than men were likely to agree on these inequitable attitudes, which highlights how gender inequality is ingrained in both genders and hampers progress of equality. Therefore, churches need to work with both women and men to challenge expectations and harmful norms regarding femininities and masculinities. Overall, the reason why women supported most of the inequitable GEM statements requires further investigation. There is an opening for churches to join hands and use their positions as social institutions that establish and enforce social norms to respond to this challenge.Women and men with more education, single, and with supportive attitudes for more gender equality had high GEM scale scores. The association between higher educational levels and equitable norms seems to reinforce the importance of education. The church can encourage education, assist youths who have to leave school for income-generating activities, and provide different educational opportunities, especially those that integrate promotion of gender equity. The churches also need to reach young women and men with messages and role models that promote healthy, non-violent and gender equitable lifestyles among church youths.Churches need to acknowledge the critical responsibilities that women and men can have as key partners in strengthening the response to gender equality and ensure that church staffs, policies and programmes seek to facilitate and advocate for their meaningful involvement.Churches should create, support, and reinforce gender equitable norms, as well as foster alliances with other stakeholders working towards a more gender-equitable future.The focus of church-youth interventions can be broadened to achieve gender-equitable attitudes and practices not just at the individual and family levels but to exercise a wider influence also on community and institutional levels across DRC.Our study findings indicate the importance of the churches in developing policies and programmes that address gender inequality with particular focus on young people."}
+{"text": "To determine the presence of low back pain and the associated factors in operating room nurses.The population of the descriptive study consists of 133 operating room nurses working in the operating rooms of five major hospitals located in Istanbul, and the study sample consists of 96 operating room nurses who are not on leave or sick leave between July-2016 to February 2017. Data were collected via a question form prepared by the researchers.It was determined that more than half of the operating room nurses forming the sample group had low back pain and that it is affected from the practices of operating room nurses during a shift, which may cause physical strain such as year of working as an operating room nurse, bending and staying in the same position for a long time, holding an instrument for a long time, rotational movement inadequate to body mechanics, lifting/carrying heavy medical items and pushing/pulling heavy medical equipment.Majority of operating room nurses had low back pain and it was associated with coercive movements during surgery. Anatomical, physiological, psychological and socio-cultural characteristics of healthcare professionals, along with working technique and physical characteristics of the working environment, negatively affect the musculoskeletal system. The prevalence of musculoskeletal system disorders is 5.7% in industrial workers while this rate increases to 8.8% in people working in hospitals.1Nurses, who have an important role among healthcare professionals, experience musculoskeletal system disorders, including upper back, neck, shoulder and joint pain. The most common musculoskeletal system problem observed in nurses is low back pain.5Nurses working in the operating room possess a greater risk in terms of musculoskeletal problems due to pulling and pushing gurneys, beds and other equipment, transferring the patient to the operating table or gurney, supporting an extremity for a long time and standing in the same position for a long time.3Keeping in mind those risk factors about musculoskeletal problems in operating room nurses will increase workforce loss and costs by causing functional insufficiencies in the future, determining the risk factors for low back pain in operating room nurses and taking the necessary precautions are of vital importance. The study was conducted in order to determine the rate of low back pain in operating room nurses and its associated factors.The population of this descriptive study consisted of 133 nurses who worked in the operating rooms of five major hospitals in Istanbul. Rather than sampling, census was aimed. Inclusion criteria were determined as being working in the operating rooms of the study hospitals for at least six months, not being on leave/sick leave between July 2016 and February 2017 and volunteering to participate in the study. Because some nurses had taken a leave in the defined time period and some nurses declined to participate, 72.1% of the universe was reached and the study was completed with 96 operating room nurses.Data were collected using a \u201cData Collecting Form\u201d developed by the researchers in the light of the literature.The data obtained from the study was analyzed with the Statistical Package for Social Science 21.0 packaged software. Frequency, percentage, mean, standard deviation, Chi-squared and student\u2019s t test were used when appropriate. Results were evaluated with 95% confidence interval and p<0.05 significance level.Written approval from a faculty of medicine clinical research ethics committee  and hospitals that the study was conducted in were obtained prior to the start of the study. Verbal and written consent was also obtained from nurses before data collecting tools were applied.2<BMI< 25.0 kg/m2). Total time spent standing in one shift was found to be 6.5208\u00b11.56931 hours. 76% of operating room nurses took a break during work and time worked until a break was found to be 4.0411\u00b11.16756 hours.Out of operating room nurses that participated in the study, 52.2% were between 21-35 years of age. 82.3% were female. 53.1% were married. 55.2% had a graduate degree and 47.9% had 11 years or more professional experience and 37.5% had been working has an operating room nurse for one to five years. About 72.9% did not smoke; 84.4% worked out regularly and 63.5% defined their health status as \u201cwell\u201d. About 43.8% had a responsibility for caring for a relative and/or children or that required carrying weight outside working hours. 71.9% had normal BMI  . Also, the prevalence of low back pain was significantly higher in operating room nurses with normal BMI when compared to nurses with BMI<18.5 kg/m(p<0.05) .Nurses who leaned more than five times in one shift experienced low back pain more frequently than nurses who leaned for three to five times in one shift, one to two times in one shift and never leaned. Nurses who stood in the same position for a long time three to five times in one shift had more back pain than other who stood in the same position for a long time more than five times, one to two times or never stood in the same position. Nurses who held equipment for a long time three to five times in one shift had more back pain than other who held equipment for a long time more than five times, one to two times or never held equipment for a long time. Finally, nurses who rotated backwards more than 5 times in one shift experienced low back pain more frequently than nurses who rotated backwards for 3 to 5 times in one shift, one to two times in one shift and never rotated. All differences were statistically significant (p<0.05) .Similarly, operating room nurses who lifted/carried heavy medical equipment 3 to 5 times in one shift had more back pain than other who stood in the same position for a long time more than 5 times, 1 to 2 times or never stood in the same position. Also, nurses who pulled/pushed heavy medical equipment more than 5 times in one shift experienced low back pain more frequently than nurses who pulled/pushed heavy medical equipment for three to five times in one shift one to two times in one shift and never pulled/pushed. These differences were statistically significant as well (p<0.05) .Low back pain is one of the most common musculoskeletal problems observed among nurses.Age is among the individual risk factors that may cause low back pain. There was no statistically significant association between age and the prevalence of low back pain in this study. Even though there are studies in the literature indicating that age variable does not impact the prevalence of low back pain11Marital status variable did not cause a difference in the frequency of low back pain in the present study. Despite there are studies, which found that single individuals experience more low back pain than married individuals15As the years worked increase, total exposure to risks in the workplace also increase, increasing the risk of associated health deterioration. In the present study, individuals who had been working for an operating room nurse were found to have more low back pain. Even though many studies performed with healthcare professionalsSmoking is reported to be a risk factor that increases the incidence of low back pain.Regular exercise affects the individual positively both physically and mentally. It can especially prevent the development of musculoskeletal problems by strengthening the muscles. Similar to our study, \u00c7il-Ak\u0131nc\u0131 et alBody mass index another individual factor that impacts the musculoskeletal system. In the present study, nurses with normal BMI were found to have more low back pain than overweight nurses. Even though Daraiseh et alRepetitive movement, inappropriate posture and excessive force exertion are important factors causing musculoskeletal problems.In conclusion, it is recommended that operating room nurses should be trained about body mechanics, necessary steps should be taken to ensure adequate number of nurses in order to minimize the frequency of practices that may cause physical stress, acceptable break time in a shift should be provided to nurses when increasing the number of nurses is not possible, operating rooms should be arranged ergonomically and regular exercise programs should be organized to increase the endurance of lumbar muscles of operating room nurses.This study was presented (poster presentation) at \u2018\u20188th EORNA Congress\u2019\u2019 4-7 May 2017, in Rhodes, GREECE.IC, AK, YO, AO: Conceived, designed and did statistical analysis & editing of manuscript.IC, AK, YO, AO: Did data collection and manuscript writing.IC, AK, YO, AO: Did review, final approval of manuscript and are responsible for integrity of study."}
+{"text": "Blocking the interaction between cell-surface receptors and their ligands is a proven therapeutic strategy. Adhesion G protein-coupled receptors (aGPCRs) are key cell-surface receptors that regulate numerous pathophysiological processes, and their large extracellular regions (ECRs) mediate ligand binding and function. The aGPCR GPR56/ADGRG1 regulates central nervous system myelination and melanoma progression by interacting with its ligand, tissue transglutaminase 2 (TG2), but the molecular basis for this interaction is largely undefined. Here, we show that the C-terminal portion of TG2 directly interacted with the GPR56 ECR with high-nanomolar affinity, and used site-directed mutagenesis to identify a patch of conserved residues on the pentraxin/laminin-neurexin-sex-hormone-binding-globulin-like (PLL) domain of GPR56 as the TG2 binding site. Importantly, we also show that the GPR56-TG2 interaction was blocked by previously-reported synthetic proteins, termed monobodies, that bind the GPR56 ECR in a domain- and species-specific manner. This work provides unique tools to modulate aGPCR-ligand binding and establishes a foundation for the development of aGPCR-targeted therapeutics. For example, the programmed death-1 (PD-1) receptor on T-cells is the target of pembrolizumab and nivolumab, both cancer immunotherapeutic monoclonal antibodies that block the aberrant activation of PD-1, and thereby potentiate the immune system\u2019s ability to attack cancer cells5. Though blocking receptor-ligand interactions has been successful in multiple contexts, developing blocking agents that target a specific receptor, or its specific ligand-binding domains, remains a nontrivial obstacle.Cell-surface receptors communicate signals across the plasma membrane. Vital to this process is the extracellular interaction between a signaling molecule and its receptor, which initiates a multi-step cascade, and ultimately results in a cellular response. Aberrant signaling can have catastrophic consequences, prompting the development of therapeutics to block the interactions between signaling ligands and their receptors9. In addition to their seven-pass transmembrane helix bundle (7TM) that transduces signals across the plasma membrane10, aGPCRs characteristically contain large extracellular regions (ECRs), which comprise multiple adhesion domains involved in ligand binding6. Recent work has also shown that aGPCR ECRs play a direct role in the regulation of receptor function and downstream signaling19. Though most aGPCRs are still orphan receptors, the ligands of several aGPCRs have been identified as other cell-surface proteins or soluble extracellular matrix (ECM) proteins22. However, mechanistic studies of aGPCR-ligand interactions to identify binding sites and binding affinity have not yet followed the identification of these structurally complex protein ligands. Furthermore, strategies for modulating these interactions remain untested.Adhesion G protein-coupled receptors (aGPCR) are cell-surface receptors that are involved in key pathophysiological processes including neurodevelopment, immunology, and tumorigenesis27, major depressive disorder28, peripheral immunity30, CNS immunity31, skeletal muscle development34, pancreatic beta-cell function36, and cancer progression43. We have shown that the GPR56 ECR is composed of an N-terminal pentraxin and laminin/neurexin/sex hormone-binding globulin (PLL) domain and a juxtamembrane GPCR autoproteolysis-inducing (GAIN) domain and that the ECR plays a receptor-autonomous regulatory role in G protein signaling12. Furthermore, we have developed synthetic proteins, called monobodies, that bind to the GPR56 ECR and modulate G protein signaling, thus acting as allosteric agonists and allosteric inverse agonists13. Additionally, we identified a conserved, surface-exposed patch of the PLL domain that is necessary for GPR56-mediated myelination in zebrafish12. Though we speculated that this patch may be important for native ligand binding, we had no definitive information regarding the identity of such a ligand nor tools to probe such an interaction.GPR56/ADGRG1 belongs to the aGPCR family and plays a critical role in diverse pathophysiological processes such as brain development44. Studies have also implicated TG2 in murine models of melanoma progression and suggested that GPR56 inhibits melanoma progression in a TG2-dependent manner37. Additionally, more recent work has elucidated a complex array of interactions between neuronal GPR56 and microglial TG2 in the proliferation of oligodendrocyte precursor cells, leading to myelination in the central nervous system (CNS)45. Thus, there is strong evidence to support the importance of the GPR56-TG2 interaction in normal physiology and disease processes. TG2 is an enzyme that plays an important role in cross-linking proteins in the extracellular matrix (ECM). It has four domains (D1-D4), the second of which (D2) catalyzes a Ca2+-dependent transamidation reaction that crosslinks proteins in the ECM47. TG2 canonically plays roles in apoptosis, cell\u2013matrix interactions, and tissue stability, as well as in pathologies including autoimmune disorders, neurodegenerative conditions, and cancer49. Though the GPR56-TG2 interaction was first identified over a decade ago, neither the affinity of TG2 for GPR56 nor its specific binding site have been determined. In early experiments, the region of GPR56 later identified as the PLL domain was shown to be necessary for TG2 interaction44. However, as experiments were not performed with purified proteins, it remained unclear whether the GPR56-TG2 interaction was a direct, binary interaction that might be suitable for targeting by pharmaceutical agents.Tissue transglutaminase 2 (TG2) was reported as a putative ligand for GPR56 using unbiased pull-down experiments in 2006In this study, we used purified proteins to biochemically determine that the GPR56-TG2 interaction is indeed direct with binding affinity in the high-nanomolar range. We then utilized structure-guided mutagenesis analysis to identify the TG2 binding interface on GPR56. The binding interface is centered around a histidine residue that is critical for the in vivo myelination function of GPR56. Finally, we demonstrated that PLL-binding monobodies specifically block TG2 binding, paving the way for pharmacological disruption of native ligand binding to aGPCRs.12, we expressed mouse and human GPR56 ECR constructs as well as mouse TG2 (mTG2) constructs in High Five insect cells. Our TG2 constructs corresponded to full-length mTG2 (mTG2 FL) and the C-terminal D3D4 domains  resulted in a GPR56 loss of function phenotype in a zebrafish modelKD) of 440\u2009\u00b1\u200920\u00a0nM and 330\u2009\u00b1\u200915\u00a0nM, respectively  constructs were expressed in HEK293T cells and binding of purified mTG2 D3D4 was quantified using flow cytometry Fig.\u00a0.13 to quantify the surface expression level of GPR56. Then, we used the \u03b23 staining to gate a subset of the cells that fell within a standardized narrow range of GPR56 surface expression, within which we quantified the degree of mTG2 D3D4 binding  that resulted in decreased mTG2 D3D4 binding signal Figs.\u00a0. These r12, so as to remove a previously identified N-linked glycan at Asn148 that is positioned immediately adjacent to His89  and form a contiguous surface Fig.\u00a0B,D,E. Wes89 Fig.\u00a0. Our resWe found that the H89A\u2009+\u2009Y93A\u2009+\u2009R104D triple mutant completely abolished mTG2 binding without affecting cell-surface expression , abbreviated \u03b27, binds the human PLL domain but not the mouse counterpart. By contrast, Mb(mGPR56_\u03b212), abbreviated \u03b212, binds the mouse but not human PLL domain, supporting the utility of these two monobodies as excellent controls in our experiments  compete with each other for binding to the GPR56 ECR, we immobilized the first protein of interest on M280 beads, incubated with unlabeled human GPR56 ECR, and stained with a fluorescently labeled version of the second protein of interest Fig.\u00a0C. In thi44. Thus, with the ultimate goal of modulating this interaction as a potential therapeutic strategy for multiple diseases, there remains great interest in advancing a fundamental biochemical understanding of GPR56-TG2 binding.The interaction between GPR56 and TG2 has been implicated in remarkably diverse biological processesKD) suggests a conserved interaction interface between TG2 and GPR56 in human and mouse. Though previous studies using immunoblotting have suggested that human GPR56 interacts more strongly with mTG2 than human TG2, these experiments do not necessarily reflect affinity of native proteins55. Future studies may elucidate the conservation of the interaction between GPR56 and human TG2.We first established a quantitative binding assay to measure the affinity of mTG2 for GPR56 in their native states. We found that mTG2 has similar affinity to human and mouse GPR56. The small difference  is at the core of much of the mechanistic understanding of TG2 function. However, the active site of TG2 is not present in the GPR56-binding fragment of TG2  and incubated with Ni\u2013NTA sepharose resin for 3\u20135\u00a0h at 4\u00a0\u00b0C with constant stirring. Proteins were biotinylated before eluting from resin as previously described18. Briefly, the beads were washed and incubated with purified BirA biotin ligase\u2009+\u2009biotin\u2009+\u2009ATP for 1\u00a0h at 27\u00a0\u00b0C in 50\u00a0mM Bicine pH 8.3\u2009+\u2009150\u00a0mM NaCl\u2009+\u200910\u00a0mM\u00a0Mg acetate. The biotinylated TG2 was then eluted in 10\u00a0mM HEPES pH 7.2\u2009+\u2009150\u00a0mM NaCl\u2009+\u20091\u00a0mM TCEP\u2009+\u2009200\u00a0mM imidazole. The eluent was filtered and injected into a Superdex 10/300 Gel Filtration column equilibrated in 10\u00a0mM HEPES pH 7.2\u2009+\u2009150\u00a0mM NaCl\u2009+\u20090.5\u00a0mM TCEP for TG2 FL and 10\u00a0mM HEPES pH 7.2\u2009+\u2009150\u00a0mM NaCl\u2009+\u20090.5\u00a0mM TCEP\u2009+\u20091\u00a0mM CaCl2 for TG2 D3D4. Peak fractions were pooled and frozen in liquid nitrogen.Full length mTG2 was provided as kind gift from Lei Xu (University of Rochester). The TG2 FL and the TG2 D3D4 constructs (residues T471-A686) were cloned into the vector pVL1393. N-terminal 6xHIS and AVI-tags were added to each construct to facilitate purification and biotinylation, respectively. Baculoviruses were generated for cytosolic insect cell expression as previously described2. Transient transfection was performed with cells at 50\u201360% confluence as follows: 2\u00a0\u00b5g cDNA was diluted into 50 \u00b5L final volume of serum-free DMEM, while 3 \u00b5L LipoD293 transfection reagent (SignaGen) was added to 47 \u00b5L serum-free DMEM. Diluted LipoD293 was then added to diluted cDNA, and complexes were allowed to form by incubation at room temperature for 10\u00a0min. The transfection complex mixture was then added dropwise to each well. After 48\u00a0h incubation at 37\u00a0\u00b0C/5% CO2, the cells were detached using citric saline solution (135\u00a0mM KCl\u2009+\u200950\u00a0mM sodium citrate) and washed with PBS\u2009+\u20092% BSA.HEK293T cells were cultured in 6-well plates in Dulbecco\u2019s modified Eagle\u2019s medium  supplemented with 10% FBS (Sigma), at 37\u00a0\u00b0C in 5% COHEK293T cells were transfected with wt or mutant GPR56 constructs as described above and co-stained with monobody \u03b23\u2009+\u2009neutravidin-488 tetramers and TG2\u2009+\u2009neutravidin-650 tetramers. Tetramers were independently prepared in the excess of free biotin and soluble unlabeled neutravidin before mixing together to avoid the possibility of forming \u03b23\u2009+\u2009NAV650 and TG2\u2009+\u2009NAV488 tetramers. To normalize for differential expression of GPR56 mutants, TG2 binding signal was normalized to a particular bin of \u03b23 binding signal Figure . Thus, oHEK293T cells were transfected with GPR56 constructs (wt or mutant), incubated with 500-fold molar excess of unlabeled tetramerized monobody competitor (\u03b27 or \u03b212), and then co-stained with 500\u00a0nM monobody \u03b23\u2009+\u2009neutravidin-488 tetramers and 1\u00a0nM TG2\u2009+\u2009neutravidin-650 tetramers. Binding signal was normalized to GPR56 expression as described above Figure . Data we12. In short, M280 beads were coated with TG2, following which, purified and biotinylated GPR56 fragments were incubated with beads at various concentrations. Neutravidin-650 was then added to detect the GPR56 fragments. Binding signal versus GPR56 fragment concentration was plotted to calculate the apparent dissociation constant, KD. Data were collected on an Intellicyt flow cytometer, initially processed in FlowJo, and standard 1-to-1 binding curve-fitting was done in Prism.M280 bead-binding assay was carried out as previously described18. In short, Hi Five insect cells (Trichoplusia ni) were cultured in Insect-Xpress medium (Lonza) supplemented with 10\u00a0mg/mL gentamicin at 27\u00a0\u00b0C and were used for production of recombinant proteins. HEK293T mammalian cells (Homo sapiens) were cultured in Dulbecco\u2019s modified Eagle\u2019s medium  supplemented with 10% FBS (Sigma) at 37\u00a0\u00b0C in 5% CO2 and were used for cell-surface expression assays and flow cytometry binding assays.Insect cell culture and mammalian cell culture were both performed as previously describedSupplementary information."}
+{"text": "The original version of this article  unfortunThe first and corresponding author was unfortunately presented as Mercedes Rodriguez Romano. The correct author name is Mercedes Romano.The correct author name has been included in the author list of this Correction article."}
+{"text": "Non-human primates (NHPs) play an important role in biomedical research, where they are often being re-used in multiple research studies over the course of their life-time. Researchers employ various study-specific screening criteria to reduce potential variables associated with subsequent re-use of NHPs. However, criteria set for NHP re-assignments largely neglect the impact of previous exposures on overall biology. Since the immune system is a key determinant of overall biological outcome, an altered biological state could be predicted by monitoring global changes in the immune profile. We postulate that every different exposure or a condition can generate a unique global immune profile in NHPs.Changes in the global immune profile were evaluated in three different groups of rhesus macaques previously enrolled in dengue or malaria vaccine studies over six months after their last exposure. Na\u00efve animals served as the baseline. Fresh blood samples were stained with various immune cell surface markers and analyzed by multi-color flow-cytometry to study immune cell dynamics in the peripheral blood. Serum cytokine profile in the pre-exposed animals were analyzed by mesoscale assay using a customized U-PLEX NHP biomarker panel of 12 cytokines/chemokines.Pre-exposed macaques showed altered dynamics in circulating cytokines and certain innate and adaptive immune cell subsets such as monocytes, HLA-DR+NKT cells, B cells and T cells. Some of these changes were transient, while some lasted for more than six months. Each group seemed to develop a global immune profile unique to their particular exposure.Our data strongly suggest that re-used NHPs should be evaluated for long-term, overall immunological changes and randomly assigned to new studies to avoid study bias. Non-human primates (NHPs) are a long-lived, sentient species which can be trained to perform certain cognitive and behavioral tasks . Most ofWhen re-assigning NHPs in various research protocols, each study typically implements their own study-specific, screening criteria to exclude previously exposed animals to a similar disease or similar environmental stimulus. As a standard practice, in most cases, NHPs are re-assigned to new studies after a short resting period. There have been no reports clearly defining the scientific basis of length of the resting period between repeated exposures of NHPs to different study protocols. Majority of NHP re-assignments assume that effect of pre-exposure can only be specific to a particular study or a disease, mostly neglecting their impact on overall biology of the animals. Since the immune system is a key determinant of overall biological outcome, changes in the global immune profile could represent an altered biological state of the animal. Here, we propose that global changes in immune system thus, could be a useful inclusion/exclusion criteria to re-use NHPs in different protocols or to determine their resting period.The immune system continuously cross-talks with the rest of the organs in the body, modulating the immune profile accordingly, not only during diseases directly affecting the immune system, but in diseases related to other systems such as neurological or reproductive system . LikewisIn order to understand whether each exposure could have resulted in a distinctive overall immune profile, we studied three different groups of rhesus macaques previously enrolled in infectious disease research protocols. Pre-exposed animals were monitored over 6 months after their most recent exposure, whereas na\u00efve macaques served as the baseline. To avoid any technical bias, flow cytometric monitoring of immune cell frequencies and automated leukocyte counting were performed using fresh whole blood. We found long-term, exposure-related changes in circulating immune cell subsets and cytokines, leading to distinctive overall immune profiles in pre-exposed rhesus macaques.Guide for Care and Use of Laboratory Animals\u201d, Institute of Laboratory Animals Resources, National Research Council, National Academy Press, 2011, the Public Health Service Animal Welfare Policy, and the policies of WRAIR. Adult purpose-bred rhesus macaques of Indian origin were housed at the WRAIR animal facility for the duration of their studies. Animals were socially pair-housed and fed a commercial diet , provided free access to water, and supplemented with a variety of fresh fruits and vegetables. Environmental enrichment was provided in accordance with standard operating procedures of the WRAIR animal facility. Animal cages were cleaned daily and sanitized bimonthly. Automatic lighting was provided through a 12:12\u00a0h cycle.Blood and serum samples reported in the present study were received as part of a tissue-sharing program from the protocols reviewed and approved by Walter Reed Army Institute of Research (WRAIR)/Naval Medical Research Center (NMRC) Institutional Animal Care and Use Committee in compliance with all applicable federal regulations governing the protection of animals and research . Experiments reported herein were carried out in compliance with the Animal Welfare Act and per the principles set forth in the \u201cMacaca mulatta, three to six years old) were selected based on their most recent study exposures. Since all the assessments were made on fresh whole blood samples, age matched, na\u00efve rhesus macaques  = 3, Males (M) = 6) served as the control group for all the comparisons described in the present study. Experimental group one consisted of rhesus macaques  that had been primed with bivalent or tetravalent dengue purified inactivated vaccine conjugated with alum adjuvant  and boosted with tetravalent dengue live attenuated virus  followed by a challenge with dengue virus-2 . Day of the DENV-2 challenge was considered as the day of the last exposure for group one. Experimental groups two and three consisted of rhesus macaques that received a cell based, 293-gp96-Ig-PfCSP-PfAMA (gp96-Ig-PfCA)  and NMRC-M3V-D/Ad-PfCSP-PfAMA (D/Ad-PfCA)  candidate malaria vaccines, respectively. The gp96-Ig-PfCA malaria vaccine had contained irradiated human embryonic kidney (HEK) 293 cells transfected with a mixture of plasmids encoding a secreted form of heat shock protein gp96 (gp96-Ig), Plasmodium falciparum circumsporozoite (PfCSP) protein and P.\u00a0falciparum apical membrane antigen (PfAMA). The vaccine had been administered subcutaneously in three doses at 0, 5 and 25 weeks. In D/Ad-PfCA heterologous prime-boost vaccination, macaques in the group three had been primed with three intramuscular doses containing a mixture of two DNA plasmids encoding PfCSP and PfAMA at 0, 4, 8 weeks and boosted once intramuscularly with a mixture of two non-replicating recombinant human serotype 5 adenovirus vectors expressing PfCSP and PfAMA antigens at 25 weeks. The day of last exposure for group two and three were therefore, 25 weeks post-last vaccination/boost.Three groups of rhesus macaques  were processed within four hours of collection and stained cells were immediately acquired by multi-parametric flow cytometry for immunophenotyping. Serum samples (75\u00a0\u00b5l) were sub aliquoted into polypropylene micro tubes and stored at \u221280 \u00b0C, and later used to determine the concentration of circulating cytokines and antigen-specific antibodies. Time points were determined based on availability of samples within each experimental group.Multi-parametric, flow cytometry was used to determine the phenotype and frequency of innate and adaptive immune cell subsets in the peripheral blood. Cell frequencies for experimental group one were measured at 1,2,3,4 and 6 months post-DENV challenge. Cell frequencies for experimental group two and three were measured at day 6, day 20, 2.5, 4, and 6 months post-last vaccination/boost. The nine color NHP immunophenotyping panel consisted of following surface antibodies from BD biosciences, San Jose, CA: CD45 , CD3 , CD8 , CD16 , HLA-DR ; following antibodies from Miltenyi Biotec, San Diego, CA; CD20 , CD4 , CD159a , CD14 ; and CD11c . One hundred microliters of whole blood sample was stained with a cocktail of fluorochrome-conjugated antibodies (seven color panel for group one and nine color panel for group two/three), incubated for 10 min at room temperature and diluted in sheath fluid for immediate acquisition. Acquisition was limited to cells expressing V450 fluorochrome/CD45 (trigger) at a particle cut-off size (FSC) of 4000 and 50,000 events/sample were acquired at a medium flow rate by 17-color, LSRII Fortessa flow cytometer at the ImmunoCore facility using the FACS DIVA software. Gating strategy used to analyze the phenotype and frequency of each immune cell subset is provided in the Concentration of white blood cells in EDTA-preserved whole blood samples was determined using a Luna Dual Fluorescence Cell Counter within an hour of sample collection . Ten microliters of whole blood was diluted in 990\u00a0\u00b5L of 1x PBS (1:100), mixed by vortexing and 2\u00a0\u00b5L of acridine orange/propodium iodide dye mix  was added to 18\u00a0\u00b5L of diluted blood sample. Ten microliters from the mixture was loaded into Luna photon-slide disposable hemocytometer , and the concentration of fluorescent white blood cells was determined using the Luna dual fluorescence automated cell counter. Frequency of each individual cell subset determined by flow cytometry was used to calculate their absolute counts (cells/\u00b5L) as a proportion of the total white blood cells (CD45hi cells) (or thereby as a frequency of the parent cell population). The parent population of each immune cell subset described in the manuscript is shown in \u03b3, IL-1\u03b2, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/IL23p40, MCP-1, MIP-1\u03b1, TNF-\u03b1)  was used to analyze serum cytokine/chemokine levels per manufacturer\u2019s instruction. All cytokines for experimental group one were measured at day 7, 2 months, and 6 months, post-DENV-2 challenge. Cytokines for experimental group two and three were measured at day 6, day 20, 2.5 months, and 6 months post-last vaccination/boost.A customized U-PLEX NHP biomarker panel of 12 cytokines/chemokines  was used to generate correlograms  and dendograms. Agglomerative hierarchical clustering with Pvclust was used to plot dendograms. AU p-values were reported on the dendograms.Data from the na\u00efve group were collected independently from the three experimental groups . Therefore, series of unpaired T-tests (two-tailed) were carried out to compare between the na\u00efve group and each of the experimental groups at different time points post-exposure . Welch\u2019s correction was applied with unpaired T-test, when To study innate immune cell dynamics, we monitored frequencies and absolute cell counts of circulating monocytes (CD45+CD14+ or CD45+CD14+HLA-DR+), natural killer (NK) cells (CD45+CD3-CD16/CD159a+), NK cells expressing HLA-DR (CD45+CD3-CD16/CD159a+HLA-DR+), natural killer T(NKT) cells (CD45+CD3+CD16/CD159a+), NKT cells expressing HLA-DR (CD45+CD3+CD16/CD159a+HLA-DR+) and dendritic cells (DC)s (CD45+CD14-CD16/CD159a-CD3-CD20-CD11c+HLA-DR+) in whole blood by flow cytometry and automated cell counting  and S1H.n\u00a0=\u00a010) that had been primed with bivalent or tetravalent dengue purified inactivated vaccine (DPIV) and boosted with tetravalent dengue live attenuated virus (TDENV-LAV) followed by a challenge with dengue virus-2 (DENV-2). Experimental group two had received three doses of a cell based malaria vaccine, containing irradiated HEK 293 cells secreting heat shock protein chaperon, gp96-Ig, and the two malaria antigens, circumsporozoite protein and apical membrane antigen-1 (gp96-Ig-PfCA) (n\u00a0=\u00a05). Experimental group three had been primed thrice with a mixture of DNA plasmids expressing the same two malaria antigens as in the experimental group two, and had been boosted once with non-replicating recombinant human serotype 5 adenovirus (Ad5) plasmids expressing the same antigens (D/Ad-PfCA) (n\u00a0=\u00a05). The pre-exposed animals were monitored at multiple time points, over 6 months since their most recent exposure . A group of na\u00efve monkeys (n\u00a0=\u00a09)  were used as the controls.Experimental group one consisted of rhesus macaques  and 3 months  post DENV-2 virus challenge compared to the na\u00efve animals (3.9\u00a0\u00b1\u00a01.8). This transient expansion of monocytes were subsided to the levels present in the na\u00efve macaques by 6 months post-challenge  were found to be similar between rhesus macaques vaccinated with gp96-Ig-PfCA and na\u00efve animals over the course of 6 months . In contWe did not observe any noticeable changes in NK cells, HLA-DR expressing NK cells, DC or NKT cells in the two malaria vaccine groups (data not shown). However, we noted more permanent alterations to a specific subset of NKT cells expressing HLA-DR activation marker . NKT celThe frequency of HLA-DR expressing NKT cells were notably lower in most of the individual macaques pre-exposed to gp96-Ig-PfCA at 2.5 months and 4 months after the last vaccination . HLA-DR+In the D/Ad-PfCA malaria group, there was a persistent reduction of HLA-DR+NKT cell frequency through 2.5 months, 4 months and 6 months post-Ad5 boost compared to the na\u00efve animals . SubsequIn summary, dynamic changes in circulating innate immune cell subsets appeared to be unique for their past exposures (ex: vaccination strategy or pathogen). Also, the data indicate that these changes can either be transient or more permanent, which could last up to 6 months.\u03b3, IL-1\u03b2, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/IL-23p40, MCP-1, MIP-1\u03b1, TNF-\u03b1). Most of the cytokines were either non-detectable or present at levels less than 2 pg/mL, except MCP-1, MIP-1\u03b1 and IL-12/IL-23p40.To determine changes in circulating cytokine milieu at multiple time points over 6 months post-last exposure, we used a customized U-PLEX NHP biomarker panel of 12 cytokines (IFN-\u03b1/CCL3 (MIP1-\u03b1) are two major chemokines (or a chemotactic cytokines) critical for leukocyte migration and infiltration  to the site of inflammation (P\u00a0=\u00a00.059) by 6 months compared to na\u00efve animals (133.9 \u00b1 47.1)  and macrophage inflammatory proteinltration . MIP1-\u03b1/ammation . We foun \u00b1 47.1) . In contificance . Both MCp period  and 3C.\u03b1 dynamics, more pronounced persistent alterations were noted in circulating concentrations of interleukin (IL)-12/IL23-p40 cytokines in pre-exposed macaques. IL-12 and IL-23 are heterodimers that share the p40 subunit, mainly produced by dendritic cells and macrophages. IL-12 mainly regulates differentiation of T helper 1 (Th1) cells and interferon gamma (IFN-\u03b3) cytokine production. IL-23 is crucial for maintenance and expansion of Th17 cells (P\u00a0=\u00a00.007), 2 months  and 6 months  post-DENV-2 challenge, than the na\u00efve animals (115.7 \u00b1 52.3) (P\u00a0=\u00a00.01) and 20 days  post-Ad5 boost (P\u00a0=\u00a00.12), subsequently returning to the levels found in the na\u00efve group (115.7 \u00b1 52.3) by 6 months   . Slower  \u00b1 52.3) . We obsed5 boost . This waP\u00a0=\u00a00.8) . Rhesus P\u00a0=\u00a00.8) .Dynamics of IL-12/IL-23p40 seemed to follow a trend unique to each exposure, when shifting from altered state to steady or na\u00efve state. This transition may take up to 2.5 months or longer than 6 months post-last exposure, depending on the type of the exposure. Our findings agree with the fact that different exposures can induce either transient or persistent changes, uniquely altering the dynamics and composition of the circulating cytokine milieu.In order to evaluate global impact on the humoral arm of the adaptive immunity, we determined the cell frequency and counts of circulating B cells. Interestingly, a persistent reduction in the overall CD20+B cell frequency was seen after DENV-2 challenge . AlthougAs shown in Based on the differential ability of DPIV/TDENV-LAV, D/Ad-PfCA and gp96-Ig-PfCA vaccination strategies to elicit antibodies  and S2B,T cells are the central players of cell mediated immunity, which can modulate the function of many immune cells, activate host defense mechanisms and lyse pathogen infected cells. To understand global changes in circulating T cells following exposure to different protocols, we determined the frequency and cell counts of various T cell subsets. CD3+ T cell subsets were analyzed on CD3+ gated population \u2013S1F.P\u00a0=\u00a00.003), compared to the na\u00efve group (26.6 \u00b1 2.0)   . This co9 \u00b1 4.0) . FrequenP\u00a0=\u00a00.02) and cell counts  of CD3+ gated CD4+ T cell population were transiently decreased by day 20 post-Ad5 boost, subsequently returning to the levels seen in na\u00efve animals  by 2.5 months. Nevertheless, CD4+ T cell counts seemed to continuously increase over 6 months post-Ad5 boost , rather than remaining in a steady state . dy state . CD3+ T We then analyzed the dynamics of CD3+ T cell subsets expressing HLA-DR, a well-known marker for activated T cells . We obseAs shown in Here we show that each individual exposure could alter the global balance of cell mediated adaptive immune responses in the peripheral blood, irrespective of the anticipated vaccine-focused, antigen-specific, cell-mediated immune responses.We next sought to determine whether the observed changes in individual immune cell subsets could subsequently affect the overall immune profile of pre-exposed rhesus macaques . We usedCorrelation coefficient of cell counts was computed for all possible combinations (pairs) of immune cell subsets within each group to generate a correlation matrix and was visualized by a correlation plot (correlogram) using R studio software . Each correlogram represents negative and positive relationships between the cell counts of all pairs of immune cell subsets tested within each group . CorreloRepeated exposure to various physical and mental stimuli could modulate overall biology of NHPs, with possible implications on re-using those animals between various research protocols. However, so far, we have lacked proper scientific evidence to justify the widely accepted, arbitrarily implemented, 4\u20136 weeks rest-period between re-assignment of NHPs in different research protocols. One way to understand the impact on overall biology due to a certain exposure is to study dynamics of key biological systems of the exposed NHPs. Changes in the immune system in fact, are better indicators of overall biological impact, due to its continuous cross-talk with the rest of the body compartments . Here, w\u03b1 and IL-12/IL-23p40 cytokines, leading to a long-term suppression of their secretion  and Toll-like receptor (TLR) signaling pathways in innate immune cells  and cytokines in rhesus macaques previously exposed to different antigenic stimuli. We observed exposure-specific, transient and persistent changes (up to 6 months), in different immune cell populations as well as in the cytokine profile. Our data suggest that overall biological changes represented by exposure-related, global immune imprinting should be carefully evaluated before re-assigning NHPs to new studies to avoid study bias. Further research is warranted to understand whether such changes could lead to \u201ctrained-immunity\u201d or \u201cimmune tolerance\u201d and subsequent impact of NHP re-use on the experimental outcome.10.7717/peerj.10955/supp-1Data S1This file containes raw data for Figure 1-7.Click here for additional data file.10.7717/peerj.10955/supp-2Table S1Click here for additional data file.10.7717/peerj.10955/supp-3Table S2Click here for additional data file.10.7717/peerj.10955/supp-4Table S3Click here for additional data file.10.7717/peerj.10955/supp-5Table S4Click here for additional data file.10.7717/peerj.10955/supp-6Figure S1Acquisition was limited to cells expressing V450 fluorochrome/CD45 (common leukocyte antigen) (trigger) at a particle cut-off size (FSC) of 4000. Diluted whole blood samples were acquired at a medium flow rate  by 17-color, LSRII Fortessa flow cytometer at the WRAIR Flow Cytometry core facility. (A) After excluding doublets, leukocytes were gated on side-scatter and CD45. Of the two CD45 populations shown in S1A here, only the CD45hi population expressed all the other lineage markers for innate and adaptive immune cells subsets. Therefore, CD45hi population was selected for further characterization of following cell subsets (B) CD45+ gated CD4+ T cells and CD8+ T cells (C) CD45+ gated CD20+ B cells and CD45+ gated CD3+T cells (D) CD3+ gated activated HLA-DR+CD3+ T cells (E) CD3+ gated CD4+ T cells, CD3+ gated CD8+ T cells, CD4-CD8- double negative (DN) T cells (F) CD3+ gated, activated HLA-DR+CD4+ T cells and HLA-DR+CD8+ T cells (G) CD45+ gated CD14+ monocytes and CD45+ gated HLA-DR+CD14+ monocytes (H) CD45+ gated CD3-CD16/CD159a+ NK cells, CD45+ gated CD3-CD16/CD159a+HLA-DR+ NK cells, CD45+ gated CD3+CD16/CD159a+ NKT cells, CD45+ gated CD3+CD16/CD159a+HLA-DR+ NKT cells. Gating strategy for dendritic cells (DCs) is not shown here. CD45+ leukocytes co-expressing HLA-DR and CD11c were considered as DCs after excluding the cells expressing CD14 and CD16/CD159a, CD3 and CD20 (CD45+CD14-CD16/CD159a-CD3-CD20-CD11c+HLA-DR+).Click here for additional data file.10.7717/peerj.10955/supp-7Figure S2Plasmodium falciparum (Pf) full-length circumsporozoite (CSP) protein (PfCSP) in gp96-Ig-PfCA vaccinated animals (n=5) and D/Ad-PfCA vaccinated animals (n=5) at 6 days, 20 days and 2.5 months post-last vaccination. Data represent mean and the standard deviation.(A) Specific to each of the four dengue serotypes (DENV 1-4) in DPIV/TDENV-LAV vaccinated animals (n=10) at 2 months and 6 months post-DENV-2 challenge (B) specific to Click here for additional data file."}
+{"text": "Coumarins are naturally occurring molecules with a versatile range of activities. Their structural and physicochemical characteristics make them a privileged scaffold in medicinal chemistry and chemical biology. Many research articles and reviews compile information on this important family of compounds. In this overview, the most recent research papers and reviews from 2020 are organized and analyzed, and a discussion on these data is included. Multiple electronic databases were scanned, including SciFinder, Mendeley, and PubMed, the latter being the main source of information. Particular attention was paid to the potential of coumarins as an important scaffold in drug design, as well as fluorescent probes for decaging of prodrugs, metal detection, and diagnostic purposes. Herein we do an analysis of the trending topics related to coumarin and its derivatives in the broad field of drug discovery. Coumarins are molecules that belong to a very special family. Their conjugated double ring system makes them interesting molecules for different fields of research. Coumarins can be found in industry as cosmetics and perfume ingredients, as food additives, and especially in the pharmaceutical industry in the synthesis of a large number of synthetic pharmaceutical products . This laDipteryx odorata). It can also be found in sweet woodruff , vanilla grass (Anthoxanthum odoratum), and sweet grass (Hierochloe odorata), among others. This explains the great interest in the extraction and characterization techniques of natural coumarins, and in the synthesis of their derivatives. In addition, the simplicity of its chemical backbone is very attractive, as well as the reactivity of the benzene and pyrone rings. Conjugated double bonds are responsible for an electronic environment that plays a very important role in this family of compounds.Coumarin  is foundThis review is based on the most relevant literature that comprises new data from recent research articles and overviews on the development of new therapeutic solutions and fluorescent probes based on the coumarin scaffold. The research articles and reviews organized to prepare this manuscript have been compiled from various electronic databases, including SciFinder, Mendeley, and PubMed. The latter was the main source of information, due to its specificity in the biomedical field.Searching for the word \u201ccoumarin\u201d in Mendeley, PubMed, and SciFinder in early November 2020, and filtering by year \u201c2020\u201d, more than a thousand references appeared. Another search was conducted in late December to include as much information as possible in this manuscript. A diversity of journals from different fields publishes research articles and reviews related to the biological interest of both plant extracts containing coumarins and/or synthetic molecules based on this scaffold. Hybrid molecules containing different pharmacophores  like pipI) of aldo\u2013keto reductase (AKR) presenting an iminocoumarin scaffold, with activities between 25 and 56 nM, have been described for the treatment of prostatic cancer [II) has allowed the preparation of new multitarget mitogen-activated protein kinase (MEK) inhibitors and nitric oxide (NO) donors, both with antiproliferative properties [III  exhibiting a hydroxamate structure similar to HDACi vorinostat (SAHA) has been published [Other important targets for cancer treatment, especially lymphomas, are histone deacetylases (HDACs). A series of coumarins (ublished . The comV). This molecule is an inhibitor of thioredoxin reductases (TrxRs), presenting high antitumor activity (IC50 = 3.6 \u03bcM), and is also used as a diagnostic agent [In addition, it is worth highlighting the design of hybrids in which one part of the molecule provides fluorescent properties, and another provides therapeutic action (theranostic). Such is the case of the fusion of a 7-aminocoumarin fluorescent ring with a chalcone fragment (ic agent . CoumariVI) has also been described, in which the chlorambucil pharmacophore is incorporated into more complex carbazole\u2013coumarins , carriers of a mitochondrial triphenylphosphonium ligand. This system allows, by irradiation, the controlled release of the chemotherapeutic agent [The preparation of photo-triggered drug delivery systems , and radioactive iodine-resistant advanced thyroid carcinoma) were studied . The samVII and VIII), especially active on Gram-positive and negative bacteria according to substitution patterns [IX) that show activity on methicillin-resistant Staphylococcus aureus (MRSA) [There is also an abundant bibliography related to the interest of coumarins as antimicrobials. Most of the projects are still inspired by the classic antibiotic novobiocin. There are several works in which antibacterial activity is found due to the presence of an azole ring introduced in different positions of the coumarin system. Articles have been published recently on the antibacterial activity of azole\u2013coumarins, as well as 3/4/7 substituted arylcoumarins  .X (I), active against MRSA in nanomolar concentrations [XI, a coordination of coumarin\u2013quinoline hybrids with Cu(I), with activity against Flavobacterium psychrophilum, a Gram-negative bacterium that causes significant septicemia in fish, causing devastating economic problems in aquaculture [Interestingly, coumarin metal complexes also show antibacterial activity. Such is the case of 3-arylcoumarins that present general structures X , coordintrations ; or the aculture .Leishmania was described [XII). Derivatives of this natural product have been prepared and substitutions at positions 6 and 8, as well as the phenyl ring at position 4, turned out to be mandatory for the studied activity. This structure\u2013activity relationship (SAR) study led to the synthesis of the simplest and most lipophilic analogue XIII  [In addition to the antibacterial activity, in a recent and comprehensive review on coumarins, activity against protozoa of the genus escribed . The mosgue XIII , the mosin (XIV) .XV) [para position with a trifluoromethoxy group that originated compound XVI  inhibitor. With this in mind, dual inhibitors of HIV-1 RT and protease (PR) have been designed, in which the coumarin fragment responsible for RT activity is linked to the fragment of the antiretroviral darunavir, active against PR of the HIV, through different amide, carbamate, or amine linkers (XV) . The secound XVI  with IC5ectively .XVII), hybrids that can therefore be used in the treatment of immunomodulatory diseases.Although we have found very few publications related to these activities, in some cases the antioxidant activity of coumarin derivatives of both natural  and syntXVIII) as inhibitors of the Kallikrein-related peptidase 9 (KLK9) involved in inflammatory processes of the skin is reported [XIX) gives rise to new inhibitors of cyclooxygenase-2 (COX-2) with activities comparable, in many cases, to indomethacin [Regarding the anti-inflammatory activity, it is worth mentioning a review on the coumarins of natural origin  with a detailed anti-inflammatory activity due to the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2 factor) that protects cells against stress oxidative . In othereported . Finallymethacin .XX) have been described as antagonists of hA3 receptors, showing a high affinity (in the low nanomolar range) and selectivity for this subtype [XXI) have been described as dual hA1/hA3 antagonists in the low micromolar range [The affinity of the coumarin system for adenosine receptors has also been published recently. The 3-arylcoumarins  [XXIII (50 = 12.98 mM) [The activity of coumarin derivatives on \u03b1-glucosidase was also reviewed in 2020 , in a stXXII  being a 3.87 mM) . This ac) [XXIII  being de2.98 mM) .IX and XII. These are coumarins that incorporate arylacrylamide substituents at position 3 (XXIV) that showed inhibitory activity in the nanomolar range [XXV) [Closely related to these structures, works have been published on coumarin derivatives with inhibitory activity on carbonic anhydrase ar range . In othear range  that thege [XXV) .XXVI), both fragments with demonstrated tyrosinase inhibitory activity [These last structures are closely related to others that present tyrosinase inhibitory activity in the sub-micromolar range. This is the case of coumarins  that incorporate a kojic acid fragment through a triazole linker at position 4 (activity .XXVII) that presents a high inhibitory activity of the steroid sulfatase (best compound of the series with IC50 = 0.13 \u03bcM), which is of interest in the treatment of hormone-dependent breast cancers [Likewise, the introduction of a sulfamate group in the coumarin scaffold originates a hybrid prototype  is another case of hybrid structures as selective MAO-B inhibitors [XXX) allowed obtaining derivatives with significant inhibitory activities of AChE/BuChE and beta-secretase 1 (BACE1) [XXXI) with iron-chelating properties was incorporated [The role played by coumarin derivatives as agents that exhibit biological activities associated with neurodegenerative diseases, such as Alzheimer\u2019s disease, is very important. Throughout this year, a large number of manuscripts related to this field have been found. Due to the multidirectional nature of these diseases, there are also many works on hybrid coumarins directed at different pharmacological targets, such as monoamine oxidase B (MAO-B) or acetylcholinesterase (AChE), amyloid aggregation, or oxidative stress, among others. Hybrids of general structure XXVIII  have beehibitors . The inc (BACE1) . In otherporated .XXXII) and carbazole\u2013coumarin (XXXIII) hybrids [XXXIV) [Other multitarget structures are the benzotriazole\u2013coumarin  . Compoun [XXXIV) .XXXV) have recently been published for their potential against monoamine oxidase A (hMAO-A), hMAO-B, hBACE1, hAChE, and butyrylcholinesterase (hBuChE) [hMAO-B inhibitors in the nanomolar range; six turned out to be hMAO-A inhibitors in the low micromolar range; one showed inhibitory activity of hBACE1, and another one hAChE inhibitory activity, both in the micromolar range. In addition to the enzymatic inhibition, all of the studied molecules proved to be non-cytotoxic to neurons in the motor cortex. As a main conclusion, results suggest that by modulating the substitution pattern at position 7 of the scaffold, selective or multitarget molecules can be achieved.The 7-amidocoumarins ((hBuChE) . The reshMAO inhibitors, in 2020 a theoretical work was published comparing different QSAR models and docking calculations, in order to predict the hMAO-B activity of the 3-arylcoumarins [hMAO-A and hMAO-B, and the most promising models were validated. Selective activities were found in the low nanomolar range against this isoenzyme for 6 and 8 methyl-substituted 3-arylcoumarins, also presenting methoxy groups or bromine atoms in different positions of the 3-phenyl ring (XXXVI). These advancements may represent robust tools in the design of potent and selective derivatives.The 3-arylcoumarins are a family of compounds with proven activity on different targets related to neurodegenerative diseases, especially Alzheimer\u2019s and Parkinson\u2019s diseases, the two most prevalent. In the last decade, several manuscripts described very promising activities of this scaffold, both as selective and multitarget compounds. SAR studies were performed, and important conclusions were drawn based on the substitution patterns in the main scaffold. Due to the large number of molecules based on this scaffold currently synthetized and studied as oumarins . Based oXXXVII) as novel and promising agents against Parkinson\u2019s disease have been described [hMAO-A and hMAO-B, antioxidant profile, neurotoxicity in neurons of the motor cortex, and neuroprotection against hydrogen peroxide production were studied. The in vivo effect on locomotor activity was also evaluated by an open field test (OFT) for the most potent, selective and reversible hMAO-B inhibitor of the series: 3-(4\u2032-bromothiophen-2\u2032-yl)-7-hydroxycoumarin (IC50 = 140 nM). In reserpinized mice pre-treated with levodopa and benserazide, this molecule exhibited a slightly better in vivo profile than selegiline, currently a therapeutic option for Parkinson\u2019s disease. The results suggested that the 7-position substitution of the coumarin scaffold is interesting for enzyme inhibition. Furthermore, the presence of a catechol at positions 7 and 8 exponentially increases the antioxidant potential and the neuroprotective properties.Analogues of 3-phenylcoumarins were also published during 2020. The discovery and optimization of 3-thiophenylcoumarins (escribed . This stII), and upregulating the expression of NF-\u03baB inhibitor (I\u03baB-\u03b1). An attenuation of cognitive dysfunction by these compounds was observed. This effect can at least be attributed to the inhibition of AChE and the reduction of oxidative stress, neuroinflammation, and neuronal loss, opening a new door for these classic coumarins.The neuroprotective effects of xanthotoxin and umbelliferone on streptozotocin (STZ)-induced cognitive dysfunction in rats were evaluated . AlzheimThe classic anticoagulant effect of specific coumarin derivatives, based on acenocoumarol and warfarin, also remains one of the classic applications for this family. During 2020, a review on this topic was published .In addition to the interest of coumarins as a versatile scaffold in drug design, the important role that this scaffold plays as fluorescent probes to detect metals, enzymes, and biomaterials, among others, should be highlighted ,57,58,59XXXVIII) [XXXIX) [XL) [XLI) [XLII) [Coumarins are being used in the selective detection of metals such as copper (XXXVIII)  or its d [XXXIX) ,62. A re [XXXIX) . Other wIX) [XL) . In the L) [XLI) . In some) [XLII) .XLIII) used in the determination of cysteines [XLIV) [XLV) [In some cases, these metal complexes serve as probes for the detection of biothiols, as in the case of copper complexes with benzothiazoles  . In otheV) [XLV) .XLVI) [XLVII) [In addition to the determination of metals, in many cases coumarin derivatives are used as fluorescence probes for the detection of hypochlorite, as in the case of coumarin\u2013thiophene complexes (XLVI)  or of 2- [XLVII) .XLVIII) [XLIX and L), and can be used to photochemotherapeutically target the mitochondria in the treatment of cancer [Coumarins can also be used as photocleavable linkers in the controlled release of drugs or biomaterials. This is the case of the in vivo photolysis of the microtubule inhibitor 4-pyridinomethylcoumarin (XLVIII) . 7-Hydrof cancer ,73.LI) [LII) [LIII) [Other reviews report on the use of 7-hydroxycoumarin and its derivatives in determining the activity of cytochromes P450 (CYP) enzymes  as well LI) . The useI) [LII) . Finally) [LIII) ,78.Coumarins are privileged structures for biological applications. Their conjugated double ring system allows different spots for chemical modifications, and a large number of derivatives can be obtained. Therefore, structure/activity studies appear to be the hottest emerging topic. During the year 2020, more than a thousand research articles and reviews related to coumarins could be found. This highlights the great potential that these molecules can have in different fields of research. For simplicity, our overview focused on the potential of coumarins in medicinal chemistry. The most relevant studies were included. The range of applications described in this document, and some others outside the scope of this general description  and diagnostics.Due to the length of this general overview, synthetic strategies for obtaining new coumarins have not been discussed in detail. To find information on the most recent synthetic pathways, we recommend the manuscripts by Molnar and co-authors  and KovaCoumarins belong to a privileged family for biomedical proposes. Their simplicity, chemical properties, and the efficiency of the synthetic routes to obtain a wide range of substitution patterns make these compounds highly attractive and versatile for medicinal and biological chemists. To date, and during 2020, more than a thousand research articles and reviews containing information on coumarins appear in the PubMed, SciFinder, and Mendeley databases. The number of research groups working on this scaffold, and the impact of the results, highlight the potential of these molecules. Special attention has been paid to the potential of coumarins in drug design, as well as to fluorescent probes. This last application seems to be the most promising field of research for the next few years, since several coumarin derivatives have shown great potential in the decaging of prodrugs (drug release) and for diagnostic purposes."}
+{"text": "In Bolivia, before 1982 there were no records of visceral leishmaniasis (VL) cases that would allow us to review and describe the temporospatial occurrence of VL by ecoregions in provinces and departments of Bolivia to evaluate its impact on public health, risk of outbreaks, or dispersion.  This update on VL in Bolivia is based on research, reviews, and retrospective literature analyses of online data and libraries and institutional reports, from 1939 to the present. Lu. longipalpis vector. VL cases from seven departments, involving 12 different ecoregions were located within the Amazon and Plata basins. In Bolivia, 56 cases of VL have been reported. Until 2014, only three endemic departments had been identified . Since then, further cases have been recorded in Pando, Cochabamba, and Beni, and in Chuquisaca in 2015. In Yungas, a VL focus was confirmed by isolating and comparing parasites from human and dog cases, and from the Lutzomyia longipalpis is its main vector (currently dispersed in six departments). The primary vectors in areas where Lutzomyia longipalpis is absent are Migonemyia migonei and Lutzomyia cruzi.  We confirmed that dogs are its primary reservoir, and  Official attention to neglected tropical diseases (NTD) in Bolivia, including leishmaniasis, has been limited in focus due to the misconception of \"highland country\" (Andean country); Authorities, Visceral leishmaniasis (VL) researchers, and health care professionals need to better understand the vulnerability of the native population, which inhabits an endemic tropical area that covers 60% of the nation\u2019s territory. Our purpose is to contribute evidence to broaden public awareness of the population\u2019s exposure to Visceral leishmaniasis in BoliviaBolivia is located at 10\u00b0 to 23\u00b0 south latitude. It is divided into nine departments covering a surface area of 1,098,581 km\u00b2. Bolivia is also divided into three physiographic regions: an Andean Zone, a subtropical Zone, and a tropical Zone. VL  is transmitted along the river basins and certain ecoregions of the latter two zones: the Subtropical zone in the center-south of the State covers 13% of the territory , with altitude decreasing from 2,500 to 900 m (from west to east), mild to warm climate, from 15 to 25\u00b0C. The tropical Zone in the north, northeast, east, southeast and south of Bolivia covers 59% of the national surface . It is a vast plain with low plateaus covered with widespread forests at altitudes of 900 m and below. The zone has an average annual temperature of 22 to 25\u00b0C. Both zones house two river basins, the Amazon, which covers 65.9% of the country, and the Plata, which covers 20.9%.,Lu. longipalpis (the main VL vector) inquiries were carried out in the departments of La Paz, Beni, Santa Cruz, Pando, and Tarija during the periods described above.In Bolivia, phlebotomine (sandflies) are found in a wide array of habitats spanning altitudes of 170 to 2700 mLeishmania sp. parasites. In Yungas, there are no wild dogs, or they are extremely rare within the focus. Therefore, various attempts have been made in La Paz, Beni and Tarija departments to define the main VL reservoirs. Captured wild mammals, and clinically suspicious dogs were studied by autopsy. Samples of liver, spleen, and bone marrow were taken, and used to seed culture media and inoculate hamsters.In the literature, various species of medium and small wild mammals  have been found infected with -,,VL diagnosis is the Programa Nacional de Control de la Leishmaniasis\u2019s (PNCL\u2019s)The PNCL-recommended therapy used in Children's Hospital is Meglumine Antimoniate  with intravenous or intramuscular injections at a dose of 20 mg SbV/kg/day for 30 days, which generally yields good resultsLutzomyia longipalpis, which is a complex species whose intra-specific taxonomic level is under discussionLu. longipalpis include Lu. cruzi,Migonemyia migonei,L. infantum vectors in VL areas where Lu. longipalpis is not present, or in scenarios in which two or more species display sympatric presence.To date, 121 species of Phlebotominae have been recorded in BoliviaLutzomyia longipalpis  acts as a primary vector. In 1973, Jorge Velasco reported that 78% of catches in Yungas were Lu. longipalpis specimens. Le Pont F. captured Lu. longipalpis in forest and domestic, but mainly in peri-domestic zones in Yungas, finding it to be the most competent anthropophilic vector among 14 species studied in or near dwellingsLeishmania sp. infection rates determined by dissection and direct observation were between 0.8 and 4.2%, according to capture site, incriminating Lutzomyia longipalpis as Yungas\u2019 VL vectorLu. longipalpis morpho-types had been described: The 1S phenotype in the aforementioned focus in La Paz, and the 2S phenotype found in Santa Cruz and in two Andean areas between 1,800 and 2,700 m in the towns of Chuquisaca and Potos\u00ed-Lu. longipalpis presence was recorded in Assis town, Acre state, tri-national border Brazil, Bolivia, and PeruLu. longipalpis phenotype 1S dispersion in these two regions (Pando and Tarija), necessitating further studies.Prior to 2017, four distribution regions in Bolivia, and two Lutzomyia cruzi (Mangabeira 1938). In 2010, in El Carmen town, Busch province, Lu cruzi presence was describedLu. cruzi as the most abundant species in the same department  and in small towns near the Pilcomayo River  in Tarija. Both its presence, and its current or potential importance as a VL vector should be defined in the Bolivian Pantanal of the department of Santa Cruz\u2019s border with BrazilMigonemyia migonei. This species was recently associated with natural L. infantum infection in the VL endemic zone of Pernambuco, BrazilLu. Longipalpis vector.Leishmania infantum DNA in the insect digestive tract. L. infantum DNA sequences have been obtained by PCR-RFLP in Ny. neivai and Ny. whitmaniOther species. Phlebotominae species have been described as potential vectors based on their presence in disease foci, their high anthropophilicity, and identification of Leishmania hertigi variantOur investigations of wild animal disease reservoirs did not reach a conclusive result. We only isolated one enzymatic In 2001, in the department of Tarija, 148 specimens of 9 wild mammal species were captured that could not be processed to detect the disease, because of a health quarantine declaration due to the presence of Hanta virus presence in the area during that periodL. infantum was isolated from five dogs (Canis lupus familiaris) from the Yungas area with serological confirmationLu. longipalpis (vector). In Yungas, most dogs with the disease die around two to three years-old. Dogs five or more years-old are infrequently foundRegarding domestic reservoirs, in 1982, Indoor use of insecticides widely used for 62 years (1956-2018) for \"malaria control\" displays a marked chronology of the introduction and retirement of different chemical agents: Organochlorines 1956-1993, Pyrethroids 1993-2013; Carbamates 2014-2018. These treatments, coupled with other vector control initiatives decreased endemic malaria municipalities from 148 to 19 by 2018. Many of the 129 municipalities, where insecticide is no longer being fumigated on walls, was endemic to malaria and leishmaniasis. Thus, a potential collateral benefit to reducing abundance of vectors other than anophelines has been eliminated. 2) in Sud Yungas assessing the impact of the intervention in phlebotome. He recorded a pretreatment average of 16.7 sandflies/night/CDC light trap, and managed to eliminate Lutzomyia longipalpis from houses and animal shelters by 9 and 10 months post-treatment, respectivelyLe Pont in 1989 conducted a residual spray study with deltamethrin  of IBBA (Instituto Boliviano de Biolog\u00eda de la Altura), the LNPE-INLASA , the PNCL , health related documentation, and previous publications. This search allowed us to find and review all recorded VL cases up to 2010, the year in which notifications began to be registered by SNIS-VE .Ecoregions dissemination :,Bolivian internal human migration from highlands to lowlands (colonization) beginning in the 1980s and linked to socio-economically motivated development projects, caused important anthropic impacts on the environment. This activity continues the degradation and invasion of natural ecosystems, leading to significant change. One unforeseen consequence was an increase in CL incidence at the national level, with more than 3,000 cases/year and a trend toward VL dispersionTo better understand these trends, CL and VL investigations were carried out in different ecoregions, in four of the seven departments, using native VL case reports.The area of La Paz has three ecoregions . Santa Cruz has two zones, an old endemic zone in Germ\u00e1n Busch province, and the southeast of Bolivia , formed by three ecoregions. In two of these (cerrado chaque\u00f1o and bosque seco Chiquitano) seven cases have been reported. One study focused on the two provinces in the department of Tarija: Arce and Gran Chaco, to the south of Bolivia in Salta province border, Argentina, where six cases have been reported within the potential transmission area. Furthermore, the SNIS-VE has reported VL cases in some provinces of the departments of Cochabamba, Beni, and Pando (2014), and in Chuquisaca (2015).The first three historical reports are: Gatti et. al. (1939); Barros and Rosenfeld (1942); and Arruda et. al.(1949); these provided no certain tracing of the origins of their infections, but we add them to the record of general VL cases in Bolivia.Leishmania sp. Infections. Most cases were in malnourished children from rural areas found in 18 provinces (15 ecoregions where the transmission cycle can be developed) in seven departments. Patients with VL have been reported in areas endemic for cutaneous leishmaniasis, mainly with L. (V.) braziliensis. Families that had some knowledge of cutaneous leishmaniasis and the economic resources to transfer their patients to a main city, did so, so that they could be diagnosed and treated in reference centers. Seventy nine VL cases have been reported spanning 36 years (1982 - 2018). Fifty six of these had Visceral Leishmania, and 23 had Our investigations .Leishmania infantum was isolated and typed. He was recognized as the \"First indigenous case of Visceral Leishmaniasis in Bolivia\". , Cochabamba (eleven cases), and Beni (one case). In 2015, one case came under study by the ministry of health in Chuquisaca. A Medical Records review of La Paz city Children's Hospital covering the last ten years (2009 to 2018) idetified four additional diagnosed VL cases thath were not included in the SNIS-VE records. Based on SNIS-VE notifications, we consider Arani provinces in Cochabamba, and Oropeza provinces in Chuquisaca to have extremely low to no likelihood of vectors. This conclusion is supported by notification records from large city hospitals located in the Andean zone where there is no transmission .One hundred and eighteen years after the discovery of the first VL case in South America, this disease is currently one of the most neglected, and mainly affects disadvantaged human populations, such as those living in developing countries with poor sanitary conditions, and populations involved in anthropic environmental transformations in subtropical and tropical areas. Both of the above characteristics frequently manifest in Bolivia. Therefore the main objective of the present study is to contribute compiled evidence from 19 national references, five national meetings of leishmaniasis officials/representatives with local and regional representatives, and technical reports, to broadly disseminate knowledge of the state of VL transmission throughout the Bolivian Health System.L. infantumLu. longipalpis,Lu. cruzi and Mg. migonei), in addition to a number of homogeneous ecological areas shared with neighboring countries with well known established VL foci, including Mato Grosso do Sul, Brazil,,,In Bolivia, there is a significant presence of dogs infected with Lu. longipalpis genetic structure along these borders suggested possible hybridization of a recently arisen urban dispersive population with ancestral rural populations, thus permitting a disease with rural endemic and scattered transmission to become epidemic and urban. We have identified three outbreaks of VL prior to 2009, and between 2014-2015 national health authorities have reported four other outbreaks . These regional health events happen in the context of vector urbanization and dispersion of VL parasite transmission mechanisms, which began in the 1970s in northeastern Brazil. Between 2000-2013 it was already generating outbreaks in southern Brazil, northern Argentina, Paraguay, and Uruguay. At the same time, along Bolivian borders with these countries, transmission and presence of vectors was being reported. Lu. longipalpis records in six departments, four of these with human VL cases , show a new VL epidemiological scenario in Bolivia.Current The recently created Unified Health System  in Bolivia (2019) faces as its main challenge the sustained implementation of surveys and laboratory tests over medium and long terms, being necessary to strengthen health promotion and risk prevention efforts among populations living in endemic areas, improving health population coverage , strengthening the quality of diagnosis at all levels, and treatment and epidemiological surveillance.Considering the experience obtained during the last years building \"regional reference centers\", the human resources training needed to form multidisciplinary teams that can develop operational research programs funded by sustainable financing sources is a requisite to confirm new outbreak notifications, reduce relevant knowledge gaps, and assure timely provision of diagnostic supplies (mainly VL diagnosis), medications, and appropriate case management."}
+{"text": "Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step for the precise regulation of gene expression. Despite the apparent close relationship between promoter-proximal pausing and nucleosome, the role of chromatin remodeler governing this step has mainly remained elusive.Saccharomyces cerevisiae using PRO-seq experiments. This non-uniform distribution of RNAPII exhibits both similar and different characteristics with promoter-proximal pausing in Schizosaccharomyces pombe and metazoans. Interestingly, we find that Ino80p knockdown causes a significant upstream transition of promoter-proximal RNAPII for a subset of genes, relocating RNAPII from the main pausing site to the alternative pausing site. The proper positioning of RNAPII is largely dependent on nucleosome context. We reveal that the alternative pausing site is closely associated with the +\u20091 nucleosome, and nucleosome architecture around the main pausing site of these genes is highly phased. In addition, Ino80p knockdown results in an increase in fuzziness and a decrease in stability of the +\u20091 nucleosome. Furthermore, the loss of INO80 also leads to the shift of promoter-proximal RNAPII toward the alternative pausing site in mouse embryonic stem cells.Here, we report highly confined RNAPII enrichments downstream of the transcriptional start site in Based on our collective results, we hypothesize that the highly conserved chromatin remodeler Ino80p is essential in establishing intact RNAPII pausing during early transcription elongation in various organisms, from budding yeast to mouse.The online version contains supplementary material available at 10.1186/s13059-021-02500-1. Hsp genes using various in vivo analyses [Emerging evidence indicates that promoter-proximal pausing is a decisive step in transcription that supports the precise control of gene expression in metazoans . In the analyses \u20135. Theseanalyses  and firsEarly biochemical studies in a purified system identified several critical factors that regulate the establishment and release of paused RNAPII. These studies examined the ability of protein factors to confer the sensitivity to 5,6-dichloro-1-\u03b2-D-ribofuranosylbenzimidazole (DRB), which has been known to inhibit the synthesis of full-length transcripts. In this system, the two critical factors, DRB sensitivity inducing factor   and negaD. melanogaster [Initial genome-wide studies based on the chromatin immunoprecipitation (ChIP) method revealed the widespread occurrence of RNAPII accumulation near the transcription start site (TSS) and implicated its role in poising of gene expression in nogaster \u201312 and mnogaster , 14. Advnogaster \u201317 or senogaster , 19. Parnogaster  and precnogaster , is ableS. cerevisiae, it has been traditionally believed that much of the transcription regulation occurs during the recruitment step of RNAPII to a gene promoter [S. cerevisiae promoter regions [C. elegans [S. pombe [S. cerevisiae examined that well-expressed genes exhibit a modest accumulation of read density downstream of TSS [S. cerevisiae.In promoter , 21. Accpromoter , 23. How regions . Further regions , 26, sug elegans  and S. pS. pombe . In addim of TSS , resultem of TSS , implyinspt6-1004). It was accompanied by a striking defect in nucleosome architecture [Nucleosome poses a strong barrier for RNAPII passage at various stages of transcription, and cells benefit from employing highly conserved chromatin remodelers to overcome these physical barriers . Biochemitecture , furtherHtz1 for H2A [S. cerevisiae [S. cerevisiae [The chromatin remodeler, Ino80p, has been shown to play a key role in the regulation of RNAPII at transcribed genes through its remodeling activity . Ino80p  for H2A , 38. Subrevisiae , 40. Howrevisiae , 42. Aparevisiae , 44. Sevrevisiae \u201347 or atrevisiae . In addirevisiae , 48\u201350, revisiae . NeverthS. cerevisiae using PRO-seq experiment that detects only elongation-competent RNAPII but not inactive RNAPII such as arrested or unstable form [S. pombe and metazoans. Using the auxin-inducible degron (AID) system [S. cerevisiae. Genes, whose pausing sites are regulated by Ino80p, exhibit RNAPII pausing at the alternative pausing site even in the physiological condition, and Ino80p is critical in facilitating the utilization of the main pausing site in a nucleosome context-dependent manner. Furthermore, we observe the shift of RNAPII pausing toward the alternative pausing site upon INO80 knockdown also in mESCs, suggesting the essential role of Ino80p in regulating RNAPII pausing in various organisms from budding yeast to mouse.Here, we reveal a non-uniform distribution of elongating RNAPII in ble form . We exam) system , 53, we S. cerevisiae, we performed PRO-seq [S. pombe spike-in control. We used Ino80p-AID cells [\u03c1  data (GSM1974983) exhibited a generally similar order of signals to those of our data  between two data  cells and found a strikingly similar pattern around TSS with Ctrl data  showed the strikingly accumulated signal downstream of TSS, resembling the distribution of RNAPII pausing  and our PRO-seq data generated in spt4\u0394  [wt (ED665) PRO-seq data showed a highly correlated pattern with the previously reported data generated in wt (972) cells (GSM1974985) [S. cerevisiae data, we classified more paused genes in our data than the previous data in S. pombe    was used1974985)  within bS. cerevisiae, we focused on analyzing distribution in Ctrl PRO-seq data at RNAPII-transcribed protein-coding genes. We also performed precision nuclear run-on 5\u2032 cap sequencing (PRO-cap) with 2-Biotin run-on (biotin-11-CTP and UTP) [To further examine the genome-wide significance of these apparent pausing-like features in and UTP)  in the Cand UTP) , 35. Thuwt Rpb3p NET-seq\u00a0[N = 5697; see \u201cMethods\u201d). Both NET-seq and ChIP-exo data exhibited the enriched signal near TSS similar to PRO-seq data  of PR intensity might be because BioGro data was generated based on the tiling array, which could not show the high resolution as much as PRO-seq.We first tested whether the Ctrl PRO-seq signal around TSS was consistent with the previously published  NET-seq\u00a0 and ChIP NET-seq\u00a0, indepen NET-seq\u00a0. We geneS. cerevisiae was thus higher than that previously observed in S. pombe (28%) [D. melanogaster (63%) [We next classified protein-coding genes as being paused or not paused as described above. We identified 2599 (45.6%) high-confidence paused and 1990 (34.9%) not paused genes among 5697 filtered protein-coding genes   but loweS. cerevisiae. First, we generated heatmaps of PRO-seq, PRO-cap, and existing data of MNase-seq [S. pombe [Next, we sought to identify the features of the PRO-seq pattern in Nase-seq  and ChIPNase-seq . PRO-seqNase-seq . We alsoS. pombe .S. cerevisiae was lower than generally in humans. Indeed, we found that about half were not paused genes in the group of genes with the lowest gene activity, whereas about one-third or a quarter was not paused genes in the groups of middle and highest gene activity. Based on our collective results, we propose that RNAPII distributions at the 5\u2032 end of genes in S. cerevisiae are an aspect of early transcription elongation that showed similar characteristics with PR pausing in S. pombe and metazoans.At protein-coding genes, gene activity, which was determined by PRO-seq GB density , generalS. cerevisiae compared to metazoans. The peak of average PRO-seq enrichment within PR regions was located ~\u2009100\u2009bp downstream of TSS . It confirmed that the addition of a small volume of ethanol to YPD did not affect elongating RNAPII distribution across genes.The highly conserved chromatin remodeler Ino80p is expected to have a putative role in regulating PR RNAPII distributions given that it has been not only shown to localize around the 5\u2032 end of genes  and but Interestingly, we found that PRO-seq experiments under Ino80p knockdown (Ino80p-KD) caused an upstream skewed pattern of the peak within PR regions Fig. a. The exp value for those genes in an effort to focus on the most significantly changed peak upon Ino80p-KD (77 genes out of 353 shift-to-5\u2032 genes and 12 genes out of 150 no-shift genes showed multiple increasing peaks). We designated these selected increasing peaks in Ino80p-KD as P2. To compare the significance of increase at P2 for shift-to-5\u2032 and shift-to-3\u2032 genes, we displayed heatmaps of the PRO-seq log2 fold change around P1  between shift-to-5\u2032 genes upon Ino80p-KD and shifted to 5'\u00a0genes in arp5\u0394  or not paused (N = 2072) among the 16,068 protein-coding genes . To analyze the transition of RNAPII pausing under INO80-KD, we defined P1 and P2 in the same manner as in S. cerevisiae. However, results obtained from these analyses differed from those observed in S. cerevisiae. We found significantly increasing 2962 peaks for 1890 genes upon INO-KD and examined that increasing peaks were mainly located downstream of P1 . Heatmaps of the PRO-seq log2 fold change around P1 showed that downstream increasing P2 was more robust and frequent than upstream increasing P2  was located downstream of the +\u20091 nucleosome  containing 10% knockout serum replacement , 1% non-essential amino acids , 1% sodium pyruvate , 0.1\u2009mM \u03b2-mercaptoethanol , 1% FBS , 0.5% antibiotic-antimycotic , and 1000\u2009units/ml LIF .E14Tg2a mESCs were maintained under feeder-free conditions at 37\u2009\u00b0C with 5% COThe siRNAs against EGFP (5\u2032-GUUCAGCGUGUCCGGCGAG-3\u2032) and INO80 (5\u2032-GGCUUAUCUGUAAAGGCACAAUUGA-3\u2032) were synthesized and annealed by Bioneer. mESCs were seeded to plates, incubated for 24\u2009h, and transfected with the indicated siRNAs  using DharmaFECT I  according to the manufacturer\u2019s protocol. Briefly, 50\u2009nM of siRNAs and DharmaFECT I diluted in Opti-MEM  were separately incubated for 5\u2009min at 25\u2009\u00b0C, and further mixed and incubated for 20\u2009min at 25\u2009\u00b0C, and then used for transfection. The culture medium was replaced at 24\u2009h of transfection, and cells were harvested at 48\u2009h of transfection.Whole-cell lysates of Ino80p-AID cells were prepared using a standard bead-beating protocol, and proteins were eluted by boiling at 100\u2009\u00b0C for 5\u2009min in 2\u00d7 SDS sample buffer . The utilized antibodies included anti-FLAG M2  and anti-\u03b2-actin . Anti-FLAG M2 was used to detect 9xFLAG-tagged Ino80p, and \u03b2-Actin was used as a loading control. The Ino80p-AID cells were a gift from the Friedman lab as described\u00a0above .mESCs were washed with PBS  and detached from the dishes by incubation with trypsin-EDTA  at 37\u2009\u00b0C for 2\u2009min. The trypsin was inactivated by the addition of GMEM with 1% FBS, and 0.5% antibiotic-antimycotic, and the cells were harvested, washed with PBS, and resuspended in EBC300  containing protease inhibitors. Whole-cell lysates were prepared by vigorous vortexing the cell mixture for 30\u2009min followed by centrifugation for 30\u2009min at 4\u2009\u00b0C. Proteins were eluted by boiling at 100\u2009\u00b0C for 5\u2009min with 5\u00d7 SDS sample buffer. The utilized antibodies included anti-INO80  and anti-Tubulin . Tubulin was used as a loading control.2O. Cell pellets were resuspended in 10\u2009ml of 0.5% sarkosyl  and incubated for 20\u2009min on ice. Cells were spun down at 400\u00d7g for 5\u2009min at 4\u2009\u00b0C and resuspended in storage buffer  to an optical density (OD) of 5 per 200\u2009\u03bcl. The solutions were flash-frozen using LN2 and stored at \u2212 80\u2009\u00b0C.Yeast cells were permeabilized as described  with som6 plated mESCs were washed once with PBS and detached by incubation with trypsin-EDTA at 37\u2009\u00b0C for 2\u2009min. The trypsin was inactivated by the addition of GMEM with 1% FBS and 0.5% antibiotic-antimycotic, and the cells were harvested and washed twice with ice-cold PBS. The cells were resuspended in 5\u2009ml of ice-cold swelling buffer  for 5\u2009min on ice. Lysis buffer  was added, and the cell pellets were resuspended by gentle pipetting using an end-cut tip. The cells were centrifuged at 1000\u00d7g for 5\u2009min at 4\u2009\u00b0C, and the cell pellets were resuspended in 1\u2009ml of freezing buffer . The pelleted nuclei were transferred into a new 1.5-ml tube and were resuspended in a freezing buffer at ~\u20095 \u00d7 106 nuclei per 100\u2009\u03bcl. The solutions were flash-frozen using LN2 and stored at \u2212 80\u2009\u00b0C.mESCs were transfected with the indicated siRNAs, and nuclei were isolated as previously\u00a0described , 81 withS. pombe (ED665) cells was added to each 5 OD of permeabilized S. cerevisiae sample (or vice versa) before the nuclear run-on reaction was performed. Combined yeast cells were spun down at 400\u00d7g for 5\u2009min at 4\u2009\u00b0C. Nuclear run-on reactions were conducted with 25\u2009\u03bcM biotin-11-UTP , 25\u2009\u03bcM biotin-11-CTP , 125\u2009\u03bcM ATP , and 125\u2009\u03bcM GTP  in run-on reaction buffer  with 0.5% sarkosyl. The reaction mixtures were incubated at 30\u2009\u00b0C for 5\u2009min. For the isolated nuclei of mESCs, nuclear run-on reactions were performed with 25\u2009\u03bcM biotin-11-UTP , 25\u2009\u03bcM biotin-11-CTP , 125\u2009\u03bcM ATP , and 125\u2009\u03bcM GTP  in run-on reaction buffer  with 0.5% sarkosyl. The reaction mixtures were incubated at 37\u2009\u00b0C for 5\u2009min.Nuclear run-on reactions and RNA extractions were performed based on the published protocol  with minRNA was extracted from the run-on-reacted cell pellets using either a standard hot-phenol method (for yeast samples) or TRIzol LS . Next, the respective library was generated, followed by the published PRO-seq or PRO-cap protocols  for the http://hannonlab.cshl.edu/fastx_toolkit/) as follows: Adaptor sequences (5\u2032-TGGAATTCTCGGGTGCCAAGG-3\u2032) were removed, the reads were trimmed to a maximum length of 36\u2009bp and, for PRO-seq, the reads were reverse-complemented. Next, reads that mapped to rRNA sequences were depleted using SortMeRNA [S. cerevisiae (sacCer3) and S. pombe (SpombeASMv2), and then uniquely aligned reads from each genome were parsed for downstream analysis. The processed reads of mESCs samples were mapped to the M. musculus mm10 genome. The coverage of the aligned reads was generated using the genomecov function of BEDtools [Sequence alignment and data processing were performed based on the publicly available alignment pipelines of GitHub, as used in the previous study  with minortMeRNA , and reaortMeRNA : The proBEDtools . Only thBEDtools . The dowBEDtools .N = 6692) were initially used for S. cerevisiae samples. The observed TSS was defined as the single nucleotide with the most PRO-cap reads within the 250\u2009bp upstream and downstream of the annotated TSS, in a similar manner to that used in the previous report [S. cerevisiae, the regions from the observed TSS to downstream 250\u2009bp (TSS to TSS + 250\u2009bp) were used as the PR regions, and those from TSS + 250\u2009bp to the annotated TES were used to calculate the GB signals. For mESCs, the regions from upstream 100\u2009bp to downstream 200\u2009bp of the 5\u2032-peak were used as the PR regions, and the regions downstream 1\u2009kb from the 5\u2032 peak to the annotated TES were used as the GB regions. To classify the genes as being paused or not paused, we calculated the significance of PI. Significant enrichments of the signals from PR regions compared to GB regions were evaluated using Fisher\u2019s exact test with Bonferroni\u2019s correction as in the previous study [p value was lower than 0.01 and as being not paused if the p value was higher than 0.99, as determined using combined replicates. A gene exhibiting p value <\u20090.05 or p value >\u20090.95 for both biological replicates was further assigned as a high-confidence paused or high-confidence not paused gene, respectively. All average profiles centered on the indicated point in this work were generated using a bootstrapped estimation. Briefly, 1000 random gene sets were taken as each representing 10% of the total genes, and the median and confidence intervals were calculated using each average of the subsample. In the relevant figures, the thick line represents the median value and shaded regions indicate the 12.5th and 87.5th percentiles.Protein-coding genes based on the annotated data in the Saccharomyces Genome Database . For each gene, we then identified consensus peaks present across all samples in the data set and filtered out non-systematic peaks with 3-folds lower than the mean value of promoter-proximal signals. The peak showing the highest mean smoothed read count for two replicates in the control condition  was designated as P1. We calculated the smoothed read count ratios of the other peaks to P1, resulting in a n x m read count ratio matrix for m samples and n consensus peaks identified from all genes. To identify the increasing peaks, we next applied an empirical statistical test previously reported [n \u00d7 m read count matrix. Briefly, for each gene, we calculated t-statistic values for the observed ratios of consensus peaks in the samples between two conditions . We then generated an empirical null distribution for t-statistic values by calculating t-statistic values after randomly permuting the samples in the n \u00d7 m read count matrix 1000 times. The adjusted p value was calculated by performing the left-sided test for its observed read count ratio using the empirical distribution. Finally, we selected the increasing peaks as the ones with a p value <\u20090.1. We further filtered out false-positive peaks with log2 median ratios lower than a cutoff after estimating an empirical null distribution of log2 median ratios for the smoothed read count ratios between the two conditions during the above random permutations. The cutoff was determined as the mean of 10th and 90th percentiles in the empirical distribution. To select genes with the unchanged peaks upon knockdown or deletion, we first excluded genes containing the selected increasing peaks as described above. We then sorted the remaining peaks in descending order of p value and selected n peaks from the top to set the number of genes similar to those of the genes with increasing peaks to be compared.We first smoothed each PRO-seq data with a Savitzky-Golay smoothing method  using threported  to the nS. cerevisiae sacCer3 genome or to the M. musculus mm10 genome using Bowtie, which trimmed the 3\u2032 bases to 36\u2009bp (if the raw reads were longer than 36\u2009bp), allowed two mismatches, and for paired-end data, restricted the maximum insert size to 200\u2009bp. BEDtools was used to covert the aligned BAM files to BED formats\u00a0[S. cerevisiae or the observed 5'-peak in mESCs was assigned as the +\u20091 nucleosome. The +\u20091 dyad was defined as the mid-point between the start and the end inflection, and 75\u2009bp around the +\u20091 dyad was referred to as the +\u20091 nucleosome position. To discard false-positive nucleosome positions, nucleosomes that did not overlap the H3K4me3 ChIP-seq enrichment calculated from the existing data [S. cerevisiae (sacCer3) and S. pombe (SpombeASMv2) was used, and unique reads from each genome were parsed for downstream analysis. MACS2 [Raw sequencing reads of the indicated accession numbers were downloaded from NCBI GEO unless otherwise noted. For MNase-seq, raw reads were uniquely mapped to the  formats\u00a0. The BED formats\u00a0 to detering data , 86 wereing data . The Gaus. MACS2  was useds. MACS2 .Additional file 1 : Fig. S1. PRO-seq analysis in S. cerevisiae upon the loss of Spt4p. Fig. S2. PRO-seq analysis in S. pombe.Fig. S3. PRO-seq analysis in mESCs. Fig. S4. PRO-cap detects the precise transcription initiation sites genome-wide. Fig. S5. PRO-seq is highly correlated with Rpb3p NET-seq and ChIP-exo in S. cerevisiae.Fig. S6. Correlation of promoter-proximal PRO-seq pattern with nucleosome architecture and gene activity. Fig. S7. AID system is employed to investigate the immediate effect upon Ino80p knockdown. Fig. S8. The transition of RNAPII in Ino80p knockdown is independent of both TSS usage and H2A.ZHtz1. Fig. S9. The Ino80 complex is essential for RNAPII pausing site determination associated with the +\u20091 nucleosome. Fig. S10. INO80 knockdown yields RNAPII pausing site determination defect in mESCs. Table S1. Summary of PRO-seq reads and reproducibility obtained in this study. Table S2. List of S. cerevisiae\u00a0strains used in this study.Additional file 2. Review history."}
+{"text": "This study aimed to explore the ability of radiomics derived from both MRI and 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) images to differentiate glioblastoma (GBM) from solitary brain metastases (SBM) and to investigate the combined application of multiple models. The imaging data of 100 patients with brain tumours (50 GBMs and 50 SBMs) were retrospectively analysed. Three model sets were built on MRI, 18F-FDG-PET, and MRI combined with 18F-FDG-PET using five feature selection methods and five classification algorithms. The model set with the highest average AUC value was selected, in which some models were selected and divided into Groups A, B, and C. Individual and joint voting predictions were performed in each group for the entire data. The model set based on MRI combined with 18F-FDG-PET had the highest average AUC compared with isolated MRI or 18F-FDG-PET. Joint voting prediction showed better performance than the individual prediction when all models reached an agreement. In conclusion, radiomics derived from MRI and 18F-FDG-PET could help differentiate GBM from SBM preoperatively. The combined application of multiple models can provide greater benefits. The differentiation of GBM and MET faces great challenges in imaging diagnosis because of the sharing imaging features, such as cystic necrosis, ring enhancement, and obvious peripheral oedema, and some METs appear solitary, while GBM may sometimes be multifocal3. In particular, it is challenging to differentiate MET from GBM, while MET appears as SBM. However, the differentiation necessary for the treatment of these two tumours is entirely different4. Histopathological examination is still the gold standard for qualitative diagnosis. However, the accuracy of pathological diagnosis will also be affected by various factors6. Sometimes, a biopsy is unavailable for specific reasons, such as the patient being too weak to undergo surgery, the tumour being involved, or being too close to an eloquent area. Therefore, noninvasive and highly accurate differential diagnosis methods are of great significance.Glioblastoma (GBM) and metastatic tumours (MET) account for a large proportion of brain tumours, especially in the elderly8; however, it is not very practical by traditional research methods based on qualitative features and parameters from MRI9. Radiomics is an efficient research method for extracting many quantitative features from medical images11, providing more information than human eyes can recognize. It offers good performance in assessing the pathophysiology of tumours and distinguishing tumour characteristics13. In recent years, radiomics has made considerable progress in tumour and nontumor disease diagnosis17. There have been several studies on the differentiation of GBM and SBM by radiomics derived from MRI. For example, radiomic features extracted from peritumoral oedema areas in T1-weighted contrast-enhanced imaging (T1C) and T2-weighted imaging (T2) were used to differentiate GBM from SBM19. The above studies have shown good potential, with limited radiomics effectiveness based only on MRI. An 18F-FDG-PET examination can reflect the metabolic characteristics of tumours at the molecular level, and it plays an essential role in tumour detection, staging, and efficacy evaluation20. With the precision and personalization of clinical treatment, the application value of 18F-FDG-PET in tumours has been increasingly recognized and promoted. Therefore, it is necessary to incorporate 18F-FDG-PET into brain tumour radiomics research. Radiomics based on 18F-FDG-PET has been used to differentiate lymphoma and glioma of the central nervous system21. In a previous study by Zhang22, different combinations of conventional MRI (cMRI), including T1C and T2, diffusion-weighted imaging (DWI) and 18F-FDG-PET images were explored to establish different radiomic models to differentiate SBM and GBM and found that the integrated model based on cMRI, DWI, and 18F-FDG-PET had the highest discriminative power between the two tumours. However, in the clinic, advanced sequences such as DWI are not as readily available as cMRI. Therefore, we hypothesize that the radiomics features derived from cMRI and 18F-FDG-PET can also better differentiate the two tumours than MRI alone. Some previous studies on radiomics have shown that each classifier has advantages and limitations24. It is difficult to choose an absolute optimal model. Therefore, we hope to build a variety of models and jointly apply these models to obtain greater benefits.MRI plays a vital role in distinguishing brain GBM from brain SBM25, the MRI images of all cases were acquired from only one MRI scanner and the same as the PET.We retrospectively collected the imaging data of brain tumours in 100 patients  who underwent MRI and 18F-FDG-PET/CT scanning in the First Affiliated Hospital of Chongqing Medical University from April 1, 2016, to March 10, 2021. This study complies with the Declaration of Helsinki, and research approval was granted from the Biomedical Research Ethics Committee of Chongqing Medical University with a waiver of research-informed consent. To avoid the inconsistency of the acquired image acquisition and scanning parameters, which may affect the radiological characteristics and quantitative analysisThe inclusion criteria of patients were as follows: (a) glioblastoma or metastasis confirmed by surgery and pathology; (b) preoperative cranial MR imaging, including T2 and T1C, and preoperative cranial 18F-FDG-PET examination; and (c) the interval between preoperative MRI examination and 18F-FDG-PET examination was no more than two weeks. The exclusion criteria were as follows: (a) multiple tumours; (b) a history of brain tumour biopsy or treatment before MRI and 18F-FDG-PET examination; and (c) unqualified image quality with artefacts or tumour size less than 1\u00a0cm. The patient selection process flowchart is shown in Fig. MR images were obtained from the 3.0\u00a0T MRI system  with an 8-channel head coil. The main parameters of the T1C sequence were as follows: repetition time (TR)\u2009=\u2009750\u00a0ms, echo time (TE)\u2009=\u200915\u00a0ms, slice thickness\u2009=\u20095\u00a0mm, and slice interval\u2009=\u20091\u00a0mm. The main parameters of the T2 sequence were as follows: TR\u2009=\u20098,000\u00a0ms, TE\u2009=\u2009140\u00a0ms, flip angle\u2009=\u200990\u00b0, slice thicknesses\u2009=\u20095\u00a0mm, and interval\u2009=\u20091\u00a0mm.A PET/CT scanner (Philips Gemini TF 64 PET/CT scanner) was used for 18F-FDG PET data acquisition. The participants fasted for at least 4\u00a0h before 18F-FDG , administered injection intravenously at a dose of 5.55\u00a0MBq/kg and then rested in a quiet, dim room for 40\u201360\u00a0min before PET/CT scanning. A PET/CT scan of the head was performed for a one-bed position (5\u00a0min/bed position) with a slice thickness of 2\u00a0mm. The 18F-FDG-PET images acquired from the PET/CT system were calibrated on the PET/CT workstation, on which the interpolation of the 18F-FDG-PET image in DICOM format was performed to double the physical resolution of the image.https://nrg.wustl.edu/software/dicom-browser) for data desensitization, and the desensitized images were loaded into 3D-Slicer  for registration. T1C and 18F-FDG-PET images were registered separately based on the T2 images. A radiologist with 5\u00a0years of experience delineated the tumour and the oedema area around the tumour on the T2 images. After all delineations were complete, a neuroimaging doctor with 10\u00a0years of experience modified and determined the final delineated area. The region of interest (ROI) was copied to the corresponding layers of the registered T1C and 18F-FDG-PET images. In this way, the mask data for each of the three sequences were formed. The two doctors were unaware of the pathological types of all cases.First, MRI and 18F-FDG-PET data were imported into DicomBrowser software (26. Referring to the Image Biomarker Standardization Initiative (IBSI), the radiomics features of T2, T1C, and 18F-FDG-PET were obtained by using Python's PyRadiomics package. All features were extracted from the original and derived images. The latter was processed by a wavelet filter (Wavelet) and Laplacian of Gaussian filter (LoG). The t test was performed on the features extracted from the GBM and SBM cases to eliminate features with no significant difference. The features selected by t test were then used to determine effective features using five dimensionality reduction methods as follows: linear discriminant analysis (LDA), principal component analysis (PCA), partial least squares regression (PLS), near-collar component analysis (NCA), and least absolute shrinkage and selection operator (LASSO)27. Both LDA and PCA are linear dimensionality reduction methods that transform the original n-dimensional dataset into a new dataset through an orthogonal transformation. The partial least squares method uses the basic relationship between the independent and dependent variables to model the covariance structure in the two-variable space to achieve dimensionality reduction. NCA uses the Mahalanobis distance as the distance measurement. The conversion matrix was obtained through the dimensionality reduction in original data and learned by continuously optimizing the classification accuracy. LASSO dimensionality reduction uses the L1 regularization linear regression method to perform dimensionality reduction and to zero part of the learned feature weight, thereby achieving feature sparseness and reducing the data dimensionality. Five classification algorithms were chosen: support vector machine (SVM), logistic regression (LR), K nearest neighbours (KNN), random forest (RF), and adaptive boosting (AdaBoost). SVM classification performance is excellent in a small sample of machine learning tasks28. The logistic regression (LR) classifier runs faster and has higher requirements for feature engineering29. The idea of the KNN classification algorithm is simple and effective, but there is also a large number of calculations during the classification process, which requires considerable memory30. Random forest (RF) reduces the risk of overfitting by averaging decision trees. It is virtually a stable classification method, but the calculation is complex and requires more time to train the model31. Adaptive boosting (AdaBoost) is a vital ensemble learning technology that enhances a weak learner with a prediction accuracy only slightly higher than random guessing into a strong learner with higher prediction accuracy32. However, the disadvantage of this classifier is that it is more sensitive to outliers.For all MRI data, the hybrid white-stripe method was used to perform signal intensity normalization to avoid data heterogeneity biasThe entire dataset was split into a training cohort  and a validation cohort  by stratified sampling using computer-generated random numbers at a ratio of 8:2, and 25 models were generated by applying fivefold cross-validation with the five dimensionality reduction methods and the five classification algorithms. Nomenclature was adopted by combining the names of the dimensionality reduction method and the classification algorithms, e.g., \"LASSO_LR\": a combination of the LASSO dimensionality reduction method and the LR classification algorithms. Three model sets were built, in which 25 models with 18F-FDG-PET and MRI data were regarded as the integration set, 25 models with isolated MRI data were regarded as the MRI set, and 25 models with 18F-FDG-PET data alone were regarded as the PET set.After the three model sets were built, the receiver operating curve (ROC) of each mode was drawn, and the area under the receiver operating curve (AUC) was also calculated. The differences in the average AUC of the three model sets were compared. The model set with the highest average AUC was selected and then ranked the 25 models according to the AUC level. To verify the performance stability of models of different AUC levels, 15 models with three levels of AUC were selected and equally divided into three groups. To present a certain level of difference in AUC value between the models of the three groups, we defined 5 models with the AUC ranking of 1-5th as Group A, 5 models ranked 11-15th as Group B, and 5 models ranked 21-25th as Group C.33 were used to explore the combination performance of the five models in each group. During this process, each model was regarded as a specialist and provided with the same weight in the diagnosis. The final diagnosis was made according to the simple majority rule35. For instance, a case was determined to be GBM when more than three of the five models predicted it to be GBM. According to the consistency of voting results, three agreement patterns were obtained: 3A pattern referring to 3 models reaching an agreement that a case was predicted as GBM or SBM by three of the five models; 4A pattern referring to 4 models reaching an agreement; 5A pattern referring to 5 models reaching an agreement. Accuracy, sensitivity, and specificity were used to evaluate the performance of individual and joint voting prediction.Individual and combined application of five models in the three groups were performed. The same weighting and a simple majority vote methodThe entire workflow of our research is shown in Fig.\u00a0www.ibm.com/products/spss-statistics). Delong\u2019s test was performed with Python 3.8 (https://www.python.org/downloads/release/python-380) for the difference in the AUC values of the models. All statistical tests were two-sided, and the statistical significance level was set at 0.05. P values of less than 0.05 were considered to be statistically significant.Pearson's chi-square test was used to compare the sex difference between GBM and SBMS in the entire data, training cohort, and validation cohort. Student's t test was applied to compare the age difference between GBM and SBM. The Mann\u2013Whitney U test was used to compare the differences in the distribution of AUC between each two of the three model sets. All statistical analyses above were carried out with SPSS 19.0 statistical software  in clinical practice. The collaboration between specialists in clinical practice is significant for making comprehensive and correct decisions40. In this study, the 5A pattern of the joint voting has improved accuracy, sensitivity, and specificity to varying degrees compared with the individual prediction. The performance of the 4A and 3A patterns all shows a downwards trend, and the average level of individual prediction outperformed the 3A pattern, which is similar to the results of Dong41. In the combined application of multiple models, it was interesting that the five models in Group A, with higher AUC values than Groups B and C, were more likely to reach an agreement . In Groups B and C with lower AUC values of the models, the application of the 5A pattern improved the prediction performance more significantly. Similar results can also be found in the MRI and PET sets . This is consistent with the research results of Qians Tables \u2013S5. BothOf course, there are some limitations to this study. First, the image data were obtained by the same MR and PET/CT scanner. Therefore, the samples we obtained were relatively few. Although the results performed well, the generalization ability of each model still needs a large sample size for further verification. In practical work, it is difficult to obtain image data with consistent scanning parameters in different medical institutions or even in the same institution. In addition, the simple voting method with the same weight is adopted in the joint application of multiple models, and more joint application methods and comparisons with different methods can be further explored.Radiomics derived from cMRI and 18F-FDG-PET can help differentiate GBM from SBM preoperatively, which may achieve greater benefits in clinical practice. Multimodal radiomics based on MRI and 18F-FDG-PET is expected to become a powerful research method for the differentiation of intracranial tumours. The combined application of multiple models inspired by MDT can generate extra benefit, especially when the performance of the model is mediocre. The combined application of multiple models can be used as a new method in radiomics research.Supplementary Information."}
+{"text": "The ongoing COVID-19 pandemic is characterized by different morbidity and mortality rates across different states, cities, rural areas, and diverse neighborhoods. The absence of a national strategy for battling the pandemic also leaves state and local governments responsible for creating their own response strategies and policies.This study examines the content of COVID-19\u2013related tweets posted by public health agencies in Texas and how content characteristics can predict the level of public engagement.All COVID-19\u2013related tweets (N=7269) posted by Texas public agencies during the first 6 months of 2020 were classified in terms of each tweet\u2019s functions , the preventative measures mentioned, and the health beliefs discussed, by using natural language processing. Hierarchical linear regressions were conducted to explore how tweet content predicted public engagement.The information function was the most prominent function, followed by the action or community functions. Beliefs regarding susceptibility, severity, and benefits were the most frequently covered health beliefs. Tweets that served the information or action functions were more likely to be retweeted, while tweets that served the action and community functions were more likely to be liked. Tweets that provided susceptibility information resulted in the most public engagement in terms of the number of retweets and likes.Public health agencies should continue to use Twitter to disseminate information, promote action, and build communities. They need to improve their strategies for designing social media messages about the benefits of disease prevention behaviors and audiences\u2019 self-efficacy. COVID-19 is a new infectious disease that is caused by SARS-CoV-2, a new and potentially deadly coronavirus. The ongoing COVID-19 pandemic is characterized by different morbidity and mortality rates across different states, cities, rural areas, and diverse neighborhoods. The absence of a national strategy for battling the pandemic also leaves state and local governments responsible for creating their own response strategies and policies . Thus, iPublic health agencies have been actively using platforms such as Twitter and Facebook to communicate with their stakeholders during public health crises. The accumulating literature on organizational social media use has identified the following three primary functions: information, action, and community . The infAccording to the Health Belief Model (HBM), a person\u2019s decision to adopt a recommended health behavior is influenced by their desire to avoid an illness and their belief that the recommended behavior can help prevent an illness . The folThe original HBM was a psychological model that was created to predict an individual\u2019s health behaviors. It has recently been used to guide the design of health messages for effectively promoting health behaviors and evaluate the presence or absence of elements in media content that might contribute to people\u2019s health beliefs . UnderstIn addition to behavioral outcomes, public engagement is another indicator of the effectiveness of public agencies\u2019 crisis communication efforts. Public engagement refers to the various forms of communicative interaction between the public and government agencies, such as the public sharing or replying to governmental agencies\u2019 messages . Public We adopted Johnson and Taylor\u2019s  conceptuThis study focused on public agencies in the state of Texas. Texas was chosen because this state became one of the disease epicenters following the enforcement of Governor Abbott\u2019s state reopening measures in April 2020. At the time of data collection (mid-July 2020), Texas was facing the second peak of COVID-19 cases and had the highest 7-day average number of daily new cases  . In addi=15,382).We conducted the following steps to select the sample tweets for analysis. First, we identified all of the active Twitter accounts of public health departments and Office of Emergency Management (OEM) organizations at the city, county, and state levels in Texas. To identify public health departments, we obtained a list of health department directories from the Centers for Disease Control and Prevention and the US Department of Health and Human Services. Additionally, a list of local-level health agencies was obtained from the National Association of County and City Health Officials. In this step, we identified a total of 26 Texas public health departments that actively tweeted during the studied period. We also used a list of Texas city and county names to conduct searches on Twitter and identified an additional 56 official OEM organization Twitter accounts, which yielded a total of 82 organizations. Second, we created a list of 25 COVID-19\u2013related keywords . All tweets from the 82 organizations that contained at least one of these keywords and were published between January 1 and June 30, 2020, were downloaded using Twitter\u2019s developer application programming interface  handwashing, (2) social distancing, (3) mask wearing or face covering, (4) staying at home or sheltering in place, (5) getting tested, (6) learning more information, and (7) other behaviors.Finally, HBM variables, including severity (any reference to the magnitude and seriousness of COVID-19), susceptibility , benefits , barriers (the difficulties associated with adopting or implementing the recommended behaviors), and self-efficacy (one\u2019s ability to engage in recommended behaviors) were coded. TThe coding of these health beliefs was adapted from Tang and Park , respectSeveral rounds of training sessions were conducted to assist two coders with understanding each item in the codebook. Afterward, around 20%  of the tweets were used for the development of a training data set. Two coders coded 150 tweets that were randomly selected from the remaining 80%  of the tweets. These tweets achieved satisfactory intercoder reliability . Two items (barriers and self-efficacy) were dropped from the codebook because they were nearly completely absent from the collected tweets. Afterward, each coder independently coded half of the training data set.Data cleaning was conducted by following the steps laid out by Du et al . The bidPrecision, recall, and overall F1 score  were calculated for each variable. We also calculated the microaveraging F1 score and macroaveraging F1 score to evaluate variables\u2019 performance in each classification task. We summed up all of the individual true positives, false positives, and false negatives for the microaveraged score. For the macroaveraged score, we used the average of the F1 scores of different categories. Overall, our model achieved good results . AfterwaHierarchical linear regressions or stepwise linear regressions were used to answer RQ3 . This method enabled the assessment of separate effects from different blocks of variables. Since both variables for measuring engagement were highly skewed, we adopted the standard practice of log-transforming these metrics before they were entered into regression models. In the two regression models, the independent variables consisted of the following three blocks: (1) the information, action, and community message types; (2) the dichotomous thematic categories, which included social distancing, face covering, sheltering in place, getting tested, information seeking, and other behaviors; and (3) the health belief variables, which included severity, susceptibility, and benefits. To control for the effect of account popularity (popular accounts were more likely to promote greater public engagement), we entered the log-transformed number of followers as a control variable in each model.A total of 7269 tweets were related to COVID-19. Of the 82 public health and OEM agencies, only 61 tweeted about COVID-19. These organizations tweeted about COVID-19 for an average of 119 times (SD 203.09).RQ1 asked about the functions of tweets. Sharing information was the most prominent function of the tweets posted by public health agencies , followed by the action function . Community building was the least salient function, as only 10.19% (741/7269) of the tweets promoted the engagement of community members and provided emotional support.RQ2 asked about the types of actions that tweets promoted and the health beliefs that tweets mentioned. Of the behaviors recommended by agencies, learning more information was the most recommended action among the tweets , followed by getting tested , staying at home or sheltering in place , social distancing , face covering , and handwashing . RQ3 was proposed to examine the relationship between the content of tweets and public engagement. Overall, the public\u2019s engagement with the tweets posted by public health agencies was relatively low, as each tweet had an average of 13.05 retweets (SD 43.16) and 19 favorites/likes (SD 59.97). P<.001). Additionally, severity and susceptibility significantly promoted retweeting tendencies .In terms of promoting public sharing or retweeting behaviors, tweets that fulfilled the information and action functions were more likely to be retweeted. Tweets that contained mentions of covering one\u2019s face, sheltering in place, getting tested, and seeking COVID-19\u2013related information were also more likely to be retweeted, whereas those containing handwashing information were significantly less likely to be retweeted .In terms of predicting the number of favorites that tweets received, the results showed slightly different patterns. Tweets that were primarily about promoting action and building community were more likely to receive favorites from the public compared to those about providing information. Furthermore, content that included information about social distancing, sheltering in place, and getting tested were more likely to be favorited, whereas tweets that mentioned handwashing behaviors had a consistently low chance of being favorited by the public. Consistent with the other engagement indicator, the severity and susceptibility health beliefs also significantly predicted the chance of being favorited by the public  and individuals  in and beyond the state of Texas.This study only examined public health agencies\u2019 tweets from a single state in the United States, and our data only covered the first wave of the COVID-19 outbreak in the United States. According to the Crisis and Emergency Risk Communication Model, the public has different informational and emotional needs during different stages of an outbreak . It is iThis study examines the content of COVID-19\u2013related tweets that were published by the public health agencies in Texas during the first 6 months of 2020. We found that although public health agencies mostly used Twitter to disseminate pandemic-related information, they could use the Twitter platform to further promote preventative actions, since in this study, the public positively responded to tweets that promoted actions. Furthermore, the public was most likely to engage with tweets that described people\u2019s susceptibility to contracting COVID-19, as such information helped them to understand the risk of the disease. However, there was a lack of information that convinced the public of the high feasibility of proposed preventative behaviors and increased the public\u2019s confidence. Public health agencies can vastly expand their reach during public health crises by steadily building up their follower bases."}
+{"text": "The intestinal microbiota is thought to be involved in the occurrence of inflammatory bowel disease in remission with irritable bowel syndrome (IBS)-type symptoms, but the specific distinct profile of these bacteria remains unclear. This cross-sectional study aims to investigate the fecal microbiota profiling in patients with these diseases.+) or without IBS-type symptoms (CDR-IBS\u2212), ulcerative colitis patients in remission with IBS-type symptoms (UCR-IBS+) or without IBS-type symptoms (UCR-IBS\u2212), IBS patients and healthy controls, were collected and applied 16S ribosomal DNA (rDNA) gene sequencing. The V4 hypervariable regions of 16S rDNA gene were amplified and sequenced by the Illumina MiSeq platform. The differences in the sample diversity index in groups were analyzed with R software.Fecal samples from 97 subjects, including Crohn\u2019s disease patients in remission with IBS-type symptoms (CDR-IBSP\u2009<\u20090.05). The observed species index in the CDR-IBS+ group was higher than that in the CDR-IBS\u2212 group . No difference was found in alpha diversity between UCR patients with IBS-type symptoms and those without related symptoms. At the genus level, the number of Faecalibacterium in CDR patients with IBS-type symptoms increased significantly, while Fusobacterium decreased versus those without such symptoms . However, compared with the UCR-IBS\u2212 group, the number of Faecalibacterium in the UCR-IBS+ group decreased, while the number of Streptococcus increased, but there was no significant difference in the genus structure. The abundance and composition of the microbiota of IBS patients were not distinct from those of healthy controls.The richness of the intestinal microbiota in the CDR-IBS group was markedly lower than those in the control and IBS groups based on the analysis of observed species and the Chao index (Faecalibacterium and a decrease in Fusobacterium. The IBS-type symptoms in UC patients in remission cannot be explained by changes in the abundance and structure of the intestinal microbiota.The IBS-type symptoms in CD patients in remission may be related to an increase in The online version contains supplementary material available at 10.1186/s12876-021-02015-w. Changes in the intestinal microbiota can result in the loss of intestinal homeostasis, which has been found in a variety of intestinal disorders, including inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) . IBD is Irritable bowel syndrome (IBS) is a functional bowel disease characterized by recurrent abdominal pain, bloating, and altered bowel habits. IBD patients in the active stage often have abdominal pain, diarrhea, bloody stools and other uncomfortable symptoms. Some IBD patients in remission (IBDR) have persistent gastrointestinal symptoms, including abdominal pain, diarrhea and abdominal discomfort. For IBDR, these symptoms meet the criteria for IBS and can be defined as IBS-type symptoms \u20138. AccorAlthough IBS and IBD are functional and organic diseases, respectively, they have some common etiologies, especially the intestinal microbiome , 12. EmeTherefore, we hypothesized that the IBS-type symptoms of IBD patients in remission would be closely related to alterations in the intestinal microbiota. To clarify this hypothesis, we performed a cross-sectional study to initially explore the alterations in the intestinal microbiota of IBD patients in remission with IBS-type symptoms.All participants with IBD had an established radiological, histological, or endoscopic diagnosis of CD or UC according to the criteria of the European Crohn & Colitis Organization (ECCO) . In this. Patients with confirmed IBD had symptoms of abdominal pain and changes in bowel habits, and these symptoms met the Rome IV criteria P value lower than 0.05 was considered statistically significant.All the data were analyzed using Statistical Package for Social Sciences version 25.0 . Age parameter data are expressed as the mean\u2009\u00b1\u2009standard deviation. Unless specifically explained, the majority of microbiota data were nonnormally distributed, and the data are expressed as the median . Kruskal\u2013Wallis one-way analysis of variance was used to compare the microbiota data. Partial graphs were drawn using GraphPad software 8.0 . A A total of 97 subjects were enrolled in the study, including 34 IBS patients, 45 IBD patients in remission, and 18 healthy controls. All subjects who met the enrollment criteria from the First Affiliated Hospital of Nanjing Medical University were recruited from August 2018 to September 2019. The mean ages were 42.9\u00a0years in the IBS group, 30.9\u00a0years in the CDR-IBS\u2009+\u2009group, 29.8\u00a0years in the CDR-IBS- group, 37.1\u00a0years in the UCR-IBS\u2009+\u2009group, 42.4\u00a0years in the UCR-IBS- group and 37.9\u00a0years in the control group. The proportion of male subjects was 54.6% (53/97). However, there were more female subjects in the IBS group , which might be closely related to the obvious sex difference in the incidence of this kind of disease. Detailed demographic data and clinical characteristics of all included subjects are listed in Table +), CD patients in remission without IBS-type symptoms (CDR-IBS\u2212), UC patients in remission with IBS-type symptoms (UCR-IBS+) and UC patients in remission without IBS-type symptoms (UCR-IBS\u2212) were reduced, but only the OTUs of patients in CDR-IBS\u2212 had a significant difference , suggesting that this group of patients may have the lowest species abundance. The detailed results are shown in Table The paired-end reads were optimized to remove low-quality reads and clustered into operational taxonomic units (OTUs) for species classification at 97% similarity, and the abundance information of each sample in each OTU was counted. The abundance preliminarily explains the species richness of the sample. A total of 4,869,075 high-quality tags were obtained, and the average number of tags for each sample was 50,197. According to the 97% similar clustering principle, a total of 1118 OTUs were generated from 97 samples. Compared with the control group, the numbers of OTUs in IBS, CD patients in remission with IBS-type symptoms  plots and Venn plots . The proportion of Firmicutes displayed relative increasing trends in the CDR-IBS+ and UCR-IBS+ groups. No difference in Fusobacteria phylum abundance was determined in the current populations, although there was an increasing trend for IBDR in different types regardless of the presence or absence of IBS-type symptoms. Overall, there was no significant difference in microbiota among the IBS, CDR-IBS+ and UCR-IBS+ groups. Further analysis showed that there was no significant difference in the alteration of the microbiota community at the phylum level between the UCR with or without IBS-type symptoms and the CDR groups.As shown in Fig.\u00a0ia. Fig.\u00a0A, B. TheBacteroides, Faecalibacterium, Prevotella, Escherichia, Roseburia, Blautia, Streptococcus, Fusobacterium, Hemophilus, and Lachnospira . In addition, the changes in the microbiota community in UCR subjects were not the same as those in CDR subjects. The results indicated that the abundances of Fusobacterium and Streptococcus were increased, but the abundances of Faecalibacterium, Escherichia, and Lachnospira were slightly decreased in the UCR-IBS+ group compared to the UCR-IBS\u2212 group, although none of the differences were statistically significant. Differences were also found between CDR and UCR at the genus level. There was a significantly greater abundance of Faecalibacterium in UCR-IBS\u2212 relative to CDR-IBS\u2212 and a greater abundance of Fusobacterium in CDR-IBS\u2212 relative to UCR-IBS\u2212 . The genus differences between CDR-IBS+ and UCR-IBS+ patients were not statistically significant. The differences in the microbiota communities among the IBS, CDR-IBS+ and UCR-IBS+ groups were also analyzed. The results showed that the mean abundances of Faecalibacterium and Streptococcus tended to increase, while the levels of Prevotella and Lachnospira tended to decrease in the CDR-IBS+ and UCR-IBS+ groups compared with the IBS groups. However, further analysis of the genera among IBS, CDR-IBS+, UCR-IBS+ patients did not reveal a statistically significant difference. , diarrhea (IBS-D), or a combination of both (IBS-mixed), according to the Rome IV Diagnostic Criteria . The patOur study and a previous study  failed tIn conclusion, the obtained results from our study did not find any difference in the intestinal microbiota between IBD patients in remission with IBS-type symptoms and those without IBS-type symptoms. These results provide a certain basis for recommendations for future trials on the management of IBS-type symptoms. In the future, we still need to have a better understanding of the mechanism of IBD with IBS-type symptoms in the absence of persistent disease activity and strive to find effective treatments to relieve clinical symptoms and improve life treatment.Additional file 1: Figure S1. Rarefaction analysis of sampling by observed bacterial."}
+{"text": "In order to measure the capacity of vertical tanks more conveniently, this paper proposes a vertical tank capacity measurement method based on Monte Carlo Method. The method arranges a plurality of sensor points on the inner surface of the tank, and then performs Monte Carlo tests by generating a large number of random sample points, and finally calculates the capacity by counting the sample points that meet the criterion. The criterion for whether a sample point is located in the tank, which is the core issue, is established with the coordinates of sensor points and the distance between different sensor points along the surface of the tank. The results show that the absolute error of the measurement results of the proposed method does not exceed \u00b10.0003[m Vertical tank is widely used in the storage, transportation and measurement of liquid substances such as petroleum and chemical materials. Accurate measurement of the vertical tank capacity is directly related to the fairness of liquid substance transactions.One of the commonly methods used to measure the capacity of a vertical tank is Geometric Measurement Method (GMM). GMM uses steel tape, theodolite or other measuring device to measure tank\u2019s geometry, and use dedicated computer software to process the measured data to obtain the tank capacity \u20133. This 3 is already a very large standard metal tank, but the volume range of vertical tanks includes 20m3~700m3, even more than 700m3, and the height can exceeds 30 meters. To place the standard metal tank higher than the metal tank, this is difficult to achieve. In addition, the standard metal tank is too small compared to the vertical tank, and it takes a lot of time to pour a fixed amount of water many times. If the vertical tank is large, VM is rarely used because it takes a long time and consumes large amounts of water.Another commonly used method to measure the capacity of a vertical tank is Volumetric Method (VM). VM compares the capacity difference between a tank with higher precision and the vertical tank being measured , 4. DuriWith the development of technology, new techniques for measuring vertical tank capacity has emerged. Laser Scanner Method (LSM) reconstructs the internal volume of a vertical tank by emitting laser light to the surroundings \u20137. The pThe volumetric method, geometric measurement method, and laser scanner method are used to measure vertical tanks, with a relatively fixed cycle, about once every four years. If you want to monitor the capacity of the vertical tank in real time, it is a more feasible way to arrange sensors in the vertical tank.Monte Carlo Method (MCM) constructs a probabilistic model that approximates the performance of the system and performs random experiments on a digital computer . It is vVertical tank capacity measurement based on Monte Carlo method is to arrange sensor points on the inner surface of the vertical tank. And then, according to the coordinates of the sensor points and the distance between each point along the tank surface, criterion to decide whether a sample point is located in the vertical tank is established. Base on this criterion, the number of sample points falling in different heights is counted, and the capacity value represented by different liquid level is calculated with the number of sample points. Fig 2 is the coordinate system. Vertical tanks are generally cylindrical, and their cross-sections are generally circular. Therefore, our methods and tests are all carried out with circular cross-sections. Vertical tank is divided into upper part and bottom part. The bottom part of vertical tank is usually irregular because of ground subsidence or other reasons, and the capacity of bottom part is obtained by volumetric method or other methods. The bottom of the vertical tank generally only occupies little share of the entire storage tank, and the bottom volume is only used when the liquid is first fed or when the tank is cleared. In the daily custody transfer, the capacity table on the upper part of the vertical tank is generally used for calculation, and the amount of liquid in each custody transfer is generally not less than two meters in the height of the vertical tank. Due to the deformation of the bottom plate and other reasons, it is difficult to measure the bottom volume of the vertical tank very accurately by the geometric measurement method and the laser scanner method, and more attention is paid to the measurement of the upper part of the storage tank. Thus, this paper focuses on acquiring the capacity of upper part. The height of bottom part is Hbottom, and the height of upper part is Hupper. The inner radius of the vertical tank is R. The capacity of the vertical tank is synthesized from the upper part and the bottom part, shown in Qk is the vertical tank capacity when the liquid level is hk. Qkupper- is the capacity of upper part when the liquid level is hk. Qbottom is the capacity of bottom part.Fig 3 shows the distribution of sensor points. Sensor points are equiangularly arranged on different layers. The number of layers is Nlayer and the number of sensor points on each layer is Nmono. The projection of the sensor points of each layer on O-XY surface are coincidence. The interval between layers is hlayer and the total number of sensor points is Nsensor. Thus,Layer\u2212i are numbered as Pi,1, Pi,2,\u2026, i \u2264 Nlayer. The coordinates of sensor point Pi,j are , where 1\u2264 i \u2264 Nlayer, 1\u2264 j \u2264 Nmono. Specially, Layer\u22121 and Layer\u2212Nlayer are the lowest and highest positions of upper part respectively, and zj1, = 0, Fig 4. If sample point Psample locates in the enclosed area formed by all sensor points, we have a, b and c, makea+b+c\u22641, 0\u2264a\u22641, 0\u2264b\u22641, 0\u2264c\u22641. Pi1,j1,Pi2,j2andPi3,j3 are sensor points, 1\u2264 i1 \u2264 Nlayer, 1\u2264 j1 \u2264 Nmono, 1\u2264 i2 \u2264 Nlayer, 1\u2264 j2 \u2264 Nmono, Criterion of the Monte Carlo test is used to decide whether a sample point falls in the vertical tank. As the vertical tank is convex and the sensor points are located on the inner surface of the tank, sample points in the enclosed area formed by all sensor points must be in the tank, shown in a, b and c let Psample satisfies sample falls in the vertical tank or not. The subsequent calculation will be performed.If we cannot find Fig 5 is the positional relationship of Psample,Pi1,j1, and Pi2,j2. If Psample is between Pi1,j1, and Pi2,j2, yA = yi1,j1+\u03bb \u00b7 , zA = zi1,j1+\u03bb \u00b7 . The coordinates of Point B are xB = xi1,j1+(1-\u03bb) \u0387 , yB = yi1,j1+(1-\u03bb) \u0387 , zB = zi1,j1+(1-\u03bb) \u0387 . Then the ellipsoid equation is\u03bb is solved out, \u03bb value.As i1,j1 and Pi2,j2 along the surface of the tank are i1,j1 to Pi2,j2 along the surface of the tank is defined as li1,j1\u2194i2,j2. Since the surface continuity of the vertical tank, approximate solution to get li1,j1\u2194i2,j2 isLmajor is the ellipsoid major axis length, Lminor is the ellipsoid minor axis length. In rding to , the halChalf = li1,j1\u2194i2,j2, we can get \u03bb and the ellipsoid equation, i.e. Solving sample to A and B is shorter than Lminor, Psample is in the ellipsoid. That is, ifsample falls in the vertical tank.According to the definition of an ellipsoid, if the sum of the distance from Psample falls in the vertical tank, we haveIn summary, if a sample point Psample satisfy The coordinates of Psample do not satisfy If the coordinates of Pxmin = min{xi,j}, ymin = min{yi,j}, zmin = min{zi,j}, xmax = max{xi,j}, ymax = max{yi,j}, zmax = max{zi,j}, where 1\u2264 i \u2264 Nlayer, 1\u2264 j \u2264 Nmono, xi,j, yi,j and zi,j are the coordinates of sensor points, the coordinates of sample points would be xmin\u2264x\u2264xmax, ymin\u2264y\u2264ymax, zmin\u2264z\u2264zmax.When conducting the Monte Carlo tests, sample points are randomly generated in the smallest cuboid that contains the vertical tank. If NIN, and totally Nsample sample points are generated, the capacity of the upper part would behk, if the number of the sample points that satisfy the criterions of the Monte Carlo Test and z \u2264 hk is NkIN-, the capacity in upper part at hk ishk isAfter the Monte Carlo tests, if the number of sample points satisfying the criterions of the Monte Carlo Test is Fig 7 is the vertical tank model of the Monte Carlo test. The radius of vertical tank is R = 1.300[m], and the upper part height of the vertical tank is Hupper = 8.476[m]. The maximum capacity of upper part is \u03c0 \u0387 R2 \u0387 Hupper \u2248 45.000[m3]. Sensor points are distributed in ten layers (Nlayer = 10). Each layer has ten sensor points uniformly distributed equiangularly (Nmono = 10). Thus, Nsensor = Nmono \u0387 Nlayer = 100, i.e., there are 100 sensor points in total. Considering the size of the vertical tank, we initially set up 100 sensor points. The influence of the number of sensor points on the measurement results will be further studied in the follow-up work. Currently, 100 sensor points are used to explore the feasibility of the proposed method. The difference of heights between each layer is hlayer = 0.8476[m]. Sample points are generated in Cuboid L\u2013W\u2013Hupper, whose L = 2.600[m], W = 2.600[m] and Hupper = 8.476[m]. Thus, xmin = -1.300[m], ymin = -1.300[m], zmin = 0, xmax = 1.300[m], ymax = 1.300[m], zmax = 8.4776[m], and the coordinates of sample points satisfy -1.300[m]\u2264x\u22641.300[m], -1.300[m]\u2264y\u22641.300[m], 0\u2264z\u22648.476[m].unifrnd . Totally Nsample = 106 sample points are generated in each test. A total of 10 tests were conducted.The Monte Carlo tests are performed on Matlab, and the sample points are generated with Matlab\u2019s built-in random function \u2500 Qk is the vertical tank capacity at hk. hk, Vcuboid is the volume of cuboid, Vcuboid = L \u0387 W \u0387 Hupper.In order to discuss the measurement results of vertical tanks of different volumes, the absolute error of capacity per unit volume is calculated asFig 8 is the absolute error of capacity per unit volume of Test 1~10. It shows that there is a significant linear relationship between \u03b5k and hk in each test. The slopes of the fitting results of Test 1~10 are all around 0.0252.To further investigate the relationship between the absolute error of capacity per unit volume and the size of the vertical tank, another sixty tests are carried out for different heights and radii of tank.Fig 9 is the absolute error of capacity per unit volume with different heights of vertical tank. The heights of vertical tank are 8.476 [m], 6.781 [m], 4.238 [m], and 2.543 [m]. Each height is tested 10 times and the inner radius of vertical tank is 1.300 [m] in each test. \u03b5k. From the fitting results, even though Hupper has changed, there is still a significant linear relationship between \u03b5k and hk. The slopes of the fitting results are different. Interestingly, we find that 0.02524 \u00d7 8.476 \u2248 0.214, 0.03154 \u00d7 6.781 \u2248 0.214, 0.05047 \u00d7 4.238 \u2248 0.214, and 0.08415 \u00d7 2.543 \u2248 0.214. This shows that the product of the slope and Hupper is a constant value, which 0.214.Fig 10 is the absolute error of capacity per unit volume with different radii of vertical tank. The radii of vertical tank are 1.300 [m], 1.040 [m], 0.910 [m], and 0.780 [m]. Each radius is tested 10 times and the height of vertical tank is 8.476 [m] in each test. \u03b5k. It can be seen from \u03b5k and hk, and the slope hardly changes due to changes in radius.Hupper and R on \u03b5k, in the linear relationship between \u03b5k and hk, R has little effect on the slope, and the product of Hupper and the slope is a constant, which is 0.214. Therefore, the relationship between \u03b5k and hk can be written ashk, which is measured with the Monte Carlo method, can be compensated according to hk isBased on the above analysis of the influence of Fig 11 are devices used for vertical tank testing, including a vertical tank [R = 0.299[m]. One hundred sensor point labels are evenly affixed on the inner wall of the vertical tank, a total of 10 layers, each with 10 points. The distance between the lowest sensor point and the highest sensor point is Hupper = 0.513[m]. The distance between the sensor points of each layer is hlayer = 0.057[m]. The capacity of bottom part is Qbottom = 0.0175[m3]. A ruler is attached to the inner wall of the vertical tank for reading the liquid level with a resolution of 0.001[m]. The nominal capacity of the metal tank is 0.02[m3], indicating that it can accurately hold 0.02[m3] of liquid. The liquid in the metal tank can be transferred to the vertical tank through the plastic hose. The liquid used in the test is water. Since the nature of water is relatively stable, compared to other media, such as oil, the volume of water is less affected by temperature and volatility, so we used water to verify the accuracy of the proposed method to measure the capacity of vertical tanks.cal tank  and a mecal tank . The innFig 12 is the sequence of vertical tank test, including two parts: vertical tank test and Monte Carlo test. In the vertical tank test, the first step is to pour Qbottom = 0.0175[m3] of water into the tank, and record the liquid level as 3] of water is poured into the tank one by one, and the corresponding liquid levels are recorded. The recorded liquid levels are Qbottom from affecting the liquid level of the upper part of vertical tank, the liquid levels when the water volume in the vertical tank is 0.0375[m3], 0.0575[m3], 0.0775[m3], 0.0975[m3], 0.1174[m3], 0.1375[m3] and 0.1575[m3] are In the Monte Carlo test, a total of 10 tests are performed, and the parameters of each test are shown in Fig 13.Fig 14 is the absolute error distribution of the measurement results obtained through the Monte Carlo method, e.g. 3].Fig 15 is the relative error shrinkage curve, e.g. 3], and the relative error finally converges to less than 0.18%.A new vertical tank capacity measurement method based on Monte Carlo Method is proposed. Focusing on constructing vertical tank boundary, the method arranges a plurality of sensor points on the inner surface of the vertical tank, and performs Monte Carlo tests by generating a large number of random sample points. The criterions for whether a sample point is located in the vertical tank are established with the coordinates of sensor points and the distance between different sensor points along the surface of the tank. The results show that the absolute error of capacity per unit volume has a linear relationship with the height of the vertical tank, and has little effect with the radial size of the vertical tank. The absolute error of the measurement results of the proposed method does not exceed \u00b10.0003[mAlthough the method we proposed can effectively measure the capacity of vertical tank, there are still some limitations that need to be improved. The number of sensor points is large, and the arrangement of the sensor points is relatively neat. We will further explore the influence of the number and location of sensor points on the measurement results in the future, try to reduce the number of sensor points, and more convenient ways to arrange the sensor points, such as scattered random arrangement. In addition, this method is suitable for tanks of various shapes, such as cuboid, cylinder, etc., but the connection between any two sensor points must be inside the tank, and there can be no such connection outside the tank. At this stage, this article mainly discusses the feasibility of the proposed method. In the future application stage, there are still many problems to be solved, such as the selection of sensors and the reaction of sensors with liquids. Regarding the selection of sensors, we have made some guesses. Surface acoustic wave sensors, ultrasonic guided wave sensors, and stress-strain sensors may be suitable.S1 File(XLSX)Click here for additional data file."}
+{"text": "Physical activity during midlife (ages 45-64) plays a major role in the prevention of chronic and serious medical conditions. Unfortunately, many midlife adults struggle to be physically active in the setting of low levels of psychological well-being and the management of multiple confluent sources of stress. Effective, scalable, midlife-specific interventions are needed to promote physical activity and prevent the development of chronic medical conditions.In an initial proof-of-concept trial, we assessed the feasibility and acceptability of a new, midlife-adapted, phone- and text message-based intervention using positive psychology (PP) skill-building and motivational interviewing (MI) techniques. We secondarily analyzed post-intervention changes in accelerometer-measured physical activity and self-reported outcomes.The PP-MI intervention included six weekly phone sessions with a study trainer, with completion of PP activities and physical activity goals between calls, and in the subsequent six weeks briefer phone check-ins were conducted. Text messages over the 12-week intervention period were utilized to support participants and identify barriers to goal completion. Feasibility (session completion rates) and acceptability (participant ratings of intervention ease and utility) were assessed via descriptive statistics, and pre-post improvements in psychological, functional, and physical activity outcomes at 12 weeks were examined via mixed effects regression models.a priori thresholds for feasibility and acceptability. Participants demonstrated generally medium to large effect size magnitude improvements in accelerometer-measured physical activity, psychological outcomes, and function.Twelve midlife adults with low baseline physical activity enrolled in the single-arm trial. Overall, 76.8% of all possible sessions were completed by participants, and mean ratings of weekly phone sessions were 8.9/10 (SD 1.6), exceeding our A novel, midlife-specific phone- and text-based PP-MI intervention was feasible and had promising effects on physical activity and other clinically relevant outcomes, supporting next-step testing of this program via a randomized controlled trial. Midlife adults (age 45-64) represent over 25% of Americans and are the most rapidly growing age group in the United States , a counseling strategy designed to resolve ambivalence and enhance motivation,  common in this population. Furthermore, few programs have focused on the promotion of psychological well-being in midlife, despite the fact that PP exercises are simple to deliver and consistently enhance well-being constructs.a priori benchmarks for feasibility and acceptability, and would show small-to-medium effect size magnitude improvements in psychological outcomes and accelerometer-measured physical activity.Accordingly, using information from qualitative interviews and a small phone-based pilot study in midlife persons, . We aimed to enroll between 10\u201312 participants over a two-month period given the availability of research staff for this unfunded feasibility trial and our use of similar samples when examining initial proof of concept of behavioral interventions prior to next-step testing  and to have low self-reported physical activity ; (b) no access to a telephone or text messaging; (c) inability to communicate in English; (d) cognitive impairment, as assessed via a cognitive screen designed to assess appropriateness for research participation; ; and (f) coronary artery disease (CAD), defined as a prior acute coronary syndrome  or coronary stenosis identified through cardiac catheterization (i.e. 50% stenosis of the left main artery or 70% stenosis of another coronary artery). The age, communication, and medical condition (e.g. CAD) criteria were assessed via both the electronic record review and phone screen assessments; the remainder were assessed via phone screens. We excluded persons with CAD because one eventual goal of the program is to prevent heart disease and because individuals with CAD may have greater restrictions on moderate or vigorous activity.Exclusion criteria included: (a) a Potential participants were identified via IRB-approved searches of our medical center\u2019s electronic medical record system for midlife persons receiving care in affiliated primary care practices. Patients who had agreed to receive communications about ongoing research studies were sent opt-out letters from the study team, and those who did not opt out of contact completed a screening phone call. At the screening call, study staff described the project to potential participants and screened them for study criteria . Interested and eligible patients were mailed a study information fact sheet and were scheduled for a baseline phone study session, during which they provided informed consent and completed baseline self-report instruments. Participants were then mailed an accelerometer for physical activity assessment, wore it for one week, and then mailed it back to study staff.After confirming adequate accelerometer wear time (at least 4 days with 10\u2009+\u2009hours of wear time), a second study phone session was scheduled, and participants were mailed a written treatment manual and pedometer. During this session, participants spoke with a study interventionist, who introduced them to the treatment manual and pedometer, reviewed the program\u2019s structure and rationale, and conducted an introductory session. If the participant had inadequate wear time, the study team would have mailed the accelerometer back to the participant for further wear, and when it was returned by mail a second time (and once adequate wear time was confirmed), the second phone session would have been completed .The balance of the intervention was delivered by phone and text messages, with weekly phone contacts for 12 weeks (6 weeks of 30-minute phone sessions followed by 6 weeks of briefer [5-minute] phone check-ins) and ongoing weekly text messaging over the 12 weeks. Following the intervention, participants completed a follow-up assessment and received accelerometers by mail, wore them for 1 week, then returned them by mail..ntervention Over the first 6 weeks of the program , participants completed assigned intervention activities  on their own, then participated in 30-minute weekly phone sessions with a study psychologist interventionist. The intervention consisted of a PP component that focused on the development of skills to promote psychological well-being in daily life, along with an MI component that utilized MI concepts and specific goal-setting strategies to increase engagement in physical activity. During the calls, the participant and interventionist reviewed the prior week\u2019s activities, discussed the use of well-being skills, reviewed facilitators and barriers to physical activity, and discussed the assigned activities and rationale for the upcoming week (see IPhone sessions in the first 6 weeks. The PP program contained specific activities (see ties see  and our ties see . The fraties see . This coties see  and a prties see  by provities see . Partici: gratitude for positive events (Session 1), expressing gratitude (Session 2), recalling past success (Session 3), using personal strengths (Session 4), enjoyable and meaningful activities (Session 5), imagining the \u2018good life\u2019 (Session 6) and, planning for the future . Further details for each activity and sample pages from the manual are in During the PP portion (\u223c10\u2005min) of phone sessions during the first 6 weeks of the program, participants and interventionists reviewed the prior week\u2019s activity, discussed how skills from that activity could be utilized in daily life, and discussed the rationale for the next week\u2019s PP activity. The PP exercises were as followsThe midlife-adapted MI portion (\u223c15 mins) of phone sessions in the first 6 weeks assessed motivation to engage in physical activity, discussed barriers to activity, and set physical activity goals or used MI tools to boost motivation, depending on the participant\u2019s stage of change. This component also included a brief stress reduction module based on feedback in our prior work in midlife adults  framework Doran,  for settPhone check-ins in the final 6 weeks. The phone check-ins (\u223c5\u2005min) in the final 6 weeks of the program were structured calls with participants that focused on goals for PP skill use and physical activity completion that were created at the final phone session (Session 7) and via weekly text messaging (see below). The calls, performed between the goal-focused text messaging that occurred each week in the final 6 weeks of the program, reviewed participant PP and activity goals, checked in about progress, reviewed facilitators and barriers to completion, and modified goals if needed. Interventionists also inquired about specific sources of stress common in midlife  and referred participants to resources and strategies in the Appendix of their treatment manual, when relevant.Text messaging. Text messaging was utilized to supplement the phone sessions and written treatment manual content. In the first 6 weeks of the program, one-way messages were sent to participants that reinforced content from the phone sessions and treatment manual. For example, following Session 1 , the text messages sent to participants were:One way to increase gratitude is to deliberately take note of small positive things that happen. This week, think about and write down three good things that happened!(PP message) Barriers, such as a lack of time, can make it hard to reach your goals. If you struggle to find enough time to be active, try spreading your activity throughout the day.(MI message) The messages were sent utilizing an IRB-approved, HIPAA-compliant approach utilizing messages selected from our Amazon Web Services DynamoDB message database  who received training in both the PP and MI components of the intervention from the lead study intervention supervisor (CC), based on a protocol from our prior work , a written treatment manual, the infrastructure for sending text messages, and adequate time for conducting the weekly initial sessions (\u223c25\u2005min) in the first 6 weeks and briefer phone and text check-ins (\u223c5\u2005min each) in the next 6 weeks, along with additional time to account for missed calls and documentation.Study assessments and outcomes. Participants\u2019 baseline sociodemographic and medical characteristics were obtained via patient report and medical record review. For measures of feasibility and acceptability, session completion and participant ratings of sessions were recorded by study interventionists. Finally, participants completed self-report measures at baseline and 12 weeks via telephone and wore accelerometers at both time points for objective physical activity outcomes.Primary study outcomes: feasibility and acceptability. We measured feasibility via rates of phone session completion . The two primary feasibility measures were: (a) the proportion of all possible sessions that were completed across all participants, and (b) the number of participants completing a majority of phone sessions. We chose these to assess the overall scope of session completion and to have a clinically relevant metric, as we deemed that completing a majority of phone sessions would convey the key elements of intervention content to the participant; we have used these metrics in prior feasibility studies of behavioral interventions  of both the PP and MI activities/content each week during intervention calls over the initial 7 phone sessions, for a total of 4 weekly ratings.secondary measures of acceptability via self-report assessments at 12 weeks regarding the utility of each study component .We also collected Secondary study outcomes: Accelerometer-measured physical activity and self-report measures. This initial proof-of-concept study was not designed (and is not appropriate) to assess intervention efficacy given the small sample and lack of control condition, and for this reason the outcomes noted below are exploratory. We assessed physical activity  via the well-validated Actigraph GT3X+ accelerometer  scale,   scale,  measure  scale,  via the Medical Outcomes Study Short Form-12 (SF-12) scale    were used to report session completion rates and ease/utility scores. As a threshold for feasibility, based on our prior work.  across all participants and utilized mean scores of greater than 7.0 as our a priori threshold for acceptability, based on prior work using similar ratings for acceptability of behavioral interventions  of the change in the measure, calculated as the coefficient from the mixed model divided by the SD of the residual for the measure, rather than statistical significance, given the secondary nature of these outcomes in this small sample. In a further set of analyses, we examined correlations between number of sessions completed and change in the secondary outcomes . All analyses were conducted using Stata version 15.1 .We contacted 43 patients, and after hearing about the study and undergoing relevant screening, 19 were interested in participating. Seven screened out, and a total of 12 participants enrolled in the proof-of concept trial  completed a mean of 9.4 (SD 4.8) sessions , with 9 (75%) participants completing more than half of all possible sessions (i.e. 7\u2009+\u2009out of 12). For acceptability, participants\u2019 mean ratings of PP-MI phone session ease and utility ranged from 7.9-9.2 out of 10, with all components exceeding our a priori acceptability threshold .On additional measures of acceptability obtained at the 12-week follow-up, participants rated the overall utility of full phone sessions (first 6 weeks) 9.0/10 (SD 1.4), phone coaching check-ins  8.4/10 (SD 2.8), one-way text messages (first 6 weeks) 6.6/10 (SD 3.7), and check-in texts : 9.3/10 (SD 1.2).p\u2009=\u2009.19; ES\u2009=\u2009.62). Participants also had a small ES magnitude improvement on accelerometer-measured steps per day (mean increase of 431 [SD 1886] steps per day); p\u2009=\u2009.64; ES\u2009=\u2009.23. On self-reported physical activity measured via the IPAQ, participants had a large ES improvement in activity .Regarding the secondary study pre-post outcomes  and 5, pp\u2009<\u2009.001; ES\u2009=\u20092.81).Participants also had medium to large ES magnitude improvements  and similar but slightly higher scores on measures of feasibility and acceptability. This project extends that prior work, as it includes half the number of full phone sessions and is the first to utilize text messaging as an accessible means to provide additional support and content beyond the 6-week phone session period. Such work directly addresses the public health problem of low physical activity among the growing population of midlife persons, who are at risk for developing numerous chronic medical conditions during this life stage. This remote intervention approach might be particularly well-suited to this population, as in-person interventions are likely suboptimal given that many midlife adults must manage multiple competing demands and experience substantial time pressure, and requiring fewer full (\u223c25 min) phone sessions may be beneficial in terms of patient and provider burden. A prior, non-PP based, intervention for persons in midlife was effective in the short-term but not long-term, and it used in-person individual or group sessions, which may be less feasible  in terms of completion of phone sessions, while text messaging may be less familiar to some who use this modality less, including some adults on the older spectrum of midlife. Other factors that could contribute to participation and engagement in the program\u2014either as a research project or in clinical implementation\u2014could include current levels of motivation to change behavior, self-efficacy to change physical activity, and financial and time-related factors, all of which need to be assessed in future projects.Finally, while there were small to medium improvements in accelerometer-measured physical activity, there were much larger improvements in self-reported activity and overall adherence. This could suggest that the accelerometers may not have picked up all forms of activity completed by participants (e.g. swimming), or instead that participants may have overestimated their improvements in these self-care domains. Indeed, there is substantial research examining differences in self-reported physical activity and objectively measured activity, with self-reported physical activity appearing to be overestimated in most cases  sample in this proof of concept trial and should not be used to power future studies of this intervention. Longer, larger, well-controlled studies of the intervention are required to identify the clinical effectiveness of the program in front-line clinical settings.In conclusion, in this proof-of-concept trial of a phone and text message PP-MI program adapted for inactive midlife adults, we found that the intervention was feasible, acceptable, and associated with improvements in MVPA and other clinically relevant measures. Further studies of the intervention are required to more definitively test the impact of this program, conduct rigorous analyses of mechanism and mediation of intervention effects to better understand how the program can work, and utilize implementation analyses that would be needed to estimate the scalability of the program.Time for analysis and article preparation was also funded by the National Heart, Lung, and Blood Institute through grant (R01HL155301) (CMC), and by the National Institute for Nursing Research through grant R21NR018738 (JCH). The project was also supported by the National Center for Advancing Translational Science through grant 1UL1TR002541-01. The authors have no conflicts of interest to report related to this work. This study was presented in oral form at the Academy of Consultation-Liaison Psychiatry Annual Meeting on November 11, 2021."}
+{"text": "Human immunodeficiency virus type 1 (HIV-1) epidemic in China is featured by geographical diversity of epidemic patterns. Understanding the characteristics of regional HIV-1 epidemic allows carrying out targeted prevention and controlling measures. This seven-year cross-sectional study was conducted in Heilongjiang, one province of Northeast China, where newly diagnosed infection is fast increasing yearly, but temporal HIV-1 epidemic trend is largely unknown.gag and env gene sequences. Recent infection was determined by Limiting-Antigen Avidity assays. Comparison analyses on the median ages, CD4 counts, proportions of stratified age groups and CD4 count groups, and rates of recent HIV-1 infection among different population and sampling times were performed to understand temporal HIV-1 epidemic features.Information of 1,006 newly diagnosed HIV-1-infected participants were collected before antiretroviral therapy during 2010\u20132016 in Heilongjiang province. HIV-1 genotype was identified based on the viral Homosexual contact among men who have sex with men (MSM) was the main transmission route and CRF01_AE was the most dominant HIV-1 genotype. During 2010\u20132016, the HIV-1 epidemic showed three new changes: the median age continued to decline, the cases with a CD4 count more than 500 cells/\u03bcl (CD4hi cases) disproportionally expanded, and the recent HIV-1 infection rate steadily increased. MSM cases determined the temporal trend of HIV-1 epidemic here. Increase of young MSM cases (aged <30 years) made the main contribution to the younger age trend of MSM cases. These young MSM exhibited a higher median CD4 count, a higher proportion of CD4hi cases, and a higher rate of recent HIV-1 infection than cases aged 30 years and more. MSM infected by CRF01_AE virus mostly affected HIV-1 epidemic patterns among MSM population.Young MSM have become a new hotspot and vulnerable group for HIV-1 transmission in Heilongjiang Province, Northeast China. The rapid increase in the number of young MSM cases, mainly those with CRF01_AE infection, changed temporal HIV-1 epidemic pattern here. Measures for prevention and control of HIV-1 infection among this population are urgently needed in the future. Human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic in China becomes increasingly severe and complicated. By the end of 2018, the reported absolute number of Chinese people living with HIV-1 was approximately 0.89 million, but the real number was estimated at more than 1.25 million . One bigHeilongjiang, one province in Northeast China, lies on the border between China and Russia. Since first HIV-1 infection was identified in 1993, annually reported HIV-1 diagnoses in Heilongjiang continued to increase slowly until the year 2008 when an expansion of HIV-1 infection among men who have sex with men (MSM) occurred . Since tIn this study, we collected clinical information and samples from more than 1,000 new HIV-1 diagnoses in Heilongjiang Province and made a 7-year cross-sectional study. This paper reported that MSM was driving HIV-1 epidemic trend in Heilongjiang Province and highlighted the critical role of young MSM, especially those infected by CRF01_AE virus, on the change of HIV-1 epidemic pattern here.All the participants were recruited in the Fourth Affiliated Hospital of Harbin Medical University from January 2010 to December 2016. Peripheral blood samples were collected immediately after diagnosis for HIV-1 genotyping. The information including sex, age, the way to acquire HIV-1, and peripheral blood CD4 positive T cell count (hereinafter referred to as CD4 count) at diagnosis were also collected. The inclusion criteria of the participants included: (1) being newly diagnosed as HIV-1 infection, (2) being before antiretroviral treatment, and (3) having age and CD4 count information recorded at diagnosis. Participants who lacked a peripheral blood sample or for whom HIV-1 genotype information was not available were excluded.gag p17-p24  and env C2-C4  genes were amplified from the plasma samples by RT-PCR and subject to sequencing. The genotypes of gag and env genes were determined by the cluster distribution of genes in neighbor-joining trees constructed using MEGA 6.06 software as previously described .For each participant, 5\u2009ml of whole peripheral blood was collected, and then, plasma sample was separated for HIV-1 genotyping. HIV-1 escribed . The gagThe recent or long-term infection of each participant was determined by the Limiting-Antigen Avidity assays using HIV-1 LAg-Avidity EIA kit  as previously described . A normagag p17-p24 genes and MK702932-MK703795 for env C2-C4 genes.The nucleotide sequences identified in this study were submitted to GenBank with accession numbers of MK702086-MK702931 for p-value less than 0.05 was considered as significant unless otherwise specified.Sampling years were divided into three time phases: 2010\u20132011, 2012\u20132014, and 2015\u20132016. Mann\u2013Whitney test and Kruskal\u2013Wallis test were used for comparison of the quantitative variables across two groups and multiple groups, respectively. Chi-square test was used for comparison of qualitative variables across groups. Nonparametric Spearman correlation test was used to analyze the relationship between variables. Data analyses were performed by GraphPad Prism version 8.3.0 . The From 2010 to 2016, 1,183 newly diagnosed HIV-1 cases were collected. Of them, 177 participants were excluded because of lack of blood samples or unavailability of HIV-1 genotype information. Finally, 1,006 cases were included in our subsequent analyses. In order to ensure the exclusion of participants did not introduce a bias in data analysis, the basic information between the included cases and the excluded cases were compared. There was no significant difference in the median age, CD4 cell count, gender composition, and risk group composition between the two groups .Of the 1,006 participants in this study, the majority were male, accounting for 93.7%. Median age was 35\u2009years (interquartile range [IQR] 29\u201345), and median CD4 count was 384 cells/\u03bcl (IQR 214\u2013490). The most important mode of HIV-1 transmission was male-to-male sexual contact among MSM, followed by heterosexual contact. These two modes of sexual contacts together accounted for 85.7% (862/1006) of all cases and 98.6% (862/874) of cases with known transmission routes.env genes  and 846 gag genes  were obtained. That was, 704 samples had both env and gag genes, 160 samples had env gene alone and 142 samples had gag gene alone  to 34\u2009years (IQR 28\u201344) , suggestThe changes observed for the entire group were similar among MSM cases: median age decreased from 37\u2009years (IQR 31\u201345) to 34\u2009years (IQR 27\u201342) , the prop\u2009=\u20090.0411), while the proportions of other age groups did not significantly change . Cases aged <30\u2009years (hereinafter referred to as young cases), 30\u201339\u2009years, 40\u201349\u2009years, and above 49\u2009years accounted for 30.6%, 36.5%, 18.7%, and 14.1% of MSM cases, respectively. From 2010\u20132011 to 2015\u20132016, the proportion of young cases increased from 20.7% (17/82) to 32.1% , and 15.2% (31/204) of young MSM had a CD4 count less than 200 cells/\u03bcl, lower than that of old cases .Compared to MSM cases aged \u226530\u2009years (hereinafter referred to as old cases), young MSM cases had a higher median CD4 count (418 [IQR 307-514] versus 381 [IQR 213\u2013487]) . Additiop\u2009=\u20090.0421) (p\u2009=\u20090.0421) (p\u2009<\u20090.0001). Of note, among all CD4hi MSM cases, 90.9% (149/164) were identified as recent infections. Correlation analysis on MSM population showed a negatively correlation between age and CD4 count and a positive correlation between age and the ODn value  . On the \u20090.0421) . As expe\u20090.0421) . Of receDn value ,F.The data above suggested that young MSM seemed to be in better immune state and have a higher recent infection rate, and that the increase of young MSM cases during the study period may be result in increase of diagnoses in early stage of HIV-1 infection and the improvement in the overall immune status of MSM cases.Next, we wanted to know whether temporal HIV-1 epidemic trend in MSM cases was associated with the infection of a particular viral genotype.p\u2009=\u20090.0071).CRF01_AE, subtype B, and 07&08&C were the main genotypes circulating among MSM cases, accounting for 64.3%, 14.7%, and 12.9% of infections, respectively . There wp\u2009=\u20090.0079), and 15.0% (20/133) of young cases had CD4 counts less than 200 cells/\u03bcl, lower than that of old cases .Median CD4 count in MSM cases infected by CRF01_AE or subtype B virus was lower than that infected by 07&08&C virus . When cop\u2009=\u20090.0359). Young cases and old cases in subtype B or 07&08&C infected MSM had similar rates of recent infection , subtype B , and 07&08&C  . But younfection .These data indicated that the increase in number and proportion of young MSM infected by CRF01_AE virus made a substantial contribution to the decline in median age of MSM cases, which may lead to the improvement in the overall immune status and the increase in recent infection rate of the MSM cases. MSM infected by CRF01_AE virus mostly affected HIV-1 epidemic patterns among MSM population.This study was the first cross-sectional molecular epidemiological study based on a large sample size in Heilongjiang of Northeast China. According to our previous report and the report from the Center for Disease Control and Prevention of Heilongjiang Province, during 2010\u20132016, 8,026 HIV-1 infection cases were newly diagnosed in this province. The participants enrolled in this study represented 12.5%  of all newly reported cases and showed representative demographic characteristics  were male, which reflected the epidemiological data from Heilongjiang Province. In the official report from the Center for Disease Control and Prevention of Heilongjiang Province, the ratio of male to female cases during 2012\u20132017 was 12.3:1, that was, male cases accounted for 92.5% (12.3/13.3) of new HIV-1 diagnoses . This phIn this study, we reported that MSM cases dominated the temporal HIV-1 epidemic trend in this region: the age of cases at diagnosis was getting younger, the proportion of cases with good immune status was increasing, and the rate of recent infection was rising. We also reported that the rapid expansion of young MSM cases, especially the young MSM infected by HIV-1 CRF01_AE virus, was the key contributor to these changes.Unlike the situation in some European and American countries where HIV-1 epidemic initiated among MSM, few cases were reported among Chinese MSM until the year 2005. Since 2010, MSM population has become the group with the highest risk for HIV-1 transmission in China . One obvAs the capital of Heilongjiang Province, Harbin City is one of economic centers in Northeast China as well as one of cities that were affected by the first wave of HIV-1 expansion among MSM during 2006\u20132008 . HIV-1 pYoung people have been considered as a hot topic in HIV-1/AIDS epidemic in China for many years. But in the initial years (2005\u20132008), the clustering hotspots of Chinese young people living with HIV/AIDS (aged 15\u201324\u2009years) mainly distributed within heterosexuals and intravenous drug users in southwest provinces. After 2008, new hotspots among MSM in central and northeast provinces emerged. On the national scale, new HIV-1 cases aged 15\u201324\u2009years increased by an annual average of 35% during 2011\u20132015 . For HarIndeed, two interesting changes among MSM cases were also observed: steadily increasing proportion of CD4hi cases (CD4 count >500 cells/\u03bcl) and continuously rising rate of recent HIV-1 infections. In China, the overall improvement on immune status of HIV-1-infected persons can be partially explained by the scale-up of HIV testing. It was reported that between 2009 and 2018, the total person-times of HIV testing in China increased from 55.6 million to more than 240 million , which gHowever, it is worth noting that recently infected HIV-1 cases are usually highly infectious because of their high viral loads, but they mainly remain undetected . And, yoOne more evidence for the overall improvement of immune status of the HIV-1-infected persons was also observed in this study. As shown in CRF01_AE genotype which accounts for only approximately 5% of global HIV-1 infections concentrates in Southeast Asia and China , 2020. Ip\u2009=\u20090.0191), and 2015\u20132016 . Based on our data, the young MSM infected with CRF01_AE virus increased disproportionately during 2010\u20132014 in Heilongjiang Province, Northeast China, and the proportion of this population remained stable thereafter. There was no sign that the proportion of this young MSM population would decline. In future, more concerns should be put on the measures of HIV-1 prevention and control targeting the young MSM with CRF01_AE infection.In another study on HIV-1-infected MSM (aged 16\u201325\u2009years) from 13 provinces in northwest, eastern, and southwest regions of China, the authors reported that the proportion of CRF01_AE infections decreased from 55.4% to 43.5% between 2009 and 2014 . In the n\u2009=\u2009140). Therefore, in some statistical analyses, differences among groups or subgroups may be underestimated. Even so, several important findings were yielded. We believe that using a larger sample size will further confirm our findings. Second, the data from cases that were diagnosed in very recent years lacked. In fact, we had collected the demographical and clinical information of HIV-1-infected persons who were diagnosed in 2017\u20132019 (n\u2009=\u2009853). The median age of these cases was 33\u2009years (IQR 27\u201345) and the proportion of young cases (aged <30\u2009years) was 32.7% (279/853). This seemed that the younger age trend of local new HIV-1 infections was continuing. But because most of them lacked information on CD4 count, transmission route, and HIV-1 genotype, these cases were not included in this study. Third, HIV-1 genotyping based on partial gag and env gene regions might introduce some bias. Further analyses based on the near full-length genome of HIV-1 will be required in future. Despite these limitations, we still believe that our work could provide several clues for further research and HIV/AIDS control in Northeast China.There were limitations in this study. First, although we had made great efforts to collect samples from the clinical monitoring site, the biggest site in Heilongjiang Province, the sample size in early years (2010\u20132011) was still small . Written informed consent was obtained from each participant or participant\u2019s legal guardian.F-XW and S-LL conceived the experiments. F-XW and Q-HL designed the experiments. X-HC collected the clinical samples. Q-HL, J-YW, S-YL, S-LZ, and W-BZ performed the experiments. J-YW, Y-QZ, E-LL, and Y-RW analyzed the data. Q-HL and J-YW wrote the original draft of the manuscript. F-XW and S-LL reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundations of China (grant numbers 81971915 and 81601755) and Natural Science Foundation of Heilongjiang Province (grant number C2017043). The funders had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.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": "Considering the development of the shadow economy of 36 European and OECD countries over the period from 2003 to 2022 and the effect of the Coronavirus pandemic from 2020 onwards, the average size of the shadow economy of 36 European and OECD countries decreased from 16.48% of GDP in 2020 to 16.07% in 2021 (a decline of 0.41 percentage points). Due to a continued (forecasted) economic recovery in 2022, the average shadow economy of these 36 countries will slightly increase to 15.96% of GDP : a very modest reduction of 0.11 percentage points. In this introductory section, I begin by making some short remarks on the question of how to estimate a shadow economy (SE).Modeling the shadow economy as an unobservable (latent) variableDescription of the relationships between the latent variable and its causes in a structural model:.The link between the latent variable and its indicators is represented in the measurement model:where:In the following, the MIMIC estimation procedure .X(q\u2009\u00d7\u20091) vector of causes in the structural model.Y(p\u2009\u00d7\u20091) vector of indicators in the measurement model.\u0393(1\u2009\u00d7\u2009q) coefficient matrix of the causes in the structural equation;\u039by(p\u2009\u00d7\u20091) coefficient matrix in the measurement model;\u03b6error term in the structural model and \u03b5 is a (p\u2009\u00d7\u20091) vector of measurement error in y.The specification of the structural equation is:\u03b3i and \u03bbi are coefficients to be estimated.The specification of the measurement equation is:where The first step is that the shadow economy remains an unobserved phenomenon (latent variable) which is estimated using the causes of illicit behavior, e.g., the tax burden and regulation intensity, and indicators reflecting illicit activities, e.g., currency demand and official work time. This procedure \u201cproduces\u201d only relative estimates of the size of the shadow economy.In the second step, the currency demand method is used to calibrate the relative SE estimates into absolute figures by using two or three values of the absolute size of the shadow economy from CDA estimations.How does one proceed to get the absolute figures? Feld and Schneider  use the The following presented estimates are reached using the MIMIC and currency demand methods.3The Coronavirus pandemic caused a severe recession in almost all European and OECD countries in 2020 and to a less extent also in 2021. The recession caused a strong rise in unemployment and a sharp decline of GDP and of national income. These major causal driving forces of the shadow economy had the effect of a strong increase in the shadow economies of these 36 countries. In Tables In 2020, the worldwide Coronavirus pandemic occurred and caused a severe recession in almost all countries. One consequence of this was a strong rise in the average size of the shadow economy to 17.87% (of GDP) of the 28 EU countries.The strongest increase (by 3.13 percentage points) took place in Croatia, with a rise from 26.43% of official GDP in 2019 to 29.56% of GDP in 2020; the next strongest increase (2.81 percentage points) was seen in Bulgaria, where SE activities rose from 30.12% of GDP in 2019 to 32.93% of GDP in 2020. The weakest increase (at 0.77 percentage points) was found in Finland, where the SE rose from 10.59% of GDP in 2019 to 11.36% of GDP in 2020; the second lowest increase (0.92 percentage points) occurred in Denmark from 8.92% of GDP in 2019 to 9.84% of GDP in 2020.With the help of projections for some countries, calculations can be made of the development of the shadow economies in European and OECD countries for 2021. In 2021, \u201conly\u201d a modest decrease of the of the shadow economy from 17.87% of GDP (2020) to 17.42% of GDP  took place; hence, the average decline of the shadow economy of the EU countries will be 0.45 percentage points or 2.52%. The causes of this decline were massive public spending in infrastructure, subsidies to enterprises and special transfers to individuals which led to sizeable GDP growth combined with a decline in unemployment. The labor retention schemes applied in most OECD countries typically also partially replaced the previous labor income of workers which had effectively been laid off temporarily  to 17.29% in 2022. A decline will happen in 15 EU countries, while shadow economy activities will increase in 13 EU member countries.Turning to the development of the shadow economy in three non-EU but European OECD countries, the results are shown for the period from 2003 to 2022 in Table Next, we turn to the development of the shadow economy of the highly developed Non-European OECD countries, namely Australia, Canada, Japan, New Zealand, and the USA over the period from 2003 to 2022; results are presented in Table Finally, we consider the development of the shadow economy of all 36 European and OECD countries over the period from 2003 to 2022; the various averages are shown in Table In 2020, a strong increase of the shadow economy from 14.98% of GDP (2019) to 16.48% of GDP (2020) is observed; i.e., a 1.5 percentage points or 10% increase \u2014 the strongest increase of the average figure over the last 20\u00a0years. The main reason of this increase is the worldwide Coronavirus pandemic and the resulting severe recession which affected most countries. For 2022, a modest decline of the shadow economy \u2014 by roughly 0.52 percentage points \u2014 is forecasted. The main reason being the recovery of the official economy in 2021 and the forecasted continued recovery in 2022 (as of January 2022).Eastern, Central, and Southern European countries, such as Bulgaria, Cyprus, Czechia, Latvia, Lithuania, and Poland, have higher shadow economies in comparison to the \u201cold\u201d western European Union countries, such as Austria, Belgium, Germany and Italy. Hence, we have an increase of the size of the shadow economy from west to east.In addition, an increase in the size and development of the shadow economy can be observed from north to south. On average, the Southern European countries have considerably higher shadow economies than those of Central and Western Europe. Figures\u00a0The five non-European highly developed OECD countries  have lower shadow economies with an average size of about 8.40% of GDP in 2021 and predicted one of 8.3% of GDP in 2022, which is lower than the SE estimates for most European countries.To summarize, there are four different developments with respect to the dynamics of the shadow economy of these 36 European and OECD countries up to 2022:Since 2020, and in every country, the challenge for all governments was to undertake policy measures aimed at stimulating the official economy with strong GDP growth and a reduction of unemployment rates in order to reduce the shadow economy. The more successful such policy measures are, the more the shadow economy declines. Most countries have indeed enjoyed some success..However, the crucial question remains: \u201cIs this reduction of the shadow economy a blessing or a curse?\u201dFinally, one can make the following four policy conclusions:(i)If one assumes that roughly 50% of all shadow economy activities complement those of the official sector , the development of the total  GDP is always higher than the \u201cpure\u201d official one.(ii)A decline of the shadow economy will only increase the total welfare in a country if policymakers succeed in transferring economic activity from the shadow economy into the official economy.(iii)Therefore, policymakers have to favor and choose such policy measures as strongly increase the incentives to transfer the production from the shadow (black) to the official sector.My answer is:Hence, the conclusion of these two remarks is: The decline of the shadow economy will only be a blessing for the economy as a whole, if incentive-orientated policy measures will be applied, which I strongly recommend.(3)The massive recessions occurring in most European and OECD countries in the pandemic went along with high government deficits and rising expected long run public debt-GDP ratios. This could generate medium-term pressure in many countries to raise tax rates which in turn would make it difficult to reduce the size of the shadow economy.(4)As regards the Ukraine-Russia war which started in February 2022, many European and OECD countries will face a large wave of refugees who will face initial impediments to working in the official economy. Since standard modeling of immigration and refugee waves suggests that Eastern European countries would receive the highest shares of refugees from Ukraine, the lead position of Eastern European EU countries in terms of the size of the shadow economy in the overall EU will be reinforced in the medium term.Considering the latest developments during the fall of 2021 and from February 2022, I add two further policy conclusions:"}
+{"text": "Understanding the determinants of the shadow economy plays a vital role in formulating policies for economic growth and development, particularly for the Southeast Asian countries\u2013a new economic force for a global economy. The key drivers of a shadow economy, such as institutional quality, taxation, government expenditure, are widely examined. However, the effect of national intellectual capital, which affects macroeconomic indicators, on the shadow economy has largely been ignored in the existing literature. Our paper examines this critical link and its causality relationship for eight Southeast Asian countries from 2000 to 2017. This paper uses the dynamic ordinary least squares (DOLS) and fully modified ordinary least squares (FMOLS), which allow cross-sectional dependence and slope homogeneity in panel data analysis. Empirical findings from this paper indicate that national intellectual capital impacts negatively and significantly the shadow economy size. This finding implies that enhancing national intellectual capital reduces the shadow economy size. These two forces lead to enhanced economic growth. Our Granger causality tests confirm a bi-directional relationship between national intellectual capital and the shadow economy. As a result, policies targeted to reduce the shadow economy size can now include the accumulation of national intellectual capital, particularly for Southeast Asian Countries. Shadow, informal, or unofficial economy exists as a pervasive economic feature across nations . A shadoPrevious studies have found many determinants of the shadow economy, including unemployment , 8; tax Countries in the Association of Southeast Asian Nations (ASEAN) provide a fruitful context to examine this critical link and its causality relationship. The size of the shadow economy in these ASEAN countries is approximately equal to 30.24 per cent of the national GDP. The smallest shadow economy of 13.1 per cent of the GDP of the region belongs to Singapore, whereas the largest size of 41.9 per cent of GDP was in Thailand in 2000 and 2017 . MeanwhiThe main objective of this study is to examine the effects of national intellectual capital on the shadow economy across countries globally. To achieve our objective, we apply the index of national intellectual capital (INIC) developed by  to measuFirst, to the best of our knowledge, this is the first paper to investigate the link between national intellectual capital and shadow economy and their causality relationship in the ASEAN countries. Second, we focus on a long-term relationship between national intellectual capital and the shadow economy using the dynamic least square (DOLS) and fully modified least square (FMOLS) methods. Furthermore, pooled mean group (PMG) method is also used for robustness check. Third, we focus exclusively on the Southeast Asian nations from 2000 to 2017 to provide direct policy implications for the governments.Given the importance of national intellectual capital and shadow economy in Southeast Asian countries, this study contributes to the existing literature on the following grounds. The study is structured as follows. Following this introduction, section 2 presents and synthesizes the literature review. Section 3 discusses data and research methods. The results are presented and discussed in section 4, followed by policy implications in section 5 of the paper.The term \u201cshadow economy\u201d is often named as informal sector or economy , 24 the Most scholars agree that the shadow economy is inevitable because various economic activities cannot be counted in official accounts , 30. In The shadow economy estimation techniques can be classified into three main categories: direct, indirect, and multiple indicators multiple causes (MIMIC) . First, Tax evasion and institutional quality are considered the leading causes of the shadow economy . The theThe concept of intellectual capital was initially generated from the firm level for determining the firm\u2019s market value . HoweverThe concept of national intellectual capital has been explained in previous studies. However, there is no agreed definition of national intellectual capital due to a lack of coherent theory and dependable measurement models . NationaDespite the widely recognized importance of national intellectual capital, its assessment and measurement are tricky because of the fuzzy concept , 56. NevRecently , initiatThe current studies on the shadow economy mainly focus on employment, taxation, and institutional quality. However, previous studies have neglected to examine other factors such as national knowledge capital, especially in the knowledge-based economy and the 4.0 technology revolution. The national intellectual capital concept can be considered the endogenous economic growth theory principles, emphasising knowledge and technology in economic growth and development. Many economists have utilized intangible inputs in their academic research, including human capital ; R&D invThe above empirical studies have examined the impact of specific components of national intellectual capital on economic growth. However, the role of national intellectual capital in the shadow economy has not been investigated. We consider that national intellectual capital might be a key driver for the shadow economy for the following reasons. First, national intellectual capital is considered a significant contributor to sustainable economic growth development , 21, whiFurthermore, the nexus between national intellectual capital and the shadow economy can be explained through the structural components of national intellectual capital. Therefore, human capital is one of the critical components of INIC. In the development of the INIC index, human capital is proxied by three distinct macroeconomic indicators, including (i) school enrolment (tertiary), (ii) school enrolment (secondary), and (iii) government expenditure on education.  use humaMoreover, inward foreign direct investment (FDI) is generally used as a proxy for relational capital\u2013one component of intellectual capital. This type of capital flow might be a driver of the shadow economy. FDI inflow is an additional capital from international companies that can benefit the official economic activities  and imprOur empirical review reveals that previous studies focus on a specific type of intangible resources. No comprehensive study has been conducted to incorporate all critical aspects of national intellectual capital. This research gap warrants our analysis to provide empirical evidence on the effects of national intellectual capital on the shadow economy size and their causality relationship for the eight Southeast Asian countries.i and t represent a country and time, respectively. SE denotes a shadow economy size (a per cent of GDP) from [This study examines the impact of national intellectual capital on the size of the shadow economy in 8 ASEAN countries during the 2000\u20132017 period. The following general equation is used:DP) from  study. TDP) from  approachFor the control variables (Xj), the study employs economic growth (GDP per capita), trade openness (TR), bank credit (CE), government expenditure (GE), and inflation (INF). These variables are selected based on previous empirical analyses, including , 76.The study begins with a correlation analysis between variables for the estimation procedure. Then, the residual cross-sectional dependence check is performed to establish whether a cointegration check is required in the analysis. The panel unit root and panel cointegration tests are examined in this study. Based on the findings on the cointegration from these tests, the dynamic OLS (DOLS) and the fully modified ordinary least squares (FMOLS) estimations are used to examine the effects of national intellectual capital on the shadow economy.We also use the pooled mean group (PMG) estimation as the robustness analysis to ensure that our empirical results are robust. The main characteristic of PMG is that it allows short-run coefficients, including the intercepts, the speed of adjustment to the long-run equilibrium values and error variances to be heterogeneous country by country. In contrast, the long-run slope coefficients are restricted to be homogeneous across countries. Given that the countries in the sample are within the same regional economic bloc, the assumption of a homogeneous long-run estimate is plausible. However, these countries may be bonded by the same trade terms, laws, monetary policy, while the short-run estimates will differ due to country-specific economic and institutional differences.y) is expressed through Eq (Auto-regressive distributed lag (ARDL) model\u2019s  unrestrirough Eq .y is the size of the shadow economy (a per cent of GDP) and used as a scalar response variable, x is the vector of all the independent variables  as expressed in Eq  and t are used to denote the entities and time period, respectively. For computing the error correction model, Eq  is required to establish the stable equilibrium. The PMG model requires homogeneity in the long-run coefficient, i.e. \u03bb = \u03bbi for all the cross-section units, and the same needs to be tested empirically [Where \u03a8irically . To calcficients . The estMoreover, our study recruits the Granger causality test for panel data to examine causality between independent variables with the shadow economy.This study uses a sample of 8 ASEAN countries, including Brunei Darussalam, Cambodia, Indonesia, Malaysia, the Philippines, Singapore, Thailand, and Vietnam, from 2000 to 2017. Most of these countries are emerging markets except for a tiny country Brunei and the city-country, Singapore. Cross-sectional dependence, which may cause inefficiency in the results, frequently happens in the panel data estimation. The  test is The panel unit-root test is employed to examine the stationarity of all variables. We utilize the panel unit-root test  suggesteResults from our unit-root tests confirm that all variables used in our analysis are integrated at I(1). As such, these variables may move together in the long run. In particular, national intellectual capital and shadow economy are co-integrated. Our study uses various panel cointegration tests, including \u201385 residWe employ the panel DOLS estimator suggested by  and the In contrast, an increase in bank credit, government expenditure, and inflation appear to link with the larger shadow economy for the ASEAN countries. First, an increase in the banking sector credit tends to raise the level of liquidity, which will exert more pressure on the money quantity in circulation, resulting in high inflation . InflatiThe pooled mean group (PMG) method examines the effective long-term estimates and consistent mean values when the sample sizes are large , 90. In Our empirical results indicate that an improved national intellectual capital will reduce shadow economy size in the ASEAN countries. We now examine the causality relationship between national intellectual capital and the shadow economy. We employ a panel causality method proposed by  to detecReducing the shadow economy size is always one of the fundamental macroeconomic policies for governments worldwide, especially for the ASEAN countries, which have exhibited a significant size of the shadow economy. Previous studies have examined the effect of various macroeconomic indicators on the shadow economy. However, the effect of the national intellectual capital on the shadow economy has largely been ignored in the existing literature. This study uses a sample of eight members of the ASEAN over the 2000\u20132017 period to determine the drivers of the shadow economy and the effect of national intellectual capital on the shadow economy. Our study also examines the short-run and long-run effects and the Granger causality relationship between them.Our empirical results are two folds. The first group of results focuses on the effect of national intellectual capital on the shadow economy and their Granger causality relationship. Results in this group can be summarised as follows. First, we find that the national intellectual capital and shadow economy are co-integrated in the long run. This finding implies that the national intellectual capital and shadow economy have a relationship in the long run. Second, our empirical findings indicate that a more significant accumulation of national intellectual capital reduces the shadow economy size in the long run. Third, there exists a bi-causality relationship between national intellectual capital and the shadow economy. This finding indicates that policies targeting the shadow economy may also affect the national intellectual capital. The opposite conclusion also holds.The second group of our empirical findings focuses on the shadow economy drivers. For the Southeast Asian countries, the key drivers leading to the reduction of the shadow economy include trade openness and economic growth. Meanwhile, an increase in bank credits and government expenditures is associated with an increased shadow economy. A bidirectional causality relationship is also found between the shadow economy and critical macroeconomic factors such as economic growth, trade openness, and government expenditure. Our findings indicate that policies supporting economic growth and trade openness will also limit the expansion of the shadow economy.Policy implications have emerged based on the results of this study. First, the governments of the Southeast Asian countries should formulate and implement policies to improve the accumulation of national intellectual capital by investing in human capital and encouraging the development of intellectual infrastructure. These policies will also play an essential role in reducing the shadow economy size in the long term. Second, policies related to promoting economic cooperation, attracting foreign investment, promoting trade, and increasing trade openness should also be planned. Firms can take advantage of international trade by opening up, thereby reducing the incentive for entrepreneurs to operate in the informal sector. Hence, the size of the shadow economy can be controlled. Third, our empirical results have also shown that economic growth encourages a reduction in the shadow economy size. As such, policymakers should consider promoting economic growth and, as a result, employment for the people. Doing so is expected to reduce the size of the shadow economy because people with good income sources from the official economy will no longer be interested in working in the shadow economy. Fourth, the governments should carefully consider implementing monetary and fiscal policies with a clear understanding that an increase in bank credit and government expenditure may lead to a larger shadow economy.This study suffers limitations. Limited data is worth mentioning. Future studies may benefit from using historical data for an extended period on the shadow economy. A new theoretical framework may need to be developed to explain the channels through which national intellectual capital impacts the shadow economy. In addition, the effects of national intellectual capital on the shadow economy are currently under-examined. This effect may be relevant and important to be investigated for other countries/ regions in the future."}
+{"text": "Gold nanoclusters (Au NCs) are widely used in various types of detections due to their unique fluorescence properties. However, there are rare reports on enhanced fluorescence sensors for drug molecules. Here, we report a novel strategy for detection of sodium 2-mercaptoethanesulfonate (MES) by using a fluorescence-enhanced histidine stabilized Au NCs (His-Au NCs) probe. This fluorescence probe showed excellent selectivity and sensitivity towards MES. Furthermore, we have demonstrated that the fluorescence enhancement of His-Au NCs was attributed to ligand exchange with MES by Fourier Transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The feasibility of practical applications of this probe was further investigated by sensing the MES content in Mesna injection (Uromitexan). Schematics illustrating the MES-induced fluorescence enhancement of His-Au NCs for MES quantification because of the surface ligand exchange between His and MES. Even though the sensitivity of these instrumental analytical methods is relatively high, they are usually complicated in sample pretreatment and high in operation and instrument cost. To address these shortcomings, spectrophotometric methods for the detection of MES were established.28\u201331 Vaishnav et al.28 developed a simple colorimetric sensor based on the aggregation of spherical Ag NPs caused by MES. Ma et al.29 and Sroka et al.30 achieved the quantification of MES by measuring the ultraviolet absorbance of colored substance generated from reaction with MES. Vallvey et al.31 developed the chemiluminescence (CL) method for MES detection based on the reaction of the MES with Ce(iv). However, these spectrophotometric detection methods required extreme acidic conditions, and most of them were indirect and complicated in operation, suggesting the necessity to develop a fast, simple, and economical method for MES detection.The sodium 2-mercaptoethanesulfonate (MES), a thiol-containing drug, is mainly adopted as an antioxidant in renal protection because of the free thiol group.32 the effects of MES-stabilized Au NCs (MES-Au NCs) and histidine-stabilized Au NCs (His-Au NCs) on the propagation of pseudorabies virus were investigated. Occasionally, we found that the fluorescence intensity of His-Au NCs could be enhanced after adding MES. Inspired by this observation, herein we firstly presented novel Au NCs-based fluorescent sensors for detection of MES via linear fluorescence enhancement of His-Au NCs in the presence of MES  analysis showed no obvious difference between His-Au NCs and MES-Au NCs in morphology  scale\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>SO at 1251 and 1179 cm\u22121, strongly suggesting that MES anchors on the surface of Au NCs through the sulfur atom and Au atom by the formation of Au\u2013S bond.34,35 Meanwhile, both gold clusters exhibited the characteristic IR bands of imidazole ring  of histidine,36 confirming the partial replacement of His-ligands on the surfaces of His-Au NCs by MES. The ligand exchange could be further verified by X-ray photoelectron spectroscopy (XPS). Specifically, the XPS spectra of His-Au NCs  and X-ray photoelectron spectroscopy (XPS). Firstly, the capping ligands on the surfaces of Au NCs were characterized by FTIR Fig. S3. A peak  Fig. S4a, MES-Au  Fig. S4a and the  Fig. S4a showed tI/I0) at 500 nm as a function of MES concentration ([MES]). The dependence of \u0394I/I0 on MES concentration followed the equation: \u0394I/I0 = 0.01458[MES] + 1.01114  from MES concentration of 50 to 450 \u03bcM. The limit of detection (LOD) for MES was 4.5 \u03bcM at a signal-to-noise ratio of 3. Table S1Then the potential application of the His-Au NCs probes in the detection of MES was tested. As shown in +, Na+, Mg2+, Ca2+, Fe2+, Al3+, NH4+, Cl\u2212, Br\u2212, SO42\u2212, NO3\u2212, hydroxyproline (Hyp), glutamic (Glu), cysteine (Cys), EDTA\u00b7Na2 and DIMESNA as interferences. As shown in Furthermore, the specificity and selectivity of His-Au NCs probes were tested by several control experiments using KTable S2Our work presented a simple, economical, sensitive, and selective method for detecting MES by fluorescence-enhanced probes based on His-Au NCs. MES was found to enhance the fluorescence of His-Au NCs by replacing the His-ligands through the formation of Au\u2013S bond, leading to linear enhancement of fluorescence intensity in response to MES over the range of 50 to 450 \u03bcM with high sensitivity . To our best knowledge, this is the first report about the direct spectrometry method for MES detection using His-Au NCs probes under relatively mild condition. The His-Au NCs probes show excellent selectivity towards MES in the presence of most common interferences. Moreover, the His-Au NCs probes exhibit satisfactory recovery when used to sense the MES content in Mesna Injection, suggesting their potential application for MES determination in real samples.l-histidine (His) and glucose (glucose) were purchased from Aladdin Reagent Co., Ltd. disodium (DIMESNA) was acquired from ApexBio Reagent Co., Ltd. Cysteine (Cys) and chloroauric acid (HAuCl4) were obtained from Sigma-Aldrich Reagent Co., Ltd. Potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride hexahydrate (MgCl2\u00b76H2O), calcium chloride dihydrate (CaCl2\u00b72H2O), aluminum chloride hexahydrate (AlCl3\u00b76H2O), potassium bromide (KBr), ammonium chloride (NH4Cl), iron sulfate heptahydrate (FeSO4\u00b77H2O), sodium sulfate (Na2SO4), sodium nitrate (NaNO3), ethylenediaminetetraacetic acid disodium salt (EDTA\u00b7Na2), glutamic acid (Glu) and proline (Hyp) were acquired from Sinopharm Chemical Reagent Co., Ltd. Mesna injection (Uromitexan) was obtained from Qilu Pharmaceutical (Hainan) Co., Ltd. The deionized water was used in this study. All chemicals and solvents were of analytical grade or better and used without further purification.Sodium 2-mercaptoethanesulfonate (MES), The UV-vis absorption spectra were recorded on a UV-2450 spectrophotometer . X-ray photoelectron spectroscopy (XPS) was recorded on a VG Multilab 2000 X-ray photoelectron spectrometer . Fluorescence measurements were performed using an RF-5301 PC spectrofluorometer . Fourier transform infrared spectra (FTIR) were obtained to identify the molecular structures of Au NCs with a Nicolet Avatar-330 spectrometer  through the KBr pellet technique. The size and morphology of Au NCs were recorded on a HRTEM JEM-2100F instrument . All deionized water was obtained from a Milli-Q ultrapure water machine .25,26 Briefly, chloroauric acid  was added to the solution of histidine , then stored at 25 \u00b0C for 2 h without light. The concentration of the as-synthesized His-Au NCs was 2500 \u03bcM.(a) Au nanoclusters were synthesized by blending histidine and chloroauric acid as reported previously.(b) His-Au NCs  was mixed with MES , and maintained for reaction overnight without light.A solution of MES at a concentration of 0.01 M was separately diluted with deionized water to obtain 250, 375, 500, 750, 1500, 2250, 3000, 4500, 6000, 7000, and 8000 \u03bcM MES solutions. An aqueous solution of histidine  was added to 200 \u03bcL of different concentrations of MES solution. The final volume was 1 mL. The fluorescence intensity was measured for each set of samples after reacting for overnight 25 \u00b0C in the dark. Each set of experiments was repeated three times.2, CaCl2, EDTA\u00b7Na2, Glu, Cys, AlCl3\u00b76H2O, KBr, NH4Cl, FeSO4\u00b77H2O, Na2SO4, NaNO3, DIMESNA and MES were separately prepared at a concentration of 3000 \u03bcM. Then, 200 \u03bcL of each solution was mixed with His-Au NCs solution  with a final volume of 1 mL. The fluorescence intensity was measured for each set of samples after reacting for overnight 25 \u00b0C in the dark. Each set of experiments was repeated three times.The solutions of KCl, NaCl, MgCl(a) The Uromitexan solution  was mixed with His-Au NCs solution  at a volume of 1 mL with deionized water.(b) Two sets of the Uromitexan solution (200 \u03bcL and 2000 \u03bcM) were mixed with MES standard solutions  at a final volume of 1 mL, respectively. Then, the obtained solutions (500 \u03bcL) were separately added to His-Au NCs solution  at final volume of 1 mL with deionized water. After reaction overnight at 25 \u00b0C in the dark, the fluorescence intensity was measured for each set of samples. Each set of experiments was repeated three times.There are no conflicts of interest to declare.RA-009-C9RA01563A-s001"}
+{"text": "PMV) and Predicted Percentage of Dissatisfied (PPD) indices, according to provisions of the standard ISO 7730:2005, and comparing the results with the subjective perception of the workers revealed by applying individual questionnaires. The results of the study represent an important input element for establishing the preventive and protective measures for the analysed workplaces in correlation with the measures addressing other specific risks and, also, could serve as a model for extending and applying to other similar workplaces in future studies. Moreover, the mathematical model and the software instrument used for this study case could be used in further similar studies on larger groups of workers and in any industrial domain.Considering thermal environment aspects have a major impact not only on occupational health and safety (OH&S) performance but also on the productivity and satisfaction of the workers, the aim of the case study was to assess the thermal comfort of a group of 33 workers in an automotive industry company, starting with collecting data about the thermal environment from different workplaces, continuing with the analytical determination and interpretation of thermal comfort using the calculation of the Predicted Mean Vote ( The thermal environment has a major impact on occupational health and safety (OH&S) and also has a great influence on the productivity and satisfaction of workers ,2. OccupIn recent years, the number of studies on the effects of the indoor thermal environment on human physical and mental health has increased. The studies focused on different aspects of the theme, such as assessing the indoor thermal environment in specific locations  ,7, usingISO 7730:2005 Standard defines thermal comfort, also known as hygrothermal comfort, as \u201cthat condition of mind which expresses satisfaction with the thermal environment\u201d , which iThe main effect of cold stress is hypothermia, a gradual process that is shown in three stages: Mild, moderate and severe. Mild-stage symptoms are shivering, grogginess and poor judgment or confused thinking. The moderate stage is manifested by violent shivering, inability to think or pay attention, slow, shallow breathing, slurred speech and poor body coordination. In the severe stage, the symptoms are loss of consciousness, little or no breathing and a weak, irregular or non-existing pulse .Typical symptoms of heat stress are an inability to concentrate, muscle cramps, heat rash, severe thirst, fainting, heat exhaustion  and heat stroke  ,20.Physiological effects of thermal stress exposure determine a higher risk of work accidents, diseases and health problems, affecting productivity by presenteeism and absenteeism issues and generating additional costs for the companies ,22. FurtControlling thermal comfort can be realized by both technical and organizational measures, such as controlling the environment by adjusting its parameters  as appropriate; separating the source of heat or cold from the employee; controlling the task ; controlling clothing ; allowing the employee to make behavioural adaptations ; monitoring the employee  ,27.PMV) and Predicted Percentage of Dissatisfied (PPD) indices, according to provisions of the standard ISO 7730:2005, and comparing the results with the subjective perception of the workers revealed by applying individual questionnaires. The need for this study was raised by the finding that in the OHS risk assessment for the analysed workplaces, the risk related to the thermal environment was assessed at a low level, but some workers complained about thermal comfort, and there were no available measurements of the thermal environment parameters. The results of the study represent an important input element for establishing the preventive and protective measures for the analysed workplaces and could also serve as a model for extending and applying them to other similar workplaces in future studies.The aim of the work is to assess the thermal comfort of a group of workers in an automotive industry company, starting with collecting data about the thermal environment from different workplaces, continuing with the analytical determination and interpretation of thermal comfort using the calculation of the Predicted Mean Vote ;clt is the clothing surface temperature, in \u00b0C.quations ,13:(1)PMM is established by Annex B of ISO 7730:2005, depending on the activity type. The value of W is equal to zero for most activities [clI is provided by Annex C of ISO 7730:2005. The values of at, arv and relative humidity  are determined by measurements at the analysed workplace. The value of mrt was determined with a heat stress monitor by measuring the a, vart and the global temperature, and using the equation provided by ISO 7726:1998, Annex B [clt and ch could be determined by iterations from Equations (2) and (3).The value of tivities . The val Annex B . The valPPD index establishes a quantitative prediction of the percentage of thermally dissatisfied people who feel too cool or too warm, and is determined using the following equation [The equation :(5)PPD=1\u00ae Excel\u00ae, for the calculation of the PMV and PPD values for an assessed workplace based on the mathematical model presented above. The caption of the sheet is presented in A software instrument was elaborated by the authors, using MicrosoftThe checklist adapted from the Health and Safety Executive, shown in The case study was performed in a Romanian automotive industry company by analysing 33 workbenches from different production lines from the perspective of thermal comfort. The workers of this company were selected as the target participants of the study considering that the company was a partner in larger research on OHS, which includes the present study. Even if there is no registration of work accidents or occupational diseases caused directly by thermal stress, some of the workers raised an issue with thermal comfort. The analysed workplaces are located on the same open-space production floor. The target group is representative of the production floor, as it represents all of the workers at this production site. The measurements were performed simultaneously with 3 sets of instruments on one experimental day, in May, between 11 a.m. and 1 p.m., in sunny weather conditions with an outdoor temperature of 22 \u00b0C. The production floor is equipped with an air conditioning system, but the system was off at the moment of measurements. The thermal environment conditions were measured for each workplace using a heat stress monitor and an air velocity measuring instrument. The measurement height was 1.1 m since the workers\u2019 activity is performed in an orthostatic position. The workplaces and the relevant information about workers (age and gender) are presented in The activity performed by the employees at workbenches consists of manual handling and manual assembly of small dimensions components, using a scanner, putting the final products in boxes and using a trolley to transport boxes to the warehouse.The PPEs worn by the workers consist of safety glasses, hearing protectors, protective gloves and safety shoes.The work clothes worn by the workers consist of a cotton T-shirt, trousers, a jacket, underwear, socks and protective shoes and are similar for all the workers.at, mrt, arv and RH) were determined by measurements made with a heat stress monitor and an air velocity measuring instrument. The heat stress monitor was equipped with the following sensors: A globe thermometer (accuracy \u00b10.5 \u00b0C between 0 \u00b0C and 120 \u00b0C), relative humidity sensor (accuracy \u00b10.5% between 20% and 90%) and dry bulb thermometer (accuracy \u00b10.5 \u00b0C between 0 \u00b0C and 120 \u00b0C). The air velocity sensor accuracy was \u00b10.05 m/s, and the range was 0 to 20 m/s.For each workplace, the thermal environment parameters  of questions answered with \u201cYes\u201d. The higher the value of N, the higher the level of risk perceived by the worker related to thermal comfort issues.In the next step, using the presented software instrument, the values of The measurement results and collected data are presented in PMV ranges from 1.23 (for WP27) to 1.73 (for WP11) with corresponding PPD values of 31.28% and 48.83%, respectively. The thermal sensation is determined as \u201dSlightly warm\u201d for 21 workplaces and as \u201dWarm\u201d for 12 workplaces. These results show a moderate risk of thermal stress for the workers, from the point of view of the thermal environment parameters.For the assessed workplaces, the value of N, noted with avgN, was calculated. The results are shown in However, based on their responses to the questionnaire, the workers perceive the work environment differently from the point of view of their thermal comfort. Thus, the analysed worker group consists of 33 employees, with 14 women and 19 men aged from 20 years (for WP1) to 62 years (for WP33). The workers\u2019 responses were analysed in two separate groups: The first group is formed by the workplaces where the thermal sensation was determined as \u201dSlightly warm\u201d and the second group is formed by the workplaces where the thermal sensation was determined as \u201dWarm\u201d. For each group, the responses were clustered by age into three subgroups , and then each of these subgroups was divided in series by gender. For each series, the average value of PMV value, which is calculated based on the measured parameters and provisions in the ISO 7730:2005. Thus, for each of the two groups differentiated by thermal sensation, avgN and the level of the risk related to thermal comfort issues are lower for the young worker group (20\u201335 years) and higher for the middle-aged (36\u201350 years) and elderly workers (over 50 years). Furthermore, inside the same age subgroup, the perception of men and women is similar in most cases.The data presented in PMV values, are too high to ensure thermal comfort for most workers and show a moderate risk of thermal stress for the workers, in all analysed workplaces.The thermal sensation levels, determined by The middle-aged and elderly workers in comparison with younger workers are more susceptible to being the subject of work accidents, near-misses and occupational diseases indirectly generated by thermal stress, considering they perceive poorer thermal comfort than younger workers, as the responses to the questionnaire indicate. This perception could be the cause of errors due to premature fatigue; this is the situation for workplaces WP 02, WP 03, WP 09, WP 10, WP 11, WP 14, WP 15, WP 16, WP 20, WP 21, WP 22, WP 23, WP 25, WP 26, WP 28, WP 29, WP 30, WP 31, WP 32 and WP 33.Even if the premature fatigue due to poor thermal comfort perceived by the middle-aged and elderly workers does not generate OH&S issues, it may still be the main cause of worker errors, which could generate productivity or product quality issues; thus, the thermal comfort at the workplace not only represents an OH&S issue but equally a productivity and quality issue.PMV values and workers\u2019 responses to the questionnaire indicate the necessity to establish and implement a programme of technical and organizational preventive and protective measures to improve the thermal comfort conditions in all analysed workplaces, but especially in the workplaces with middle-aged and elderly workers.Both The findings on the thermal comfort of the workers in the analysed workplaces should be integrated into the overall OH&S risk assessment for these workplaces; the potential synergic effect of thermal comfort issues with other risks, such as neuropsychic effort determined by the work tasks or medical conditions of the workers, should be considered.For the workplaces analysed in this study case, the following conclusions could be synthetised based on the obtained results:In any work system, four specific components are involved: Executor, workload, means of work (work equipment) and work environment of the workplace/workstation. These components are in a permanent state of interdependence ,31,32,33PMV values, and subjective perception of the workers determined by questionnaires. This situation could have a negative impact on the OHS level of the analysed workplaces. Thus, from an occupational health perspective, the main effect could be premature fatigue due to the poor thermal comfort perceived, especially by middle-aged and elderly workers. Furthermore, thermal stress issues could have a potential synergic effect on other risks, such as neuropsychic effort determined by the work tasks or medical conditions of the worker. From the occupational safety perspective, even if, for analysed workplaces, the thermal stress is at a moderate level and is not susceptible to directly generating work accidents, it could still be an indirect cause of a work accident by increasing the occurrence of worker errors generated by premature fatigue. Moreover, by generating premature fatigue, the thermal stress could be the cause of productivity or product quality issues. Thus, the thermal environment represents an important part of the work environment, as a component of the work system, and plays a significant role in the workplace\u2019s OHS level by influencing the executor\u2019s behaviour within the work system. Therefore, the OHS risk assessment should also be based on the measurement of the thermal environment parameters. After performing the OHS risk assessment, a preventive and protective plan should be elaborated on, containing organizational, technical, sanitary and other measures to improve the workplace OHS level.For the analysed workplaces, the results of the study show a moderate risk of thermal stress, reflected by both thermal sensation levels determined by One of the limitations of the study is the small size of the target group, which, even if it is representative of the analysed workplaces and suitable for establishing OHS preventive and protective measures, is not big enough for extrapolation. Another limitation is determined by performing the experiment in one day, considering that the thermal comfort perceived by workers depends on psychological factors and may also be different if the experiment had been performed across different days.Monitoring the thermal environment can be performed by periodic measurements of thermal-comfort-determining parameters: Air temperature, radiant temperature, air velocity and humidity, and analysing the results in correlation with workers\u2019 personal factors, such as clothing insulation and metabolic heat. Considering the necessary time-lapse to perform the measurements and process the data, this approach does not allow real-time monitoring. This type of monitoring is suitable for applications such as this study case, where the above-mentioned parameters are relatively constant during the work shift and may vary with season. When the environmental and personal factors register important variations during the work shift, real-time monitoring may be necessary, which can be performed using wearable sensors and devices, which can integrate different parameters from the environmental, behavioural and physiological domains, while also providing real-time monitoring of the worker\u2019s health ,43.Based on the monitoring results, both technical and organizational measures should be established to improve the thermal comfort of workers. These measures should be integrated into the preventive and protective plan for the workplace, also containing measures that address other specific risks.Installing air conditioning and ventilation systems to ensure fresh air and suitable control of the temperature.Providing proper maintenance of the air conditioning and ventilation systems.Selecting and providing the PPEs by taking into account workers\u2019 thermal comfort in addition to the requirements of protection against other specific risks.For the analysed workplaces, examples of technical measures are the following:Allowing the workers to adapt their clothing to the environment temperature.Allowing the workers, and stimulating them, to report any situation of thermal stress they undergo at the workplace.Providing periodic medical control of the workers and monitoring health issues that represent a risk factor when working in thermal stress.Providing proper training to workers on thermal stress and measures to avoid it.Considering reducing or shifting working time during heatwaves.Involving workers or their representatives in consultation and participation processes regarding OH&S issues.For the analysed workplaces, examples of organizational measures are the following:The results of the study represent an important input element for establishing the preventive and protective measures for the analysed workplaces in correlation with the measures addressing other specific risks and could also serve as a model for extending and applying this to other similar workplaces in future studies. As the analysed worker groups are not big enough for extrapolation, the conclusion should be limited to this particular study case. However, the mathematical model and instruments (questionnaire and software) used for this study case could be used in further studies on larger groups of workers in any industrial domain."}
+{"text": "Monitoring the proteins and lipids that mediate all cellular processes requires imaging methods with increased spatial and temporal resolution. STED (stimulated emission depletion) nanoscopy enables fast imaging of nanoscale structures in living cells but is limited by photobleaching. Here, we present event-triggered STED, an automated multiscale method capable of rapidly initiating two-dimensional (2D) and 3D STED imaging after detecting cellular events such as protein recruitment, vesicle trafficking and second messengers activity using biosensors. STED is applied in the vicinity of detected events to maximize the temporal resolution. We imaged synaptic vesicle dynamics at up to 24\u2009Hz, 40\u2009ms after local calcium activity; endocytosis and exocytosis events at up to 11\u2009Hz, 40\u2009ms after local protein recruitment or pH changes; and the interaction between endosomal vesicles at up to 3\u2009Hz, 70\u2009ms after approaching one another. Event-triggered STED extends the capabilities of live nanoscale imaging, enabling novel biological observations in real time. Event-triggered STED is an automated approach that can initiate 2D or 3D STED imaging of specific regions in biological samples after detection of an event of interest. This approach can help maximize observations in live cell imaging and enable discovery. The temporal resolution of STED nanoscopy usually depends on the size of the region of interest to be imaged, owing to its most common single point-scanning implementation. This means that the technique can achieve high frame rate imaging (1\u201330\u2009Hz) in small regions of interest (1\u20135\u2009\u00b5m2) (refs. 4), but for larger fields of view, that is, up to 80\u2009\u00d7\u200980\u2009\u00b5m2, it takes on the order of minutes to acquire a single frame5. This illustrates the current trade-off between spatial and temporal resolution, in which fast dynamics inside cells can be followed only in sufficiently small areas that are often difficult to pinpoint due to the loss of the larger cellular context. Parallelized STED methods7 have tried to overcome this trade-off by minimizing the number of scanning steps during imaging, but they are currently limited by the camera frame rate and depletion power. Moreover, although STED nanoscopy is capable of a high temporal resolution, it is also susceptible to photobleaching and photodamage, which limits the total number of recordable frames9. Techniques often called \u2018smart microscopy\u2019 attempt to provide solutions for gentler recordings by either adapting the illumination to the sample characteristics or by switching between microscopy modalities.STED (stimulated emission depletion) nanoscopy has been successfully used to image a variety of structures in both living cells and tissues, even dynamically11 and super-resolution microscopy13 have helped to minimize the light dose and the recording time during imaging or to increase the image quality deep in tissues14. Multiscale approaches have focused so far on increasing the throughput of large-scale and relatively slow events such as cellular division17 or on screening18 with conventional methods, such as widefield or confocal imaging, or on super-resolution methods only after fixation. However, no sample-adaptive scanning approaches for live cell imaging have so far been triggered by subsecond real-time changes in the sample such as intensity spikes, local movements or morphological changes, and nor have they been automated to switch between distinct imaging modalities such as STED or other super-resolution approaches. As such, the increasingly important field of smart microscopy is still lacking a method that switches imaging scales and which incorporates nanoscopy methods that operate rapidly (on the order of tens of ms or seconds) after a stimulus.Sample-adaptive scanning approaches in conventionalIt is currently difficult to observe cellular processes at high spatial resolution (~30\u2009nm) efficiently and rapidly (up to tens of Hz) in cells, either because they are hard to localize in large cellular volumes, they happen too fast, or because the number of frames before bleaching is not high enough. However, if the user knew where and when to image the cellular process of interest, the quality, throughput, speed, and length of the observation would increase substantially, enabling the dynamics of the process to be unraveled comprehensively.Here, we present a novel sample-adaptive microscopy method called event-triggered STED (etSTED), which enables rapid two-dimensional (2D) and 3D STED nanoscopy acquisitions upon and at the site of automatic detection of subcellular events such as biosensing, local protein recruitment or vesicle trafficking in spatial proximity. It does so by combining fast (up to 20\u2009Hz) widefield imaging, which facilitates the detection and localization of events, with STED imaging, for high-resolution acquisition at the site of a detected event. The STED imaging can be performed with lateral (2D STED) or axial (3D STED) super-resolution in one or multiple frames recorded sequentially. The maximal transition between widefield and STED imaging happens in a temporal window of 40 ms from an event taking place. To detect the events of interest, the method runs a real-time analysis pipeline on every recorded widefield frame. We took special care to develop an analysis pipeline fast enough to detect the events of interest with a minimum delay of 6\u2009ms without compromising accuracy, that is, with minimal false-positive and false-negative events.The generalized implementation of etSTED enables the investigation of a diverse combination of triggering events and fine subcellular structures. The analysis pipelines developed in our implementation detect events such as intensity spikes as in local calcium or pH sensing; slower rises in intensity upon protein recruitment as in dynamin-mediated endocytosis; or the spatial proximity of vesicles during subcellular trafficking. And etSTED imaging can instead be performed on various proteins including actin, tubulin and synaptotagmin, or lipids such as cholesterol and sphingolipids enriched in the plasma membranes and endosomal vesicles. With etSTED we can observe different types of vesicle and membrane fusion events comprehensively and with an unprecedented level of detail in both neurons and cancer cell lines. This was possible only with a detection speed and multiscale approach specific to this work, which complements previously developed, slower sample-adaptive imaging methods.2 region of the sample is surveyed at any time in widefield imaging mode. These images are processed in real time with a rapid analysis pipeline, which returns a set of coordinates of any detected event. If an event, for example an intensity spike, is detected, the widefield imaging is stopped and the STED acquisition started in a small area around the detected coordinates with pre-determined image acquisition parameters. The size of the STED field of view is defined by the user, and in our applications it is always smaller than the widefield to achieve high temporal resolution (1\u201324\u2009Hz). When the STED image or timelapse is acquired, comprehensive data regarding the event are saved. This includes a widefield timelapse leading up to the detected event, the scanned STED image or timelapse, and a log file summarizing the parameters and timings of the event detection and scanning. The saved auxiliary data are important to confirm the validity of the event in post-acquisition analysis. It also enables further quantification of the event both temporally and spatially, within the larger widefield field of view. The microscope  to the vicinity of the plasma membrane during endocytosis  feedback between the two imaging modalities and their hardware components: lasers, scanners, cameras and acousto-optic modulators and tunable filters. The etSTED method is fully controlled via a widget in ImSwitch, also released as a standalone widget to facilitate its implementation in other microscope control software  and microtubules (SiR-tubulin), as timelapses over 11\u2009frames at 1.0\u2009Hz in a 5\u2009\u00d7\u20095\u2009\u03bcm22. The protein synaptotagmin-1 (Syt-1) is the calcium sensor that triggers vesicle release. The replenishment of the recycling pool is then mediated by further endocytosis, which occurs from tens of milliseconds up to 20\u201330\u2009s from the event23. Although the dynamics of synaptic vesicles upon electrical and chemical stimulation have been studied25, their concurrent behavior upon basal activity is still unexplored due to technical limitations in accessing such temporal window rapidly enough and with sufficient spatiotemporal resolution.In the presynaptic active zone the voltage-dependent, local and transient increase in intracellular calcium leads to vesicle release from the readily releasable synaptic pool26. When the synaptic vesicles are further recycled, the internalization of the labeled molecules enables imaging of the vesicle pools inside the presynaptic active zone  was 0.046\u2009\u00b1\u20090.025\u2009\u03bcm2 in the calcium-triggered timelapses and 0.030\u2009\u00b1\u20090.020\u2009\u03bcm2 in the manual timelapses   Fig. , left. F. t Fig. . Synapti2  is crucial for vesicular structures, which contain small intraluminal vesicles. They can be either sorted to lysosomes for degradation or fused to the PM for exocytosis of exosomes35. Intraluminal vesicles are highly enriched in the protein tetraspanin CD63, cholesterol and sphingolipids36. In particular, MVB\u2013PM fusion is stimulated by cholesterol nanodomains promoting a negative curvature of the membrane following the fusion event37.Once internalized, vesicles are transported along the endo-lysosomal pathway38 , 336\u2009ms (3\u2009\u00d7\u20093\u2009\u00b5m2), 41.3\u2009ms (1\u2009\u00d7\u20091\u2009\u00b5m2)). Although the STED frame acquisition time is currently the rate-limiting step, we could imagine future developments in which an even smaller and event-adapted region is imaged, which would further increase the possible STED timelapse frequency beyond the 20\u201330\u2009Hz presented here. With regards to the technical implementation, the processing time of the analysis pipelines as well as the total time from the event to the STED imaging could be further minimized with an increase in computational power and faster camera technology.In this work we outline an event-triggered method, etSTED, which enables super-resolved STED imaging within 40\u2009ms from a detected event taking place. Directing STED imaging in selected small regions around the detected events and within a specific time window does not only minimize the overall cell stress and photodamage but also enables a significant increase in the temporal resolution as compared with conventional STED imaging. The selected regions of interest can be imaged 100\u20135,000-fold faster as compared with the manual acquisition of STED timelapses in the whole investigated sample region  and has been proven to be efficient at localizing both calcium activity and local pH changes, the detection of other signaling and trafficking events might require tailored analysis pipelines. We provide two examples of such pipelines, named dynamin_rise and vesicle_proximity, which detect more slowly rising signals (~1\u2009s) and the increasing proximity of endosomal vesicles, respectively. Together this shows the potential of using diverse types of cellular dynamic events as triggers. The current implementation in the control software , and the polarization directions are temporally delayed to avoid interference. Wave plates are used to create circular polarization necessary for optimal depletion focus formation. Galvanometer mirrors are used for scanning  in a scanning system to enable constant resolution across an 80\u2009\u00d7\u200980\u2009\u03bcm2 field of view, as previously described5. Fluorescence is decoupled with a dichroic mirror. A bandpass filter  and a notch filter  are used before detection with an avalanche photodiode . For the widefield set-up, excitation of blue-shifted fluorophores was done with a modulated 488\u2009nm continuous-wave diode laser . Detection of widefield images was done through a bandpass filter  and a notch filter  with an sCMOS  camera . The widefield path is coupled into the beam path with a dichroic mirror after the scanning system. The general set-up uses a \u00d7100/1.40 oil immersion objective  and a microscope stand . The system uses a mechanical stage for lateral sample movement  and a piezostage for axial sample movement .All images were acquired on a custom-built STED and widefield set-up, based on a STED set-up previously described19 written in Python. Control of the etSTED method is performed using a custom-written widget and controller in ImSwitch, available at GitHub , which controls lasers, image acquisition, and runs real-time analysis pipelines with customizable parameters. Instructions on how to run etSTED imaging can be found in the GitHub repository of the standalone widget , while instructions on how to run ImSwitch can be found in the repository on GitHub and corresponding documentation (https://imswitch.readthedocs.io).Microscope hardware control is mainly performed through a National Instruments data acquisition (NI-DAQ) acquisition board . Hardware is controlled using microscope control software ImSwitchz-piezostage through a feedback loop, as previously described5, is used. It enables experiments to run stably for time periods longer than hours.A focus lock controlled with ImSwitch combining an infrared laser , a CMOS camera  and the The microscope control computer contains a Ryzen 7 3700X CPU (AMD) and a GeForce RTX 3060 Ti GPU (ASUS).The etSTED widget is a software module built in a generalized way to enable free choice of the lasers and detectors controlled with ImSwitch. For a thorough description of how to perform etSTED experiments with the control software, see the ImSwitch-etSTED GitHub repository readme file. The widget enables any arbitrary analysis pipeline to be used, and it additionally enables calibration of the imaging space coordinate transform.The coordinate transformation calibration is performed in a help widget into which a pre-acquired widefield image and scanning image of the same sample area can be loaded. Manual annotation of the same sample points in the images prepares fixed points for the coordinate transform calibration. A general third-order polynomial transformation with Levenberg\u2013Marquart optimization is used, enabling it to be compatible and precise regardless of any aberrations and distortions that may be present in the optical paths.Afterwards, an image analysis pipeline can be loaded, and all editable parameters of the pipeline will be loaded into the GUI . Last, to prepare for an etSTED experiment to be run, a binary mask of the sample may be recorded. It is performed with a separate functionality in which 10 frames are recorded and averaged, and a global thresholding is performed with a user-provided intensity threshold. The etSTED experiment is then started, with various options present as choices in the GUI: perform it as an endless loop or a single trigger; in visualization mode with real-time visualization of the preprocessed images to optimize analysis pipeline parameters; or in validation mode without triggering scans. While running etSTED experiments, detected event coordinates will be overlaid on the displayed widefield frames. The triggered imaging will be performed using pre-determined scanning parameters, including the choice of which lasers to use.https://github.com/jonatanalvelid/etSTED-widget). Analysis pipeline parameter values used for each experiment are listed in Supplementary Table Real-time analysis pipelines are provided as standalone python functions and must take the current widefield image, the previous widefield images and a binary mask of the considered region as input, and return a list of detected event coordinates in the widefield space. The real-time analysis pipeline additionally takes and returns a variable with any user-defined information from the analysis runs of the previous frames, for example for pipelines requiring tracking. It can additionally take any numerical parameters as input, for example various thresholds. The analysis pipelines developed in this work are provided and explained in more detail below, in Supplementary Notes 45, scipy46, cupy, opencv, trackpy47, pandas48, napari (www.napari.org) and pyqtgraph.The etSTED widget and analysis pipelines in the control software use the Python packages numpyhttps://github.com/jonatanalvelid/etSTED-widget/blob/main/analysis_pipelines/rapid_signal_spikes.py). Although it is optimized for the above-mentioned biosensors, it is also likely to perform well with similar, fast fluorescence sensors after pipeline parameter tweaking. The pipeline consists of pre-processing that uses the current frame, the previous frame, and a binary region-of-interest mask; and peak detection. The pre-processing transforms the current image into a smoothed map comparing the intensity in each pixel between the current and previous image. The peak detection compares the image with a maximum-filtered version of itself, finding local maxima as the coordinates where the two are equal. To avoid detection of noise fluctuations, the coordinate intensities are thresholded. Finally, the ratiometrically brightest peak is used throughout this work as the coordinate where the triggered STED imaging takes place. Most of the analysis pipeline runs on the GPU, significantly decreasing the runtime as compared with running it on the CPU. Altogether, the analysis pipeline runs in 6\u2009ms for the 800\u2009\u00d7\u2009800\u2009pixels widefield images used in this work.One analysis pipeline used throughout this work, rapid_signal_spikes, was developed to detect calcium events with Oregon Green 488 BAPTA-1 labeling in hippocampal neurons as well as CD63-pHluorin signal peaks in HeLa cells. It is shown schematically in Supplementary Fig. https://github.com/jonatanalvelid/etSTED-widget/blob/main/analysis_pipelines/dynamin_rise.py). The pipeline performs pre-processing with smoothing and background reduction. Then, a peak detection similar to that of rapid_signal_spikes is performed, and high and low thresholds are applied to avoid detecting noise and large clusters. Following this, the intensity in a small area around each peak is extracted. The peak positions and intensities are stored and form an extra-info parameter, and the pipeline links tracks from the peak positions. These tracks are analyzed to identify when a certain track first appears and how the intensity of that peak develops over time. An event is triggered after the appearance of a trace that stays detected for N frames (user-definable) and has an intensity that ratiometrically increases above a certain threshold ratio over those frames. The last coordinate of that trace is the event coordinate. Again, with substantial parts of the pipeline running on the GPU, it runs in 20\u201360\u2009ms for an 800\u2009\u00d7\u2009800\u2009pixels widefield image, depending on the number of tracks followed. The pipeline is likely to work after parameter tweaking for other similar event detection tasks in which local intensities are increasing over multiple frames.A second analysis pipeline used in the work is dynamin_rise, which detects slowly rising signal peaks occurring over multiple frames. We show this pipeline applied to dynamin1-GFP rising-signal-peak events in HeLa cells. It is further described in Supplementary Note https://github.com/jonatanalvelid/etSTED-widget/blob/main/analysis_pipelines/vesicle_proximity.py). Pre-processing, peak detection and track connection work similarly as in dynamin_rise, but the tracks are thereafter handled differently. The event detection is performed as a check of a set of conditions on pairs of tracks. Events are detected when five conditions are met: one track disappears; another track is close by; both tracks are consistently present; and at least one track has moved an accumulated vectorial distance and an absolute distance above certain thresholds. Each condition has a set of user-definable thresholds and ratios. Threshold values will expectedly vary with the type of vesicle investigated, given that various vesicles differ in morphology, motility, dynamics and density. Also, a substantial amount of this pipeline can run on the GPU, and the pipeline runs in 40\u2013110\u2009ms for an 800\u2009\u00d7\u2009800\u2009pixels widefield image, depending on the number of tracked vesicles.The third analysis pipeline used in this work, vesicle_proximity, detects incipient proximity-of-vesicles events, such as two endosomes approaching one another. We apply this pipeline to look at events in which multiple CD63-GFP-positive vesicles approach one another, signifying interaction. It is further described in Supplementary Note 2 were analyzed for area, aspect ratio and centroid. In the resulting data, cluster traces throughout the timelapses were connected using the centroids and a minimum Euclidean distance approach with an upper limit of 0.3\u2009\u03bcm movement per frame. Centroid traces of each cluster were extracted, and for the largest cluster in the first frame in each timelapse the trace was further analyzed to extract the mean square displacement (MSD). The MSD was calculated for a specific \u0394t as the mean Euclidean distance between each centroid position at t and t\u2009+\u2009\u0394t.Analysis of synaptic vesicle clusters in each etSTED timelapse and manual STED timelapse of active synapses was performed using the scripts provided in the Code Availability section. To extract the clusters in each frame fairly, a histogram-matching bleach correction step was performed on the timelapses. Each frame was smoothed with 1\u2009pixel Gaussian smoothing, binarized with a timelapse-constant global intensity threshold, and 1\u2009pixel eroded once. Binary objects larger than 0.015\u2009\u03bcm2 inhalation and aorta cut, and brains were extracted from the embryos. Hippocampi were dissected and mechanically dissociated in MEM . A total of 2\u2009\u00d7\u2009105\u2009cells per 60\u2009mm culture dish were seeded on poly-d-ornithine  coated no. 1.5 18\u2009mm glass coverslips , and were left to attach in MEM with 10% horse serum , 2\u2009mM l-Glut  and 1\u2009mM sodium pyruvate  at 37\u2009\u00b0C, 95\u201398% humidity and 5% CO2. After 2\u20134\u2009h the coverslips were flipped over an astroglial feeder layer  and maintained in Neurobasal  supplemented with 2% B-27 , 2\u2009mM l-glutamine and 1% penicillin\u2013streptomycin. The cultures were treated with 5\u2009\u03bcM 5-fluorodeoxyuridine at 2\u20133\u2009days in vitro (DIV) to prevent glia overgrowth. The cultures were kept for up to 24\u2009days and fed twice per week by replacing one-third of the medium per well: before DIV7 with Neurobasal complete medium, and from DIV7 with Braiphys , 1% Pen/Strep  and SM1 Supplement . Experiments were performed on mature cultures at DIV14\u201321. All experiments were performed in accordance with animal welfare guidelines set forth by Karolinska Institute and were approved by Stockholm North Ethical Evaluation Board for Animal Research. Rats were housed with food and water available ad libitum in a 12\u2009h light\u2013dark environment.Primary neuronal cultures were prepared from embryonic day 18 Sprague\u2013Dawley rat embryos. Pregnant mothers were killed with CO2 in a humidified incubator. Cells were plated on no. 1.5 18\u2009mm glass coverslips  24\u201348\u2009h before imaging.HeLa (ATCC CCL-2) cells were cultured in DMEM  supplemented with 10% (vol/vol) fetal bovine serum , 1% penicillin\u2013streptomycin  and maintained at 37\u2009\u00b0C and 5% CO5\u2009cells per well were seeded on coverslips in a 12-well plate. After 1\u2009day the cells were transfected using FuGENE  according to the manufacturer\u2019s instructions. At 24\u2009h after transfection the cells were washed in PBS solution, placed with phenol red-free Leibovitz\u2019s L-15 Medium  in a chamber and imaged.For transfection, 2\u2009\u00d7\u20091049. In brief, DNA (2\u2009\u03bcg) was diluted in TE solution . CaCl2 (2.5\u2009M in 10\u2009mM HEPES) was added to a final concentration of 250\u2009mM. The mixed solution was added to 2\u00d7 HEBS . Neurons were pre-incubated in 200\u2009\u03bcl conditioned medium from their culture dish with 50\u2009\u03bcl 5\u00d7 kynurenic acid stock (10\u2009mM dissolved in unsupplemented culture medium) in a well of sterile MW12 and placed back in the incubator until the precipitate was ready. The precipitate was then added dropwise to the cells and incubated for 3\u20134\u2009h. To stop the transfection, a 5:1 mix of Neurobasal medium without glutamate and kynurenic acid was pre-warmed. Then, 5\u2009M HCl was added until the solution turned yellow. After removal of the transfection medium, the acidic medium was added to each coverslip, which was further incubated at 37\u2009\u00b0C and 5% CO2 for 15\u201320\u2009min. After the incubation period the neurons were transferred back to the original Petri dish containing the conditioned medium and the construct was left to express for 18\u201324\u2009h at 37\u2009\u00b0C and 5% CO2.Primary neurons (DIV8\u201314) were transfected using calcium phosphate co-precipitation protocol as reported\u22121, Synaptic Systems, 105 3FB) and 1\u2009\u03bcl FluoTag-X2 anti-mouse Ig kappa light chain nanobody conjugated to Abberior STAR635P  were pre-incubated with 98\u2009\u03bcl pre-conditioned neuronal medium and incubated at 23\u2009\u00b0C for 20\u2009min. Neurons were then incubated with the Synaptotagmin-1 labeling solution for 30\u2009min in a humidified chamber at 37\u2009\u00b0C. After the incubation time the neurons were left to recover for 5\u2009min in their original medium and washed twice with artificial cerebrospinal fluid (ACSF) before imaging. Imaging was performed in ACSF at room temperature.For the labeling of active synapses, 1\u2009\u03bcl Synaptotagmin-1 antibody luminal domain  or SiR-tubulin  in 50\u2009\u03bcl anhydrous dimethylsulfoxide (DMSO). For labeling, the stock solution was diluted 1:1,000 for a final 1\u2009\u03bcM staining solution, in ACSF for neurons and in cell medium for HeLa cells. Neuronal cultures were incubated for 30\u2009min at 37\u2009\u00b0C with the dilution, and washed twice in ACSF prior to imaging. HeLa cells were incubated for 30\u201345\u2009min at 37\u2009\u00b0C with the dilution, and washed once in cell medium prior to imaging.The labeling of F-actin and tubulin was performed as previously describedFor calcium imaging, a fluorescent calcium chelator labeling was used. A total of 10\u2009\u03bcl pluronic acid F-127 solution  was added to 50\u2009\u03bcg Oregon Green 488 BAPTA-1, AM ester  for a 1\u2009mM stock solution. The neuronal culture or HeLa culture was incubated for 30\u2009min at 37\u2009\u00b0C with 1\u2009\u03bcM Oregon Green 488 BAPTA-1, AM ester , and washed once in ACSF or cell medium prior to imaging.2 with Abberior STAR RED Cholesterol-PEG(1000) . To label sphingolipids, primary neuronal cultures were incubated for 1\u2009h at 37\u2009\u00b0C and 5% CO2 with Abberior STAR RED C12 Sphingosyl PE (d17:1/12:20) . HeLa cells were then washed in Leibovitz\u2019s L-15 PBS and the neurons in ACSF prior to imaging.To label cholesterol, HeLa cells and primary neuronal cultures were incubated for 1\u2009h at 37\u2009\u00b0C and 5% CO51. pCMV-Sport6-CD63-pHluorin was a gift from D. M. Pegtel (RRID: Addgene_130901)38. CD63_OHu03119C_pcDNA3.1(+)-C-eGFP was obtained from GenScript Biotech.pEGFP-N1 Dynamin1 wild type was a gift from J. Taraska (research resource identifier (RRID): Addgene_120313)Widefield images were recorded using a 20\u2013100\u2009ms exposure time and a frame rate of 3.3\u201320\u2009Hz. The 488\u2009nm laser power used was 0.3\u20130.9\u2009mW for neurons and 0.6\u20131.9\u2009mW for HeLa cells. STED images were recorded using a pixel size of 25\u201330\u2009nm, a dwell time of 30\u201350\u2009\u03bcs, a 640\u2009nm laser power of 5\u201316\u2009\u03bcW and a 775\u2009nm laser power of 59\u2013124\u2009mW. Exact image acquisition parameters for each experiment are listed in Supplementary Table For visualization purposes, raw STED images have been smoothed with 0.5\u2009pixel Gaussian smoothing. Frames from STED timelapses have been bleach corrected using histogram matching or direct-ratio methods. 3D STED images of endocytosis events have been deconvolved, as marked by the asterisks in the images, by applying a 50\u201360\u2009nm \u2009\u00d7\u2009100\u2009nm  Lorentzian PSF and 10 iterations. Raw STED images of exocytosis have had a rolling ball background subtraction with a radius of 50 pixels applied. Frames from 23\u2009Hz STED timelapses of synaptic vesicle dynamics have been deconvolved by applying a 70\u2009nm Gaussian PSF and 5 iterations.\u221210, in Imspector (Max-Planck Innovation). For data visualization and post-acquisition analysis, custom-written scripts and JupyterLab notebooks in Fiji (ImageJ) and Python have been used (see Code Availability), equipped with additional packages such as scikit-image52, jupyterlab53 and matplotlib54.Deconvolution was performed using Richardson\u2013Lucy deconvolution, with a regularization parameter of 1\u2009\u00d7\u200910The ratios of true-positive event detections (True) and detected real events (Det), compared with the number of events, have been quantified as measures of the accuracy and precision of the rapid_signal_spikes analysis pipeline. The quantification was performed through full widefield timelapse recordings, without running the etSTED method, with the same acquisition parameters as in a full etSTED experiment. The timelapses were manually annotated for calcium events and also analyzed with the analysis pipeline. The results of the two were compared to calculate the two ratios. This quantification was performed in multiple experiments and multiple cells given that it depends on the fluorescence background, the cellular structure, the labeling density, the acquisition parameters and the user-inputted pipeline parameters. Plotted is one data point for each cell in the various experiments analyzed.P\u2009<\u20090.05 and NS indicates P\u2009>\u20090.05. Shaded confidence interval areas are chosen as the 83% level, meaning that non-overlapping areas infer a significant difference at that part of the curve.All statistical tests are two-sample two-sided Kolmogorov\u2013Smirnov tests, where the asterisk symbol (*) indicates Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41592-022-01588-y.Supplementary InformationSupplementary Notes 1\u201310, Supplementary Figs. 1\u20136 and Supplementary Tables 1\u20133Reporting SummaryPeer Review File"}
+{"text": "This study indicated that high expression of TRIP13 promoted proliferation and migration of ESCC cells and induced NDP resistance via enhancing repair of DNA damage and inhibiting apoptosis. This will provide a preliminary reference for the clinical use of NDP in ESCC treatment.Thyroid hormone receptor interactor 13 (TRIP13) plays a crucial role in poor prognosis and chemotherapy resistance of cancer patients. This present study is aimed at investigating the role of high expression of TRIP13 inducing nedaplatin (NDP) resistance in esophageal squamous cell carcinoma (ESCC) cells. High expression of TRIP13 promoted the proliferation and migration of ESCC cells performed by MTS assay, colony formation assay, wound healing assay, and transwell assay. High TRIP13 expression induced NDP resistance to ESCC based on the cell proliferation promoting/inhibition rate and cell migration promoting/inhibition rate analysis, flow cytometry assay of apoptotic subpopulations with a combination of Annexin V-FITC and propidium iodide, and Western blot analysis downregulating cleaved PARP,  Esophageal squamous cell carcinoma (ESCC) is one of the common malignant tumors with high morbidity and fatality . It remaThere are many kinds of chemotherapeutic drugs, among which platinum drugs are commonly used . CisplatRecent studies have indicated that thyroid hormone receptor interactor 13 (TRIP13) is abnormally expressed in many tumor tissues and related to poor prognosis \u201323. InteIn this study, resistance of ESCC to NDP and its correlation were detected by constructing ESCC cells with abnormal expression of TRIP13 and NDP intervention. The results showed that high expression of TRIP13 could promote the proliferation and migration ability of ESCC cells and contributed to the NDP resistance via enhancing repair of DNA damage and inhibiting apoptosis. This study predicted that TRIP13 could be used as a new therapeutic target for ESCC, laying a foundation and providing reference for the clinical use of NDP in the treatment of ESCC.2, at 37\u00b0C and 80% humidity.Human esophageal squamous cell carcinoma cell line TE1 was cultured in DMEM medium . KYSE150 was cultured in RPMI-1640 medium . TE3, KYSE30, and KYSE510 were cultured in Gibco RPMI-1640 medium . For detailed information about the cells, please refer to the literature previously published in our laboratory \u201326. The TRIP13 gene was synthesized by GenePharma  , and TRIP13 Gene primer  with ampicillin (100\u2009mg/ml)  and kanamycin (10\u2009mg/ml) , according to Biomiga EZgene\u2122 Plasmid Miniprep Kit  instruction and Biomiga EZgene\u2122 PCR/Gel Extraction Kit  instruction. We use Lipofectamine 3000  in accordance with the instruction of manufacturers to configure transfection solution and add fresh medium and mix evenly. The medium was changed after 6\u2009h, and the cells were continued to incubate for 24\u2009h.The siRNA sequence of , China) . The siR\u03b2-actin primer . Total RNA was reversely transcribed into cDNA according to HiScript\u00ae III RT SuperMix for qPCR (+gDNA wiper)  instruction, and quantitative PCR was detected according to ChamQ\u2122 Universal SYBR qPCR Master Mix  instruction. The amplification curve and melting curve of qPCR were obtained based on T method , 26.\u03b3H2A.X, caspase-3, cleaved caspase-3, Bcl-2, and Bax [\u03b3H2A.X, Bcl-2, and Bax are all from the same manufacturer . PARP (Product# 9542), caspase-3 (Product# 14220T), and cleaved caspase-3 (Product# 9664T) . Specific secondary antibodies are as follows: HRP-conjugated Affinipure Goat Antibody Mouse IgG (H+L)  and Anti-rabbit IgG, HRP-linked Antibody .Western blot was used to explore the protein expression level of TRIP13, PARP, cleaved PARP,  and Bax , 26. RIP50 and inhibition ratio were calculated using GraphPad Prism 8.0 software .ESCC cells were cultured, and cell suspensions were prepared with medium containing 10% serum. Cells were counted at an initial density of 10000 cells per well and reinoculated in 96-well plates. The cells were incubated for 6\u2009h until adherence. The medium was replaced with a freshly prepared medium containing NDP and added at the different concentration gradients shown. The cells were cultivated for 48\u2009h and treated with the MTS assay. A BioTek ELx800 microplate reader  was used to detect the absorbance of each well at 490\u2009nm. The ICFor examination of cell proliferation ability, colony formation assay was conducted , 26. TraFor examination of cell proliferation ability, MTS assay was conducted , 26. CelFor examination of cell migration ability, wound healing assay was conducted. For wound healing assay, inoculate the above-mentioned cells into a 12-well plate, 1\u2009mL per well, and after adherence for 6\u2009h, the cells were incubated in the medium supplemented with 2% FBS. Micrographs of the assigned areas were taken at 0\u2009h and after a certain amount of time by the IX73 inverted microscope . The areas of wound healing assay were analyzed by using ImageJ 1.52a .\u03bcL cell suspension and add it to the transwell chamber. Inoculate onto the Falcon Chambers  with a density of 50000 (overexpression experiment group) or 60000 (knockdown experiment group) cells per well. After 48\u2009h, the cells that migrated toward the lower chambers were stained with 0.5% crystal violet. Each assay was photographed for 5 views under the IX73 inverted microscope , and the number of cells within each chamber was counted by ImageJ 1.52a .For examination of cell migration ability, transwell assay was conducted. After cell counting of transfected cells, take 400\u2009Apoptosis was investigated by the Annexin V-FITC assay. Cells after siRNA or plasmids transfection were incubated with NDP for 48\u2009h and washed by PBS twice. After incubation, cells were extracted and resuspended with Annexin V binding solution according to the C1062 Annexin V-FITC Apoptosis Detection Kit  instruction. Accuri C6 Plus  was used to detect the apoptosis cells. The flow cytometry data for cell apoptosis was analyzed by FlowJo v. 7.6 software .t-tests were used to determine the statistical differences between independent samples. P value < 0.05 was defined as statistically significant.Statistics obtained from each assay were imported into GraphPad Prism 8  and SPSS 22.0  for graphing and analysis. All experimental results are presented as the mean \u00b1 SD. Student's https://www.oncomine.org/) and GEO , TRIP13 is highly expressed in cancerous tissue (To assess the expression level of TRIP13 in ESCC, we analyzed several data sets. In the four data sets from Oncomine (s tissue .The effect of high TRIP13 expression in ESCC cells is definitely significant, which indicates a poor prognosis of ESCC patients . We therTo evaluate the effect of TRIP13 in proliferation ability of ESCC, we transfected the TRIP13 siRNA and TRIP13 expression plasmid into ESCC cells. And the transfection efficiency was assessed by Western blot Figures a. In addApart from the proliferation ability of ESCC cells with abnormal TRIP13 protein expression, figuring out the migration ability of it is equally essential. Transwell assay and wound healing assay were used to investigate the role of TRIP13 in migration ability of ESCC. The transfection efficiency of TRIP13 expression was confirmed in Western blot Figures a. The reAlthough NDP has a good antitumor effect on ESCC \u201329, it i\u03b3H2A.X expression level of TRIP13 overexpression group was lower in KYSE150 cell and KYSE510 cell (Figures PARP is a marker of DNA damage repair process . As is s Figures , which d Figures . After N Figures . Some me Figures . After N Figures . We also Figures . On the  Figures . Also, t Figures . To sum ESCC is one of the common malignant tumors with high level of incidence and mortality, whose incidence ranked seventh and the mortality ranked sixth in the world in 2018 , 32, 33.\u03b3H2A.X. H2A.X is a marker of apoptosis. When apoptosis occurs, H2A.X is phosphorylated into \u03b3H2A.X. When the cell repair fails, \u03b3H2A.X is unable to dephosphorylate and reverts to H2A.X, resulting in the upregulation of \u03b3H2A.X expression [In chemotherapy or chemoradiotherapy, platinum-based drugs are commonly used, among which NDP has lower toxicity except bone marrow toxicity compared with cisplatin . The mecpression , 44.Nedaplatin, as the second generation platinum anticancer drug with a favorable clinical effect , 46, is TRIP13 gene, a member of the AAA+ ATPase super-family, is located in 5p15.33, encoding TRIP13 protein, which is mainly involved in cell mitosis and repair of DNA damage \u201350. ReseIn the previous research, TRIP13 could promote drug resistance to head and neck squamous cell carcinoma by enhancing the repair effect of DNA damage . Moreove\u03b3H2A.X decreased and the expression of RAD50 increased in bladder cancer cells after cisplatin treatment, which revealed that TRIP13 promoted DSB repair and reduced apoptosis of cancer cells treated with drugs [\u03b3H2A.X, cleaved caspase-3, and Bax expression decreased when the expression of TRIP13 was high. In contrast, the expression of Bcl-2 decreased and PARP, \u03b3H2A.X, cleaved caspase-3, and Bax expression increased when knocking down TRIP13. These results suggested that high TRIP13 expression-induced NDP resistance made ESCC cells easier to escape the toxicity of NDP. Therefore, ESCC cells with high TRIP13 expression exists NDP resistance may be through increasing repair of DNA damage and decreasing apoptosis. Based on the previous studies and our results, DSB repair of ESCC with high TRIP13 expression may be caused by the promotion of NHEJ. TRIP13 generally promotes the progression of tumor cells by affecting cell cycle [Several studies have suggested that the molecular mechanism of drug resistance to cancer cells is different \u201315, 21. th drugs . Since pth drugs , in our ll cycle , 54. DNAIn conclusion, high expression of TRIP13 can promote the proliferation and migration ability of ESCC cells, which contributes to the resistance effect to the NDP. And the molecular mechanisms of the NDP resistance may be through increasing repair of DNA damage and decreasing apoptosis. However, more detailed mechanisms are needed to be investigated, which may provide more evidence for the therapeutic usage of NDP in the clinical situation."}
+{"text": "The term 4D printing refers to the idea that the shape or properties of a printed object can be changed when an external stimulus is applied. In this contribution, a temperature-responsive polymer Poly (N-vinyl caprolactam) (PNVCL), which is normally prepared via radical free polymerization, was used to justify the 4D printing concept. As a result, by using a Stereolithography (SLA) 3D printer, 4D prints were successfully prepared. These prints were able to demonstrate intelligent and reversible expansion/shrinkage behaviour as the temperature increases and decreases. Additionally, in order to examine the differences in chemical structure, thermal properties, mechanical properties, and swelling behaviours of the photopolymerised and printed parts, a series of characterisation tests, including Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), goniometry, tensile test, gel fraction measurement and pulsatile swelling study were performed on this study. In conclusion, the differences between polymerisation methods are significant; despite their chemical structures and thermal properties being similar, there were significant differences with regard to tensile properties, swellability and wettability of samples. The implications of conducting this study are remarkable, not only in providing a new way of preparing NVCL, but also in demonstrating the possibility of using 4D printed NVCL for practical applications. The three-dimensional printing 3D) technique, which is also known as additive fabrication and rapid prototyping, is a fast way to create objects based on pre-designed shapes and sizes. These processes may lead to the toolless production of finished goods and the mass production of individually customised parts . Three-dD techniqStereolithography (SLA) is a solid free formation technology and was first exposed to the public in the early 1970s by a Japanese researcher, Hideo Kodama . SLA is 11EO42, due to the CEO, offers more opportunities for hydrogen bond generation to create a more stable structure, which results in a steady and sustained drug release [Poly (N-vinyl caprolactam) (PNVCL) is a temperature-responsive polymer. It is well-known for its exceptional biocompatibility, solubility, thermosensitivity, and non-ionic and non-toxic properties . Further release . Free ra release . Its fre release .By changing structures that can be converted in a pre-programmed manner in response to a stimulus, 3D printed materials can be modified to impart flexibility and boost utility. Four-dimensional printing was first initiated and labelled by a research group at MIT . It is f\u00ae 2959 Ciba Corp, New York, NY, USA), were obtained from Ciba Specialty Chemicals. H-Nu 400 IL is a broad wavelength liquid blend photoinitiator with easy addition to a free radical curable formulation. It was obtained from Spectra Photopolymers . N,N-dimethyl acrylamide (DMAAm) was purchased from Sigma Aldrich  with a molecular weight of 99.13 g/mol. The chemical crosslinker used was poly (ethylene glycol) dimethacrylate (PEGDMA) supplied by Sigma Aldrich  with a molecular weight of 550 g/mol.N-vinyl caprolactam (NVCL) was obtained from Sigma Aldrich Ireland with a molecular weight of 139.19 g/mol and a storage temperature from 2 to 8 \u00b0C. The UV light-sensitive initiators, 4-(2hydroxyethoxy) phenyl-(2-hydroxy-2-propyl) ketone . The prepolymerised mixtures were prepared by combining the desired amounts of the monomer NVCL with specified amounts of other materials and photoinitiator and 2 wt% PEGDMA for chemical crosslinking gels. The batches were placed in a 100 mL beaker and mixed using a magnetic stirrer for 20 min until a homogeneous mixture was obtained. The solutions were pipetted into silicone moulds that contained disc, jigsaw and flower impressions. Photopolymerisation was carried out for 10 min on each side, and all samples were dried prior to use for 24 h in a vacuum oven at 50 \u00b0C. The hydrogels investigated in this study were prepared by free radical polymerisation using UV light. These hydrogels were synthesised using a UV curing system . This particular irradiation chamber is a controlled radiation source with 20 UV-tubes that provides a spectral range between 315 and 400 nm at an average intensity of 10\u201313.5 mW/cm3, with a range from 25 to 300 microns per layer. The thickness of print for each layer is set at 50 \u03bcm. A range of materials can be used for printing, including standard materials, tough and durable materials, flexible and elastic materials, rigid and structural materials, dental materials, biomedical materials, castable and specialty materials. The specific resin cartridge and tank and build platform should be correctly installed in the printer before the printing begins. During the printing process, the resin can be automatically filled into the self-heating resin tank. Depending on the material, the resin would be heated and kept at a constant temperature until the print is complete. Independently developed resins can also be used on this printer, but open mode needs to be activated. In this mode, the cartridge or tank detection function, resin heating, resin wiper, and resin dispensing are disabled. Additionally, the resin needs to be filled manually in the resin tank.Form 2  is a stereolithography 3D printer developed by Formlab. Items are constructed on the platform from bottom to top in an upside-down posture. Only files in STL format can be accepted and uploaded to the printer. Based on a model designed in advance in computer-aided design software (CAD), the photopolymer resin is solidified by providing UV laser (405 nm) to form a single layer onto the surface of the photopolymer vat; then, the elevator platform descends and repeats the process to form a new layer until the actual object is built. The laser spot size of the 3D printer is 140 microns and the maximum printing size is 14.5 \u00d7 14.5 \u00d7 17.5 cmIn this study, 4D printing was achieved using the SLA 3D printer  to print homemade resin. The prototype is created layer-by-layer following the model designed in advance using computer-aided design software (CAD). Preform software is the specific app to upload the model to the printer. The orientation and numbers of the model can be adjusted in Preform software automatically or manually. The printer should be switched to open mode for third party resin use. After the model was uploaded to the printer, the homemade solution was prepared in the same way as the UV-cured hydrogels. Based on the formulations, the monomer was first placed into a 250 mL beaker and other solutions were added to the beaker using a pipette to prepare a mixture. The preparation of the H-Nu 400IL samples had to be performed in a dark environment. Therefore, aluminum foil was used to surround the beaker to avoid prepolymerisation. To achieve homogeneity, a magnetic stirrer was used to stir the mixture for 20 min at 50 \u00b0C heat. All in all, 150 g of solutions were prepared and poured directly into the resin tank in preparation for printing. After printing, the residue solution remaining on the printed objects was removed with a tissue paper containing IPA. The finished parts were placed into the UV box to post cure for 20 min. The formulations used for 4D printing are listed in \u22121, utilising a 4-scan per sample cycle and a fixed universal compression force of 75 N. Subsequent analysis was performed using Spectrum software. The tests were performed in duplicate for each sample. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was carried out on a Perkin Elmer Spectrum One FT-IR Spectrometer (C-001), fitted with a universal ATR sampling accessory. All data were recorded at room temperature (<20 \u00b0C) in the spectral range of 4000\u2013650 cmThe LCST of the chemically crosslinked hydrogels was detected by Differential Scanning Calorimetry . The DSC was calibrated with indium standards. The hydrogel samples were allowed to swell until equilibrium in distilled water at room temperature. The sample\u2019s surface was wiped with moistened filter paper to remove free water and placed into the aluminum pans and weighed out, ranging from 8 to 12 mg, using a Sartorius scale with 0.01 mg resolution. DSC measurement was performed on swollen samples from 10 \u00b0C to 60 \u00b0C at a rate of 1 \u00b0C/min, referenced against an empty pan. All samples were examined under a pure nitrogen atmosphere at 30 mL/min. The results were plotted as a function of heat flow (W/g) against temperature (\u00b0C). The tests were performed in triplicate.Contact angle goniometry is used to assess a solid substrate\u2019s capacity to resist liquids . The xerTensile testing is a destructive test process that provides information about the tensile strength, yield strength and ductility of the materials. It measures the force required to break a composite or plastic specimen and the extent to which the specimen stretches or elongates up to that breaking point . The ten0 is the weight of the xerogel after photopolymerisation, and Wd represents the dried weight of the sample after extraction of soluble parts.Gel fraction measurement may be used as a quantitative indicator of the efficiency of hydrogel network formation . The gel0) of the photopolymerised and printed samples was measured. The samples were placed into rectangular plastic containers of 120 \u00d7 80 cm2 containing 350 mL of distilled water (pH = 7.1). Samples were tested under switching conditions between ambient temperature and 50 \u00b0C. The plastic containers were first stored at room temperature and weighed after specific intervals (Wt) to determine water uptake until the swelling equilibrium was reached. Then, these samples were placed into the oven to raise the temperature to 50 \u00b0C (above LCST) and the weight of the samples was measured again. After the new equilibrium state was reached, these plastic containers were restored to room temperature. Before each measurement, the excess distilled water was dried with filter paper. The measurement was conducted in three copies for each sample. The expanding percentage for each time was calculated using Equation (2).t and W0 are the weights of the gels in the swelling state at a predetermined time and the initial dry mass of the dried state, respectively.Initially, the apparent dry weight , and 1608/1648 cm\u22121 (DMAAm). These bands are assigned to the carbonyl group C=O bond stretching vibration and the C=C bond stretching vibration. In the P(NVCL/DMAAm) spectra, only an intense peak at 1618/1615/1614 cm\u22121 in U1, U2 and P1 samples is attributed to the characteristic absorption of C=O stretching. Based on the previous studies [\u22121 in the printed and cured sample spectra, which indicates successful polymerisation and printing of the samples (C=C bonds disappearance [\u22121 (U1), 1479 cm\u22121 (U2) and 1481 cm\u22121 (P1) are attributed to the stretching of C-N bonds in both monomers [\u22121 and 3425 cm\u22121 in photopolymerised and printed samples provides further evidence that the polymerisation and 3D printing have succeeded [ATR-FTIR was used to detect the differences in the chemical structure between the photopolymerisation and 3D printing samples. When infrared radiation passes through the sample, some of the radiation is absorbed by the sample and some radiation is passed . The signal generated at the detector is a sample that represents the spectral \u201cfingerprint\" of the molecule. Different structures or chemical bonds can be distinguished following the spectral curve.  studies , only onpearance ). The bamonomers . The O-Hucceeded . As can ucceeded . For exaDifferential scanning calorimetry is a thermal analytical technique used to measure a material\u2019s heat capacity, its endothermic and exothermic transitions, and its relationship to change in temperature . The LCSThe contact angle provided the level of the hydrophobicity held by each hydrogel. The hydrophobicity of the UV-cured and printed hydrogels was determined using goniometry. The wettability of the sample surface is measured by measuring the contact angle between the water droplet and the dry xerogel surface. The more the liquid spreads across the solid surface of the material, the more attracted to the material it is. The closer the contact angle is to 0\u00b0, the greater the hydrophilicity of the sample material. Materials with a contact angle up to 90\u00b0 are considered hydrophilic, those with contact angle greater than 90\u00b0 are considered hydrophobic, and surfaces with contact angle greater than 150\u00b0 are considered to be superhydrophobic, as the liquid droplet meets the surface without significant wetting .As demonstrated in The tensile test is used to find out how strong a material is and also how much it can be stretched before breaking. The mechanical strength can be assessed by the appearance before and after water absorption. However, the behaviour of NVCL in terms of changes in mechanical properties after blending with other polymers can be more precisely analysed through tensile testing. The tensile tests were performed on the U1, U2 and P2 samples to investigate the differences between maximum load, tensile strength and elongation at maximum load. As shown in Gel fraction can be used as a qualitative indicator of the efficiency of network formation . The gre2. Based on the U1 formulation, the first part of this study aimed to investigate the swelling behaviour differences between the discs and jigsaw- and flower-shaped samples. As can be seen in The pulsatile swelling tests were carried out on both photocured and printed samples to further examine the smart 4D behaviours of the shaped samples. Taking into account the potential expanding dimensions of the samples, swelling analysis of samples was performed in a rectangular plastic container of 120 \u00d7 80 cmThe aim of the second part pulsatile swelling study was to compare the differences in dimensional expansion/shrinkage capabilities between the photopolymerised and printed samples. Comparative tests were carried out on the jigsaw and flower samples. As demonstrated in The appearances of 3D printed jigsaw and flower samples are shown in In summary, the jigsaw flower samples manufactured via UV chamber system displayed the same reversible expansion/contraction properties as the disc samples. The quality of the swollen parts can be assured. A smooth surface and excellent appearance can be maintained. However, samples with large dimensions require a longer time to reach their maximum capacity. Additionally, 4D printing has been successfully achieved in this study using P2 formulation. These prints were able to demonstrate intelligent and reversible expansion/shrinkage behaviours. In comparison, for the photopolymerised samples, the response time for both the UV-cured and printed parts was 24 h. However, the UV-cured samples exhibited a stronger swelling capacity than those prints.On the whole, by using a new photoinitiator, 4D printing was successfully achieved using an NVCL-based polymer based on the formulation S7 developed in the previous article . The 4D In this study, the potential functions of the material NVCL have been discovered. The printed and UV-cured samples can be used as a size changeable demonstrator. Additionally, by using this method, some of the more complex original 3D models could be created and become a new type of dimensional changeable toy in the future. However, the volume-changing behaviour exhibited by the hydrogel material still seems extremely homogenous compared to most other 4D printing reports.Therefore, it would be an advancement in future research if some other types of materials could be combined with hydrogel-based polymers through a multi-material 3D printer to achieve a wider variety of shape transformation behaviours. By incorporating with flexible or rigid materials, the deformation generation on the hydrogel can spur the soft material change, leading to a change in dimensions and curvature. According to this principle, a series of products such as grippers ,37, actuIn contrast to other homemade 4D printable resins prepared in the literature, this contribution exploits a unique material and rapid preparation method suitable for 4D printing. In this study, 4D printing was successfully achieved by using a temperature-responsive polymer made from NVCL. Using the Form 2 SLA 3D printer, the 4D printed jigsaws and flowers were perfectly printed and were able to show intelligent and reversible swelling/deswelling behaviour in water media after the temperature rose and fell. By carrying out FTIR, DSC, goniometry, tensile test, gel fraction measurement and pulsatile swelling studies, the effects on material properties of NVCL samples prepared via stereolithography and UV chamber polymerisation were also investigated. Overall, the differences between polymerisation methods and photoinitiator types are significant; although their chemical structures and thermal properties are similar, there were significant differences with regard to tensile properties, swellability and wettability of samples. Based on the findings of this study, the practical 4D printing concept was successfully achieved using the NVCL-based polymer. This offers not only the possibility of using NVCL in a new field distinct from biomedical applications, but also offers a fresh material for 4D printing research."}
+{"text": "To evaluate and compare the measurement accuracy of two different computer-aided diagnosis (CAD) systems regarding artificial pulmonary nodules and assess the clinical impact of volumetric inaccuracies in a phantom study.In this phantom study, 59 different phantom arrangements with 326 artificial nodules  were scanned at 80\u00a0kV, 100\u00a0kV, and 120\u00a0kV. Four different nodule diameters were used: 5\u00a0mm, 8\u00a0mm, 10\u00a0mm, and 12\u00a0mm. Scans were analyzed by a deep-learning (DL)\u2013based CAD and a standard CAD system. Relative volumetric errors (RVE) of each system vs. ground truth and the relative volume difference (RVD) DL\u2013based vs. standard CAD were calculated. The Bland\u2013Altman method was used to define the limits of agreement (LOA). The hypothetical impact on LungRADS classification was assessed for both systems.There was no difference between the three voltage groups regarding nodule volumetry. Regarding the solid nodules, the RVE of the 5-mm-, 8-mm-, 10-mm-, and 12-mm-size groups for the DL CAD/standard CAD were 12.2/2.8%, 1.3/\u2009\u2212\u20092.8%,\u2009\u2212\u20093.6/1.5%, and\u2009\u2212\u200912.2/\u2009\u2212\u20090.3%, respectively. The corresponding values for the ground-glass nodules (GGN) were 25.6%/81.0%, 9.0%/28.0%, 7.6/20.6%, and 6.8/21.2%. The mean RVD for solid nodules/GGN was 1.3/\u2009\u2212\u200915.2%. Regarding the LungRADS classification, 88.5% and 79.8% of all solid nodules were correctly assigned by the DL CAD and the standard CAD, respectively. 14.9% of the nodules were assigned differently between the systems.Patient management may be affected by the volumetric inaccuracy of the CAD systems and hence demands supervision and/or manual correction by a radiologist.The DL-based CAD system was more accurate in the volumetry of GGN and less accurate regarding solid nodules than the standard CAD system.\u2022 Nodule size and attenuation have an effect on the measurement accuracy of both systems; tube voltage has no effect on measurement accuracy.\u2022 Measurement inaccuracies of CAD systems can have an impact on patient management, which demands supervision by radiologists.\u2022  The widespread routine clinical implementation of lung cancer screening programs all over the world is directly linked to the increasing importance of standardized pulmonary nodule management. Current clinical practice guidelines rely on precise and reproducible measurements of the detected pulmonary nodules. Since different definitions of the nodule diameter can be applied , volumetry of pulmonary nodules is deemed the most accurate predictor of lung cancer risk , 2 and mIn front of an increasing number of screening examinations and the associated additional workload, careful CT reading and precise measurements are challenging for radiologists in daily clinical routine. A nodule measurement variability of\u2009\u00b1\u200925% has been demonstrated in several in vivo \u201ccoffee-break\u201d studies, in which individuals were scanned twice on the same day , 9.\u00a0A poThe influence of scanning parameters and reconstruction on nodule volumetry is well appreciated , 15. In The primary aims of this study were to evaluate the measurement accuracy of a commercially available DL-based CAD system and a standard CAD system with an in-built semi-automatic volumetry tool in comparison to the true volume of artificial pulmonary nodules and to evaluate the impact on patient management recommendations. The secondary aim was to analyze the influence of tube voltage, nodule size, and attenuation on the measurement accuracy of the CAD systems.A previously described dedicated anthropomorphic chest phantom equipped with artificial nodules was utilized in this study  , 19.3), 8\u00a0mm (268.1 mm3), 10\u00a0mm (523.1 mm3), and 12\u00a0mm (904.8 mm3). Two density types of nodules were used, nodules with a density of\u2009+\u2009100 Hounsfield units (HU) to simulate solid lesions and nodules with a density of\u2009\u2212\u2009630 HU to simulate ground-glass nodules (GGN).Nodules of four different diameters (corresponding volumes) were used: 5\u00a0mm  were analyzed. Each phantom was equipped with zero to eight nodules. A random generator decided the nodule distribution  within the simulated lung parenchyma. Exemplary nodules are depicted in Fig.\u00a0E) were 0.9\u00a0mSv, 1.6\u00a0mSv, and 2.3\u00a0mSv. The chest phantom CT data sets were used in an earlier study already [All examinations were performed on a 64-row multidetector CT scanner  with the following scan parameters: spiral acquisition mode, 24\u2009\u00d7\u20091.2\u00a0mm, pitch, 0.8; slice thickness, 1.5\u00a0mm, increment, 1.5\u00a0mm, field of view, 35\u00a0cm, and scan length, 33\u00a0cm. For image reconstruction, filtered back projection (FBP) was utilized with kernels of B30f (soft kernel) and B70f (hard kernel) and lung window setting . Each phantom arrangement was scanned with tube voltages of 80, 100, and 120\u00a0kV. Automated modulation of the tube current (CareDose4D) was used to maintain independence from body weight and body mass index (BMI) and to approximate routine scans. The mean volume computed tomography dose indices (CTDIvol) of the three voltage groups were 1.2\u2009\u00b1\u20090.6\u00a0mGy, 2.1\u2009\u00b1\u20091.0\u00a0mGy, and 3.1\u2009\u00b1\u20091.4\u00a0mGy; the corresponding mean effective doses \u2013CAD system was used in this study . The deep-learning model was principally built with two convolutional neural network (CNN) models: a DenseNet model (feature map extractor) and a Faster R-CNN\u2013based model (detector).Since the CT scans have a property of 3D image volume, the Faster R-CNN network of this system was modified to take consecutive sections as input and to form a multichannel \u201c2.5D CNN.\u201d The dimension \u201c2.5D\u201d implies that the model is not exact 3D convoluted due to the segmented axial data and their heterogeneous resolutions.In this study, instead of regular CNN, the DenseNet model was used to extract the features and back propagate them. In contrast to regular CNN, where feature maps are usually connected at one go, feature maps in DenseNet are directly connected one by one, thus forming a densely connected network with a smaller number of layers. The feature density can thus be maintained during the propagation process, and the model will possess a higher overall expressive power.The standard CAD system used is an established, commercially available software  designed to work as a second-reader tool. It automatically pre-processes and marks nodules and measures them semi-automatically offering the possibility of subsequent manual modification. An integrated software tool allowed dedicated analysis of subsolid and solid lesions.The average volume uses all measured volumes and is a first descriptive measure for the software outputs. A more detailed measure is the relative volumetric error (RVE). The RVE is calculated for each nodule and accounts for the known volume as ground truth (GT). It was calculated by using the following formula:The RVE for the different voltage and size groups was compared with descriptive measures  and additionally with the Friedman test for paired samples or, alternatively, the Kruskal\u2013Wallis test for unpaired samples.The absolute and relative volume differences  between the systems were calculated by using the following formulas:p values were interpreted in a descriptive manner, with p\u2009<\u20090.05 indicating statistical significance.The Bland\u2013Altman method with limits of agreement (LOA) was utilized to assess the variability between the volumetric measurements of the systems. In line with the literature, the RVD of each measured nodule volume was plotted against the respective average volume , 21\u201323. n\u2009=\u2009319/326). The missed nodules  could not be added to the analysis manually as this function was not implemented in the utilized version of the software. Regarding the distribution of the missed nodules, two were located in the upper lobes, three in the middle lobe/lingual, and two in the lower lobes. Regarding their size groups, there was one 5-mm nodule and two nodules from each of the remaining size groups  missed.The DL-based CAD software automatically detected 97.9% of all nodules (n = 266/326) of the nodules automatically, but the missed nodules  could be manually added to the analysis.The standard CAD system detected 81.6% (n\u2009=\u2009307/326), 97.2% (n\u2009=\u2009317/326), and 97.9% (n\u2009=\u2009319/326) for 80\u00a0kV, 100\u00a0kV, and 120\u00a0kV, respectively. The mean RVEs of the different voltage groups for DL CAD/standard CAD regarding solid nodules were 1.9/0.2%, 0.2/\u2009\u2212\u20090.02%, and 1.3/1.0% for 80\u00a0kV, 100\u00a0kV, and 120\u00a0kV, respectively  nor with the standard CAD system (p\u2009=\u20090.135).The detection rates of the DL-based CAD system per voltage group were 94.2% . However, there was no difference observed between the voltage groups among the GGN with the standard CAD system (p\u2009=\u20090.192).A subgroup analysis indicated a difference in RVE between the 80- and the 120-kV group in the volumetry of the DL-based CAD system  were analyzed by both quantification systems. The DL CAD system showed higher measurement variability regarding the solid nodules, but less variability regarding the GGN, compared to the standard CAD system. The dependent measurements of both systems are depicted as spaghetti plots in Figs. As a coincidental finding, a clustering of the 10-mm and, even more pronounced, the 12-mm solid nodules was observed for the DL CAD measurements indicating a systematic error. Hereby, one nodule cluster was measured accurately; the second cluster was underestimated in size. Further workup of this observation revealed no correlation with nodule location or the maximum number of nodules per phantom see Fig.\u00a0b.The Bland\u2013Altman method was utilized to depict the RVDs between the systems. The resulting mean RVDs (SD) were 1.3 (18.6)% for solid nodules and\u2009\u2212\u200915.2 (23.5)% for GGN with respective lower/upper limits of agreement (LOA) of\u2009\u2212\u200935.2/37.7% and\u2009\u2212\u200961.4/30.9%  of all detected solid nodules correctly; the respective value for the standard CAD system was 79.8% (n\u2009=\u2009142/178). The difference between the two systems was statistically significant (p\u2009=\u20090.004). The majority of the falsely classified nodules belonged to the 8-mm-size group; only one falsely classified nodule of each system measured 10\u00a0mm.In order to evaluate the impact of the measurement differences on patient management, the hypothetical LungRADS categories of the solid nodules were compared between the systems. The DL-based CAD system classified 88.5% (n\u2009=\u200926/174) were classified differently between the two systems  and the remaining two nodules from the 10-mm-diameter group. The 5-mm and 12-mm nodules would have all been classified in unison.14.9% of the solid nodules (3).All GGN would have been categorized correctly as LungRADS 2 by both systems since no measurement exceeded the border to the LungRADS 3 classification . Between the systems, 14.9% of the solid nodules were classified differently (p\u2009=\u20090.002). These numbers have to be interpreted with caution since most of the falsely classified nodules belonged to the 8-mm-size group, which is located right at the border between LungRADS categories 3 and 4A, implying that only minor volumetric mistakes can already lead to different classifications [Inaccurate volumetry may lead to wrong lesion management decisions, which can either delay the correct diagnosis and treatment on the one side or cause unnecessary costs on the other, especially in the context of major lung cancer screening programs. Regarding patient management, changes in lesion size and the resulting potential shift in LungRADS categorization are critical. The DL-based CAD classified 88.5% of all solid nodules correctly according to LungRADS, which was significantly higher compared to the standard CAD system . A possible explanation is that Bartlett et al excluded all nodules with vascular or pleural attachment, which are more difficult to measure.The mean RVD between the systems was 1.3\u2009\u00b1\u200918.6% for the solid nodules and \u2013\u200915.2\u2009\u00b1\u200923.5% for the GGN. Bartlett and colleagues reported a mean RVD of\u2009\u2212\u20090.9\u2009\u00b1\u200916.3% while assessing the interscan variability of 100 nodules measured twice with a standard CAD system [This study has several limitations. First, the protocol used is not state of the art for lung cancer screening according to the current guidelines by the European Society of Thoracic Imaging (ESTI), in particular referring to FBP as a reconstruction algorithm and the absence of overlapping image reconstruction . HoweverIn conclusion, the DL-based system had a higher initial detection rate of pulmonary nodules and a higher proportion of them would have been classified correctly according to LungRADS compared to the standard CAD system. Nodule size and attenuation had an effect on the measurement accuracy of both systems; there was no effect of tube voltage. Our results indicate that measurement inaccuracies between CAD systems have a potential impact on patient management, which demands careful revision and, if needed, manual correction by radiologists."}
+{"text": "Background and aimsBeing metabolically unhealthy (MU) is defined as having either hypertension, hyperlipidemia, type 2 diabetes mellitus/pre-diabetes, or fatty liver disease. We aimed to determine if MU was associated with severe COVID-19 pneumonia (severe disease).MethodsWe performed a single-center retrospective study between March 2020 and August 2021 for patients with overweight or obesity hospitalized with COVID-19 pneumonia. Logistic regression analysis was utilized to derive a risk score for severe disease. The accuracy of the model was assessed using the area under the receiver operating characteristic curve (AUROCC) and bootstrap resampling.ResultsA total of 334 of 450 patients hospitalized with COVID-19 pneumonia (74.2%) were MU. Patients who were MU had higher in-hospital mortality (10.5% vs. 2.6%) and longer length of hospitalization (median 6 vs. 4 days). MU was not associated with severe disease, p=0.311. On multivariable analysis, older age, male sex, and Asian race were associated with severe disease. Not being vaccinated was associated with doubled odds of severe disease. The AUROCC of the final model was 0.66 (95% CI: 0.60 to 0.71). The risk score at the lowest quintile had a 33.1% to 65.5% predicted risk and a 58.7% observed risk of severe disease, whereas, at the highest quintile, there was an 85.7% to 97.7% predicted risk and an 89.7% observed risk of severe disease.ConclusionBeing MU was not a predictor of severe disease, even though mortality was higher despite having higher rates of vaccination. This risk score may help to predict severe disease in hospitalized patients with obesity or overweight. External validation is recommended. COVID-19 pneumonia, caused by the SARS-CoV-2 virus, emerged in late 2019 and quickly became a global pandemic despite quarantine efforts .\u00a0AdditioThe concept of metabolic health has started to emerge within the past few years ,11. MetaAlthough obesity is an established risk factor for severe COVID-19 pneumonia, it remains unclear whether metabolic health plays a role as a risk factor. Therefore, we aimed to determine the association between being metabolically unhealthy with severe or critical COVID-19 pneumonia, and in-hospital mortality in patients with overweight or obesity. Finally, we aimed to derive risk scores to predict severe disease and critical illness from COVID-19 pneumonia in this patient population. We hypothesized metabolic unhealthiness would be predictive of severe, critical, and inpatient mortality from COVID-19 pneumonia.Patient selectionThe current study was approved by the Mayo Clinic Institutional Review Board. A waiver of informed consent was obtained. The study followed the STROBE guidelines for observational studies, which has been provided as a supplementary material .2, and 2) being immunocompromised , severe combined immunodeficiency, etc. Patients with a normal BMI were excluded to determine the impact of metabolic health, specifically, in patients with excess fat mass. A BMI >/= 30.0 kg/m2 was defined as having obesity. Class I, II, and III obesity were\u00a0defined as a BMI between 30.0 to 34.99 kg/m2, 35.00 to 39.99 kg/m2, and >/= 40.00 kg/m2, respectively. Likewise, patients who were immunocompromised were excluded as they represent a different subset of patients who likely have worse outcomes. All patients had follow-up data from admission to discharge from the hospital.Patients hospitalized with COVID-19 pneumonia between March 2020 and August 2021 were identified using our institution\u2019s registry of confirmed cases. A random selection of 450 adult patients, aged >/= 18 years of age, from the registry were enrolled. Patients were excluded from the cohort for the following reasons: 1) having a body mass index (BMI) less than 25.0 kg/mPrimary and secondary outcomesSevere COVID-19 pneumonia (onward referred to as severe disease) was defined as per the National Institutes of Health (NIH) COVID-19 treatment guidelines, including 1) oxygen saturation < 94 % on room air, 2) a P:F /FiO2 (fraction of inspired oxygen)) ratio < 300, 3) respiratory rate > 30, and 4) the presence of greater than 50% of lung infiltrates on chest imaging .\u00a0The priSecondary outcomes included in-hospital mortality from COVID-19 pneumonia, and other measures of disease severity, including admission to the intensive care unit (ICU), need for endotracheal intubation, initiation of vasopressors, development of atrial fibrillation with rapid ventricular response, initiation of continuous renal replacement therapy (CRRT), need for extracorporeal membrane oxygenation (ECMO), and length of hospital stay. The NIH guidelines define critical illness as respiratory failure, septic shock, and/or multiple organ dysfunction .\u00a0A seconRisk score development and statistical analysisAll variables were collected retrospectively. Demographic data included age at admission, sex category , race and ethnicity, smoking status, and BMI. The presence of metabolic disorders prior to admission was recorded and included hypertension, hyperlipidemia, T2DM or pre-diabetes, and the presence of fatty liver. The presence of hypertension, hyperlipidemia, and T2DM was determined through ICD-10-CM codes and a retrospective review of clinical notes. A diagnosis of pre-diabetes was determined by a hemoglobin A1C between 5.8 to 6.4%. The presence of fatty liver was determined by a review of prior abdominal imaging reports. Having at least one metabolic disorder was defined as being metabolically unhealthy (MU).The presence of pulmonary disease was recorded and included obstructive sleep apnea, asthma, chronic obstructive pulmonary disease (COPD), or other conditions. The other conditions included prior histories of lobectomy, interstitial lung disease or idiopathic pulmonary fibrosis, or pulmonary hypertension. Treatments intended to target COVID-19 were recorded and included remdesivir, convalescent plasma, immunomodulators , and corticosteroids .The selection criteria for the varied treatment options changed over time during the study period as new therapies emerged during the COVID-19 pandemic. In general, however,\u00a0remdesivir was given to\u00a0all patients without a contraindication (such as an estimated glomerular filtration rate (eGFR) < 30 mL/min or elevated transaminases), corticosteroids for patients with an oxygen requirement of > 4 L per nasal cannula, the use of convalescent plasma in patients with non-reactive antibody titers to the spike antigen, and the use of immunomodulators for refractory cases with evidence of cytokine release storm.Baseline characteristics were reported using descriptive statistics summarized as medians and interquartile ranges, or fractions and percentages for continuous and categorical variables, respectively. Wilcoxon Rank Sum and Fisher\u2019s Exact tests were utilized to determine associations with metabolic status. Multivariable logistic regression was performed to adjust for potential confounders and to identify predictors for severe disease. Variables with the highest p-values were sequentially removed until removal led to less than a 1-point reduction in the Akaike information criterion (AIC). The accuracy of the model was calculated using bootstrap resampling estimates of the area under the receiver operating characteristic curve (AUROCC). Secondary analyses for critical illness and in-hospital mortality were performed utilizing bivariable logistic regression analyses. A separate risk score for critical illness was also derived through the same method.\u00a0All tests performed were two-sided, and the statistical significance was set at a p-value < 0.05. Using an alpha of 0.05, a beta of 0.80, and a baseline incidence of critical COVID-19 pneumonia of 20% in patients with obesity, we estimated a sample size of 398 patients would be needed to show a 10% reduction in the rate of critical illness in metabolically healthy patients. SPSS Statistics for Windows, Version 28.0  was used for the statistical analyses.Patient characteristicsA total of 450 patients with overweight or obesity hospitalized with COVID-19 pneumonia were included in the derivation of the risk score\u00a0Table . 74.2% oDerivation of risk scoreAge, male sex, hypertension, T2DM, and BMI group 40.0 kg/m2\u00a0or greater were the strongest predictors of severe disease on bivariable logistic regression\u00a0Table .\u00a0In thisAccuracy of the risk score for severe diseaseThe sum of the score coefficients was subdivided into quintiles and the observed risk for severe disease was calculated for our cohort\u00a0Table .\u00a0The preIn-hospital mortality and critical illnessOlder age, being a former cigarette smoker, having a higher BMI, having been vaccinated, and having lower rates of asthma were associated with being MU. Patients who were MU had a higher rate of in-hospital mortality from COVID-19 pneumonia , and longer length of hospitalization  than those who were metabolically healthy (MH)\u00a0.\u00a0Hyperlipidemia was a significant predictor of death, p = 0.0269, whereas hypertension and type 2 diabetes approached significance, p = 0.0560 and p = 0.0605, respectively. As the number of metabolic conditions increased, the odds of critical illness  and in-hospital mortality  increased for each additional condition. Similarly, fatty liver and having four metabolic conditions were associated with an increased likelihood of the secondary composite outcome, OR = 4.13, p = 0.0108 and OR = 5.39, p = 0.0203, respectively. After adjusting for age, and BMI, the number of metabolic conditions remained significant for in-hospital mortality and approached significance for critical illness\u00a0, and MU was defined as having at least one metabolic condition [The association between metabolic status and disease severity from COVID-19 pneumonia has not been fully studied. Limitations of prior studies have included the use of national inpatient databases, homogenous populations, and variable definitions for obesity and being MU. In a recent study from South Korea, investigators concluded that metabolic health status plays a greater role in disease severity than obesity .\u00a0The stuondition .\u00a0AdditioLike these prior studies, our study found that being MU was associated with an increased risk of mortality. In contrast, these studies did not evaluate the impact of MU on severe COVID-19 pneumonia. Similarly, a dose-dependent association between the number of metabolic conditions and critical illness or mortality was not explored. Fatty liver - another metabolic condition of increasing prevalence - was also not evaluated. Compared to these studies, our study had a more heterogenous population, included patients with overweight or obesity, considered MU as having at least one metabolic condition, and considerations were made to vaccination status, treatments administered, and presence of pulmonary disease. Finally, we were able to derive risk scores for severe disease and critical illness, specifically for patients with overweight and obesity.The main finding of our study was the dose-dependent association between the number of metabolic conditions and the increased likelihood of severe COVID-19 pneumonia, critical illness, and in-hospital mortality. These associations remained for critical illness and in-hospital mortality after adjusting for age and BMI, suggesting metabolic conditions are independent of obesity. An explanation for this finding may be the concept of metabolically healthy versus metabolically unhealthy obesity . Human sA dysregulated immune response characterized by cytokine storm has been implicated as the immunopathogenesis of severe COVID-19 pneumonia -23. GiveOur study has several limitations. First, COVID-19 pneumonia was and remains a rapidly evolving disease with fast-changing treatment algorithms. Given the rapid pace of drug and vaccine development, there likely were variable treatments offered at the early onset of the pandemic compared to later in its course. Treatments, such as baricitinib and tocilizumab, and vaccinations were not available for patients before December 2021. In our cohort, less than 10% of patients were vaccinated as vaccines were only emerging by August 2021. It is unclear if higher rates and longer times from vaccination may have decreased the risk of severe COVID-19 pneumonia in this patient population.\u00a0Second, the retrospective nature of the study limited our ability to measure other measures of metabolic disease, including waist circumference, and visceral adipose tissue, and measures of inflammation, such as C-reactive protein, procalcitonin, and interleukin-6 levels. Similarly, the retrospective design prevented us from comprehensively surveying each patient for the presence of each metabolic disorder, especially fatty liver. Given the retrospective nature, we relied on the available laboratory and imaging diagnostics to determine the presence of metabolic conditions. As such, we were unable to determine the presence of liver disease in all patients given the lack of imaging. In addition, we were unable to obtain surrogates for central adiposity, such as the waist circumference, that measure\u00a0the amount of visceral adiposity.Third, the definition of MU is not well established by expert guidelines. To improve inclusivity, we chose the more inclusive definition of having at least one metabolic comorbidity to define metabolic unhealthiness. With a stricter definition, such as having two or three metabolic conditions as the cutoff for MU, it is likely we would have found a direct association between MU and severe COVID-19 pneumonia. Nonetheless, we showed that as the number of metabolic conditions increased, so did the likelihood of severe COVID-19 pneumonia.Fourth, the frequency of in-hospital mortality had low counts, which prevented us from deriving a risk score using multivariable logistic regression to predict death from COVID-19 pneumonia in patients with overweight and obesity.\u00a0Finally, the frequency of severe COVID-19 pneumonia was relatively high in our patient population compared to previous studies. The reason for this finding is unclear but may be related to selection bias as our tertiary care center cares for medically complex patients who may have additional comorbidities that were not accounted for. Nonetheless, our study has demonstrated new and novel findings related to disease severity based on metabolic health in patients with overweight or obesity hospitalized with COVID-19 pneumonia.\u00a0Obesity is a well-recognized risk factor of severe COVID-19 pneumonia. The concept of metabolic health is emerging as a novel idea to explain the discrepant observations in variable outcomes in patients with obesity. Although obesity has been recognized as a risk factor for cardiovascular disease, diabetes, and other metabolic conditions, not every patient with obesity develops these complications. In our study, we aimed to determine the association of metabolic health with disease severity in patients hospitalized with COVID-19 pneumonia. We showed that metabolic conditions have a dose-dependent association with severe disease, critical illness, and in-hospital mortality, irrespective of age and BMI.Although we did not show that being metabolically unhealthy was associated with disease severity, it is likely that an alternative definition for metabolic unhealthiness is needed. Our risk scores have several clinical implications, including reminding clinicians of the elevated baseline risk of severe COVID-19 pneumonia in patients with overweight or obesity, and further emphasizing the risk factors for disease severity. Taken together, our findings suggest clinicians should be aware that metabolic status plays an additive effect in severe disease progression from COVID-19 pneumonia. We recommend further validation of these risk scores in heterogenous populations with updated treatment and vaccination guidelines."}
+{"text": "XS), water permeability (TS), tensile strength (KL), elongation at break (DSL), fertilizer permeability (TF), and viscosity (ND), and the optimal ratio parameters of membrane material were determined. Analytic hierarchy process (AHP) combined with correlation analysis was used to construct the judgment matrix of physicochemical properties, which passed the consistency test, and to determine the weight and ranking of each index: TF (0.6144) > XS (0.1773) > KL (0.1561) > ND (0.1311) > TS (0.0775) > DSL (0.0520). The comprehensive scores of sustained-release membrane materials under different treatments were calculated based on normalized data samples and weights. It was determined that the percentage of each component in the best comprehensive performance of the slow-release membrane material was as follows: polyvinyl alcohol, polyvinylpyrrolidone, zeolite, and epoxy resin were 7.3%, 0.7%, 0.5%, and 2%, respectively.In this paper, the optimal analytic hierarchy process was used to establish a comprehensive evaluation model for the physicochemical properties of composite sustained-release membrane materials based on water absorption ( With the massive use of traditional fertilizers, environmental pollution, economic effects, food safety, and other problems are becoming increasingly obvious ,2,3. BioTF) [XS) [KL) [DSL) [KL of composite membrane reached the optimal value of 32.28 Mpa, and the DSL decreased to the lowest value of 26.21% [DSL was reduced to a lower level of 16.27% [DSL of the membrane reached the best, and the KL reached the lowest level of 0.47 Mpa [Previous studies mainly analyzed the fertility permeability (TF) , water aTF) [XS) , tensileXS) [KL) , elongatL) [DSL) , and othf 26.21% .Adding gf 16.27% . With 0.0.47 Mpa . When th0.47 Mpa . When 5%0.47 Mpa . This sh0.47 Mpa .The comprehensive evaluation method is an important step to reasonably determine the index weight and to obtain the evaluation result. Only by selecting appropriate, comprehensive evaluation methods for different problems can the evaluation results be accurate and scientific. Entropy weight method is a method to obtain information entropy and related weight according to the variation degree of information contained in each index . It has XS), water permeability (TS), tensile strength (KL), elongation at break (DSL), fertilizer permeability (TF), and viscosity (ND) of slow-release membranes. Based on the subordinate function, a comprehensive evaluation system was constructed, and the optimal membrane material with comprehensive performance was chosen to provide a theoretical foundation for the objective comprehensive evaluation of the properties of slow-release membrane materials.In this paper, the analytic hierarchy method was used to reasonably establish the weights of each index on the basis of the correlation analysis of six physical and chemical performance indices, including water absorption  were also designed as B1\u2013B4, and the ratios in solution were 0%, 0.25%, 0.5%, and 1%, respectively. The amount of epoxy resin added was 14 g, equal to 2% in the solution. A two-factor four-level comprehensive experimental design with 16 groups was adopted. The determination of XS, TS, KL, DSL, TF, and ND proceeded through reference to research methods of predecessors [The data in this study are derived from the preparation experiments of slow-release membrane materials under different ratios of water copolymer and zeolite. Four levels of water-based copolymer ratio A (PVA: PVP) were designed as Aecessors ,37,38,39Microsoft Office 365 was used for data processing and table drawing. IBM SPSS Statistics 22 data analysis software was used to analyze the correlation of physical and chemical properties of membrane materials. Yaahp 10.3 was used to determine the index weight and to construct the comprehensive evaluation model of the membrane material.XS, TS, KL, DSL, TF, and ND.Build a hierarchical model: A hierarchy diagram was constructed based on XS, TS, KL, DSL, TF, and ND was analyzed, and the paired comparison matrix was constructed by combining the evaluation criteria of the 1\u20139 scale method.Construct the optimal judgment matrix: The degree of correlation among Hierarchical ranking and consistency check: The consistency of the judgment matrix was checked by calculating the CR value.Calculate the comprehensive score of each index: The weight of each index was multiplied by the standardized value and then accumulated to obtain the comprehensive score of each treatment.The hierarchical analysis method, which was based on correlation analysis of the physicochemical properties of membrane materials, was used in this paper. The main principles and steps are as follows:XS, TS, KL, DSL, TF, and ND of membrane materials under different water-based copolymer ratios and zeolite amounts. It can be seen from 1\u2013B4, when A was decreased from A1 to A4, XS, TS, and TF increased by 51.6%, 101.1%, and 49.5%, while DSL and ND decreased by 15.7% and 61.9%, on average. XS, TS, and TF showed a positive response to the decrease of A, while DSL and ND showed a negative response. In the condition of B1\u2013B4, when A was decreased from A1 to A2, KL increased by 15.4%, and when A was decreased from A2 to A4, KL decreased by 41.9%, on average. This finding showed that the decrease in A on KL was promoted first and then suppressed. In the condition of A1\u2013A4, when B was increased from B1 to B4, KL and DSL were initially increased by 31.6% and 12.9%, and then decreased by 6.2% and 9.9%, on average. This finding revealed that the increases in B on KL and DSL were promoted first and then inhibited. Except for the A4 condition, XS was decreased by 15.5%, on average. XS presented a negative response to B increase, while the increase in B on XS was promoted first and then inhibited in the A4 condition. The ND, TS, and TF variations caused by B were 4.33%, 5.35%, 10.85%, respectively, suggesting that B increase had less effect on ND, TS, and TF. The slow-release membrane material with excellent comprehensive performance should have better water resistance, mechanical properties, slow-release property, and low viscosity [XS, TS, KL, DSL, TF, and ND reached the optimal values in A1B4, A1B2, A2B3, A1B2, A2B1, and A4B3 treatments, respectively. The above results indicated that the optimal treatment corresponding to each physicochemical property index of membrane materials is not consistent. If the optimal evaluation method based on a single index has subjective one-sidedness, the multi-index objective comprehensive evaluation method must be used. iscosity ,41,42. BXS, TS, KL, DSL, TF, and ND.XS and TF was 0.913, and there was a significant positive correlation between them (p < 0.01). TS was positively correlated with TF and XS (p < 0.01), and the correlation coefficients were 0.839 and 0.886, respectively. XS and TS were negatively correlated with ND, KL, and DSL, indicating that the higher the water absorption rate of the membrane material, the more serious the swelling inside the membrane material, and the mechanical properties of the membrane changed significantly [The correlation analysis of the six indices of the membrane is shown in ficantly .A = (ija) n \u00d7 n, and the judgment matrix must meet the following conditions: a > 0,\u00a0i,j = 1, 2, 3, \u2026, n).Taking the overall optimization of the target level as the standard. In the hierarchical structure model of membrane material, it is necessary to make a pairwise comparison of the indices at the same level to establish the judgment matrix TF was regarded as 1 according to the important principle of fertilizer permeability of sustained-release membrane material. TF is XS > TS > ND > KL > DSL. The higher correlation between two factors, the closer importance of the two factors. The values were assigned according to the degree of correlation between other indices and TF. The constructed judgment matrix is shown in The scale definition of judgment matrix is shown in CI value, which was used as the consistency index for consistency checking. The CI value was further used to obtain the CR value of the consistency index. Generally, the smaller the CR value, the more reasonable the judgment matrix and the higher the consistency. The procedure is as follows:In order to ensure the rationality of the weight distribution of each index in the comprehensive evaluation system, it is necessary to check the consistency of the judgment matrix of each level. First, the maximum characteristic root of the judgment matrix was calculated, and then the maximum characteristic root was used to calculate the max is the maximum characteristic root of the judgment matrix, \u03c9 is the weight vector, i\u03c9 is the weight of the ith evaluation index, and n is the number of evaluation index.In Formula (2): \u03bbCI is the consistency index, \u03bbmax is the maximum characteristic root of the judgment matrix, and n is the number of evaluation indices.In Formula (3): CR is the random consistency ratio, CI is the consistency index, and RI is the consistency index of matrix average.In Formula (4): CR value is less than 0.10, the judgment matrix meets the consistency test; if it is greater than 0.10, the matrix does not have consistency, and the matrix should be adjusted and analyzed until it meets the consistency requirements. After calculation, the CR values of the judgment matrices corresponding to When the ND are 0.208 and 0.131, respectively. Permeability has the greatest influence on the properties of membrane materials, followed by mechanical properties, and ND has the least influence. The weight of TF was 0.614, which accounted for the largest weight in permeability, and the ratio of TF in the total weight of the six indices was also the largest, reaching 0.406. This shows that TF is an important index to evaluate the comprehensive performance of slow-release membrane materials [KL is 0.750, which shows that KL is also important for the properties of membrane materials. The correlation analysis showed that the physical-chemical properties of membrane materials were independent and complex. The weights of all indices were in the following order: TF > XS > KL > ND > TS > DSL.After calculation, the weight distribution of each evaluation index is shown in aterials ,46. AmonXS, TS, TF, and ND were the minimum attributes, which were calculated by the following formula:Before the comprehensive score calculation, the index data were normalized by the membership function method. Among the indices of membrane materials, KL and DSL are maximum attributes, which can be calculated by the following formula:Xmin indicates the minimum value of the index, and Xmax indicates the maximum value of the indicator. In Formulas (5) and (6): U represents the membership function value of the index, The comprehensive score value of each treatment can be calculated by the following formula:iW represents the composite score of the ith treatment , liX is the membership function value of the lth index of the ith treatment , and lQ indicates the weight of each index. The comprehensive score values of different treatments are shown in In Formula (7): XS was negatively correlated with KL and DSL. You et al. [XS decreased with the increase of KL and DSL, which was consistent with the results of this paper. The entry of water molecules causes the membrane material to swell, which increases molecular distance and decreases the crosslinking degree of crosslinking groups, weakening the membrane material\u2019s mechanical properties. The proper selection of indicators is especially important when evaluating membrane materials. If the previous evaluation method based on the TF index was used [2B1 in this paper. The comprehensive evaluation method based on hierarchical analysis of six indices, including XS, TS, KL, DSL, TF, and ND, was proposed in this paper, the responses among the indices were considered, and the optimal membrane material was selected as A2B3 treatment. Data sample analysis showed that there was a difference of about 3% between A2B1 and A2B3 treatment in XS, TS, ND, and TF, indicating that the two treatments have similar performance. However, in terms of KL and DSL, A2B3 treatment was better than A2B1 treatment by 16.23 Mpa and 20.14%, respectively, showing better comprehensive performance. As a result, the results obtained in this paper by using multi-index comprehensive evaluation were more objective and reasonable.The results of this study showed that u et al.  concludewas used , the optTF and XS in the evaluation index level were 0.406 and 0.177, respectively, which showed that TF and XS were important indices to evaluate the properties of membrane materials [In the comprehensive evaluation system, the establishment of reasonable weight is very important to solve the decision problem, and it is also a key factor for the accuracy of evaluation . The optaterials . The basaterials  subjectiDSL > KL > ND > TS > XS > TF. It can be seen from DSL > ND > TF > KL > TS > XS. The entropy weight method determined that the index with the largest weight was DSL, which deviated from the more important goal of the fertilizer permeability of the slow-release membrane material. Therefore, the entropy weight method was not reasonable when determining the index weight in this paper. Following a comprehensive evaluation of membrane materials using the entropy weight method, it was determined that A1B3 was the best membrane treatment in terms of overall performance. The physical and chemical properties, such as XS, TS, and KL, of the A1B3 treatment were close to those of the A2B3 treatment, but the ND was far worse than that of the A2B3 treatment. Therefore, the entropy weight method was not reasonable in the weight establishment and comprehensive score calculation of the sustained-release membrane material indices in this paper. The index with the largest weight established by the independence weight coefficient method was DSL, which was the same as the maximum weight index established by the entropy weight method. However, the weight of TF in the independence weight coefficient method was higher than that in the entropy weight method, which made the independence weight coefficient method perform more reasonably. In this paper, the independence weight coefficient method was adopted for comprehensive evaluation of membrane materials, and it was concluded that the optimal membrane treatment was A1B3, which had the same defect as the entropy weight method. Both the entropy weight method and the independence weight coefficient method have irrationality in the weight distribution of the membrane material index, and the optimal treatment calculated by the method performs poorly in the ND index. In this paper, the optimal analytic hierarchy process was used to construct a judgment matrix combining the correlation between indices in the comprehensive evaluation, and the weight and optimal treatment obtained by combining subjective and objective methods were more reasonable.The reasonable choice of evaluation method is also crucial for whether the decision problem can be solved . The indIn order to further explore the differences between the results of the three evaluation methods, the comprehensive scores of the three evaluation methods were jointly analyzed by Spearman correlation analysis. XS, TS, KL, DSL, TF, and ND. The weights of all indices were in the order: TF > ND > TS > KL > XL > DSL. The comprehensive scores of membrane materials under different conditions were clarified, and the optimal membrane material treatment was A2B3.An improved analytic hierarchy process was used to construct a comprehensive evaluation system for slow-release membrane materials based on six indices, including"}
+{"text": "Pseudomonas sp. bacteria namely Pseudomonas aeruginosa and Pseudomonas putida were used as biocatalysts. The desulfurization pathways of DBT by the two bacteria were determined by gas chromatography (GC)/mass spectrometry (MS) and High-Performance Liquid Chromatography (HPLC). Both organisms were found to produce 2-hydroxy biphenyl, the desulfurized product of DBT. Results showed BDS performance of 67.53% and 50.02%, by Pseudomonas aeruginosa and Pseudomonas putida, respectively for 500\u00a0ppm initial DBT concentration. In order to study the desulfurization of diesel oils obtained from an oil refinery, resting cells studies by Pseudomonas aeruginosa were carried out which showed a decrease of about 30% and 70.54% DBT removal for 5200\u00a0ppm in hydrodesulfurization (HDS) feed diesel and 120\u00a0ppm in HDS outlet diesel, respectively. Pseudomonas aeruginosa and Pseudomonas putida selectively degraded DBT to form 2-HBP. Application of these bacteria for the desulfurization of diesel showed promising potential for decreasing the sulfur content of South African diesel oil.Biodesulfurization (BDS) was employed in this study to degrade dibenzothiophene (DBT) which accounts for 70% of the sulfur compounds in diesel using a synthetic and typical South African diesel in the aqueous and biphasic medium. Two  The world health organization recorded about 7 million deaths in 2012 attributed to air pollution2, associated with particulate matter. For instance, the South African government requires that the concentration of sulfur compounds in diesel should be\u2009\u2264\u200910\u00a0ppm3. Beginning in January 2006, South Africa has two types of diesel fuel available. The maximum sulfur content for standard-grade diesel is 500\u00a0ppm, compared to 50\u00a0ppm for low sulfur grade fuel. Nevertheless, Sasol has been providing 10\u00a0ppm diesel to automobiles used for business and personal purposes4. In addition, the South African government has issued rules guiding the minimum allowable sulfur content in the country\u2019s diesel fuel, which may propel the upgrading of diesel products by some oil refineries so as to meet the criteria5. This regulation will take effect from September 2023. An effective desulfurization approach can consecutively enhance the quality of the fuel, with respect to the cetane value and increase in octane number6. Therefore, the interest of researchers has been provoked in this direction to further reduce the sulfur contents in South African diesel.Diesel has been regarded as the larger and most widely used source of energy in the world. It consists of sulfur compounds that when combusted by direct method release toxic substances such as sulfur oxides  is the most commonly used technique in the refineries to reduce the sulfur content of diesel with huge success recorded. Though HDS is capable of reducing sulfur content in diesel such as heterocyclic sulfur compounds like thiols and sulfides, the technique is inefficient to desulfurize refractory sulfur compounds such as dibenzothiophene and its derivatives, especially 4,6-dimethyldibenzothiophene 9. In addition, this technology suffers from high capital and operating costs10 due to the fact that it is operated at high temperature and pressure, thereby making it energy-intensive11. Furthermore, a lot of hydrogen is used, making the process highly risky in terms of safety. This situation has provoked the thoughts of many researchers worldwide to urgently explore alternative routes for the desulfurization of petroleum distillates.Different techniques such as hydrodesulfurization, adsorption7. However, BDS has not met the industrial-scale application\u2019s requirements yet12. For BDS to attain full industrial acceptance for utilization in petroleum desulfurization, the desulfurization bacteria must possess major characteristics such as; high tolerance for solvents, high desulfurization efficiency, and wide substrate specificity13. BDS involves the use of biocatalysts for the degradation of the sulfur compound in petroleum distillate to a less harmful compound without altering the quality of the fuel. Different bacteria strains such as Rhodococcus sp.15, Pseudomonas sp.16, and Gordona sp.17 have been employed for degrading sulfur compounds in diesel oil. For instance, Pseudomonas strains used in this study are very prolific and could survive in biphasic media and metabolic diversity18. They are known to thrive in conditions with or without oxygen, although they are classified as aerobic. They are readily available since they are present naturally in soil and water.Biodesulfurization (BDS) is a promising method that could be an efficient, cheap, and less energy-intensive technique for desulfurization21. A variety of sulfur-containing compounds have been reported, but dibenzothiophene (DBT)  accounts for 70% of the sulfur compounds in diesel and it is considered a model compound for biodesulfurization in research23. Various investigations have been reported on the use of these pseudomonas strains differently, under different conditions. Al-Faraas et al.24 isolated Pseudomonas aeruginosa from Iraqi Soils for desulfurization of dibenzothiophene. The effect of different sulfur sources on the degradation efficiency of Pseudomonas spp. has been reported. However, reports on detailed desulfurization activity of the bacteria are limited in the literature24. Davoodi-Dehaghani et al.25 also investigated the desulfurization ability of Rhodococcus Erythropolis (SHT87) for the degradation of DBT. The results showed that about 3\u00a0mM DBT was totally metabolized by the SHT87 resting cells in the biphasic and aqueous systems within 10\u00a0h. The strain was able to make use of thiophene, dimethylsulfoxide, dibenzothiophene sulfone, 2- methylthiophene and DBT, as the only sulfur sources for growth at 30\u00a0\u00b0C25. Mingfang et al.26 desulfurize DBT and 4, 6-dimethyldibenzo thiophene in dodecane and straight run diesel using lyophilized cell of Pseudomonas delafieldii R-8. Results showed about 1,807\u00a0mg/L of sulfur desulfurized in the straight-run diesel oil. The specific desulfurization rate was found to be 8.75\u00a0mmol sulfur kg\u22121 (cell) h\u22121. Furthermore, Bhanjadeo et al.27 explored the desulfurized potential of Microbial Type Culture Collection (MTCC) strains on DBT via C-S bond cleavage (4-S pathway). The study revealed\u2009>\u200999% DBT desulfurization within 10\u00a0min27. As far as it could be ascertained, no recent studies have been reported on the biodegradation of dibenzothiophene in South African diesel using Pseudomonas strains.In BDS, only C-S oxidative bond cleavage takes place to release the sulfur atom as sulfate and the carbon skeleton of the thiophenic compound remains unaffected as a phenolic product. Consequently, in the BDS process, the thiophenic sulfur compound serves only as the sole sulfur source for bacteria growth, and the calorific value of the fuel is preserved by the final end product 2-HBPPseudomonas sp. Therefore, this study investigates and compares the biodegradation efficiencies of growing and resting cells of Pseudomonas aeruginosa and Pseudomonas putida on the sulfur compound (DBT) in a model diesel and typical South African diesel in the aqueous and biphasic medium.BDS is considered a complementary technique to the HDS method of desulfurization. It can also be used to develop a hybrid process (Adsorption/BDS) to remove sulfur-containing compounds from petroleum distillates. In order to achieve this goal, there is a need to investigate the effect of operating conditions for biodesulfurization using the Isolated Pseudomonas putida and Pseudomonas aeruginosa Kwik Stik, ATCC 27,853 Microbiologics, were purchased from Sigma Aldrich (Pty) Ltd., South Africa. Hydrodesulfurizer inlet feed (5200\u00a0ppm) and hydro-treated diesel (120\u00a0ppm) were obtained from a South African refinery. Dibenzothiophene, acetonitrile and dimethyl formamide  (99%) were purchased from Merck (Pty) Ltd., South Africa. Ethyl acetate was purchased from NT laboratories supply (Pty) Ltd., South Africa. Glycerol, hexadecane, and 2-hydroxylbiphenyl (2-HBP) were purchased from Sigma-Aldrich (Pty) Ltd, South Africa. All other chemicals were of analytical grade, commercially available and used without further purification. All methods were performed in accordance with the relevant guidelines and regulations.2PO4\u00b7H2O (4\u00a0g/L), K2HPO4\u00b73H2O (3\u00a0g/L), MgCl2\u00b76H2O (0.0245\u00a0g/L), CaCl\u00b72H2O (0.001\u00a0g/L), FeCl3\u00b76H2O (0.001\u00a0g/L) was prepared in the laboratory as described by Boltes et al.16. All glass wares were sterilized in an autoclave at 121\u00a0\u00b0C, for 20\u00a0min. The chemicals were dissolved in deionized water until all chemicals were dissolved. The pH of the medium was maintained at 7.0 by adding 0.5\u00a0M NaOH, drop-wisely. The solution was sterilized in autoclave at the same conditions as for the glassware mentioned earlier. The prepared BSM medium was stored at room temperature\u00a0and kept away from sunlight.Basal salt medium consisting of NaHPseudomonas aeruginosa and Pseudomonas putida each were inoculated in 50\u00a0mL Luria\u2013Bertani (LB) liquid medium in a 250\u00a0mL Erlenmeyer flask. 10\u00a0g/L of tetracycline was prepared and 150 \u00b5L was added to the inoculum to serve as antibiotics for the bacteria. The mixture was incubated for 10\u00a0days at 30\u00a0\u00b0C, and 37\u00a0\u00b0C for Pseudomonas putida and Pseudomonas aeruginosa, respectively and agitated at 130\u00a0rpm. The prepared inoculum was kept in an eppendorf tube and frozen\u00a0at \u2212\u00a080\u00a0\u00b0C.Frozen pellets of Pseudomonas putida and Pseudomonas aeruginosa previously inoculated in LB medium was used. The experiment was conducted as described by Al-Faraas et al.24 and Boltes et al.15. 0.25\u00a0mL LB-frozen stock of bacteria was put into 50\u00a0mL of BSM in a 250\u00a0mL Erlenmeyer flask. 0.1\u00a0g of DBT was dissolved in dimethyl formamide (DMF) and serially diluted to vary the concentration as required. The bacteria medium was supplemented with 1\u00a0mL of 0.25\u00a0mM (46\u00a0ppm) DBT as the only sulfur source with 150 \u00b5L tetracycline. Glycerol 20\u00a0g/L was used as the only carbon source. The flask was incubated at 30\u00a0\u00b0C and 37\u00a0\u00b0C for Pseudomonas putida and Pseudomonas aeruginosa, respectively, and agitated at 130\u00a0rpm for 10\u00a0days. The growth of bacteria was measured and desulfurization of DBT in model diesel was monitored as well.The BDS experiments were conducted in batch mode. The bacteria of Pseudomonas aeruginosa and Pseudomonas putida was performed in batch mode. The bacteria in the growing cell experiment were harvested at the late exponential phase. The bacteria medium was centrifuged at 7000\u00a0rpm for 5\u00a0min. The cells were washed thrice with potassium phosphate buffer solution. The washed Pseudomonas aeruginosa and Pseudomonas putida cells were then re-suspended into glycerol/ NaCl in the ratio of 1:1. The different cell concentrations (0.3\u20131.2\u00a0g DCW/L) of frozen resting cells of Pseudomonas aeruginosa and Pseudomonas putida in glycerol/NaCl solution were measured into 50\u00a0mL of BSM with 150 \u00b5L of tetracycline, 500 \u00b5L of glycerol and 1\u00a0mL of 500\u00a0ppm model oil. The initial concentrations of DBT varied from 250 to 1000\u00a0ppm. The mixture was incubated for 8\u00a0h at 30\u00a0\u00b0C and 37\u00a0\u00b0C for Pseudomonas putida and Pseudomonas aeruginosa, respectively, and the mixture was agitated at 130\u00a0rpm. Aliquots of samples were taken at intervals and the samples were analyzed for biodesulfurization efficiency.Biodesulfurization with resting cells of Pseudomonas aeruginosa and Pseudomonas putida were used for the degradation of real diesel in this study. About 5\u00a0mL of diesel was measured into a 250\u00a0mL Erlenmeyer flask with 5\u00a0mL of resting cell in glycerol/NaCl (1:1) into 20\u00a0mL of BSM. The solution was incubated for 8\u00a0h, 130\u00a0rpm at 30\u00a0\u00b0C for Pseudomonas\u00a0putida and 37\u00a0\u00b0C for Pseudomonas\u00a0aeruginosa, and aliquots of samples were taken at intervals for analysis.Resting cells of 28. This is explicitly described under the analytical techniques in Section \u201cHexadecane was chosen as an organic phase in the biphasic process owing to its presence in the diesel oil fraction. BSM was the aqueous medium. In this experiment, the percentage of oil-to-water varied from 0%, 20%, and 50%. The organic phase was centrifuged and extracted with ethyl acetate from the aqueous phase oil. The final DBT concentration and produced 2-HBP end product of the 4S pathway in the organic phase were determined according to Caro et al.Pseudomonas aeruginosa and Pseudomonas putida were used for degradation of real diesel in this study. Diesel sample obtained before HDS with initial DBT concentration of 5200\u00a0ppm and diesel sample obtained after HDS with initial DBT concentration of 120\u00a0ppm were supplied by a refinery in South Africa. About 5\u00a0mL of diesel was measures into a 250\u00a0mL Erlenmeyer flask with 5\u00a0mL of resting cell in glycerol/NaCl (1:1) and 20\u00a0mL of BSM. The mixture was incubated for 8\u00a0h, at 30\u00a0\u00b0C and 37\u00a0\u00b0C, and agitated at 130\u00a0rpm for Pseudomonas putida for Pseudomonas aeruginosa, respectively. Aliquots of samples were taken at intervals for analysis29.Resting cells of 660 nm), dry mass, and absorbance was determined. A calibration curve was obtained and used in the determination of the unknown cell concentration.The pH was measured using a pH meter and a Spectroquant Pharo 300 Merck, (W210324), made in the EU was used to measure the turbidity of the culture. The cell mass was determined by measuring the optical density (OD) at a wavelength of 660\u00a0nm. In order to measure the net dry cell weight (g DCW) of the biomass, 3\u00a0mL of the culture was centrifuged at 7000\u00a0rpm for 10\u00a0min. The concentrate was washed thoroughly on a pre-weighed filter paper. The filter paper containing the bacteria was dried overnight at 100\u00a0\u00b0C and the weight of the bacteria was determined by subtracting the net weight from the initial weight of the filter paper. The relationship between the optical density , equipped with an Eclipse C-18 column. Acetonitrile (55 wt%) was used as the mobile phase, with the UV detector at 254\u00a0nm, with a 1.0\u00a0mL/min flow rate, and injection volume of 10\u00a0mL for 10\u00a0min. The incubated mixture was centrifuged at 7000\u00a0rpm for 10\u00a0min and filtered. The aqueous phase was acidified with HCl in order to quench the desulfurization reaction before analyzing it with HPLC. The degraded DBT and the formed 2-HBP formed were extracted with an equal volume of ethyl acetate. In a biphasic system, the DBT and 2-HBP were extracted from the organic phase after centrifugation and analyzed using HPLC. The peak area of DBT and that of the 2-HBP of known concentrations at different elution time were used to calibrate the HPLC. The calibration curve obtained during the calibration was used to determine the unknown concentration of DBT and 2-HBP in the desulfurized samples.Detection of sulfur containing compounds in diesel before hydrodesulfurization (5200\u00a0ppm) and diesel after hydrodesulfurization 120\u00a0ppm) and the evolution of the 4S end product (2-HBP) were done by gas chromatography/mass spectroscopy (GC/MS) Shimadzu equipment with column Rx-SMX. Injection and detection temperature were set at 220\u00a0\u00b0C and 230\u00a0\u00b0C, respectively. Oven temperature at 80\u00a0\u00b0C, to 190\u00a0\u00b0C at 10\u00a0\u00b0C/min and 15\u00a0\u00b0C/min to 230\u00a0\u00b0C for 18\u00a0min in split-less mode. Helium was used as the carrier gas. To determine the calibration curve for HDS feed, and HDS outlet diesel, the diesel samples with known concentrations were diluted serially to vary their concentrations. The peak areas detected from GC/MS were plotted against known concentrations of the DBT and a calibration curve was obtained which was used to calculate the unknown concentrations of DBT in the samples. Physical and chemical properties of a typical South African diesel oil is given in Table 20\u00a0ppm anseudomonas aeruginosa and Pseudomonas putida with degradation of DBT and formation of 2-HBP as a function of time. The results showed that the growth of the bacteria increased with time. The growth of Pseudomonas aeruginosa began to decrease slightly after 120\u00a0h and the growth of Pseudomonas putida began to decrease after168 h. At 120\u00a0h, an optical density of Pseudomonas aeruginosa reached 1.0\u00a0g DCW/L and that of Pseudomonas putida was at 0.998\u00a0g DCW/L. It can be observed that as the growth of bacteria increased there was a simultaneous increase in the degradation of DBT from 46 to 0.21\u00a0ppm for Pseudomonas aeruginosa and and from 46 to 0.51\u00a0ppm for Pseudomonas putida. Like wisely, the amount of 2-HBP increased from 0 to 32.5\u00a0ppm for PA, and the amount of 2-HBP increased from 0 to 24.5\u00a0ppm for PP. All experiments revealed that the growth of the bacteria ceased before the DBT was fully converted to 2-HBP. In addition, the production of 2-HBP, as a final metabolite of the 4S pathway, was less than the consumption of DBT in both cases. These results agree with the results reported by Davodii-Dehaghani et al.25 and Caro et al.30. This could be a result of intra and extracellular accumulation of 4S compounds. The desulfurization of DBT to 2-HBP through the 4S pathway could be the reason why there was no further growth in the bacteria Pseudomonas aeruginosa and Pseudomonas putida, at 120\u00a0h for Pseudomonas aeruginosa and 168\u00a0h for Pseudomonas putida. This is due to the inhibition effect. In view of the report that the main limiting factor of BDS of dibenzothiophene is the inhibitory effect of 2-HBP.Figure\u00a022. Approximately 99.5% and 98.9% of DBT was degraded by Pseudomonas aeruginosa and Pseudomonas putida, respectively. However, 2-HBP could accumulate up to concentration of 33.06\u00a0ppm for Pseudomonas aeruginosa and 22.99\u00a0ppm for Pseudomonas putida which account for 71.9% and 50% formation of 2-HBP for Pseudomonas aeruginosa and Pseudomonas putida, respectively. The results show that the amount of 2-HBP formed was not equivalent to the amount of DBT degraded. In addition, no sulfate or sulfite accumulation was detected during growth of bacteria. Therefore, it could be assumed that the sulfur content has been assimilated by the cells.The major 4S pathway metabolite identified during the batch cultivation was 2-HBP. This was also confirmed by Rhee et al.Pseudomonas aeruginosa and Pseudomonas putida. The result showed gradual degradation of DBT from 500 to 59.17\u00a0ppm for Pseudomonas aeruginosa and from 500 to 100.17\u00a0ppm for Pseudomonas putida. Accumulation of 2-HBP also reached 250\u00a0ppm for Pseudomonas putida and 397.67\u00a0ppm for Pseudomonas aeruginosa. This accounts for 88% and 80% desulfurizing capability of Pseudomonas aeruginosa and Pseudomonas putida, respectively. The percentage of 2-HBP produced were 79.5% and 50% for Pseudomonas aeruginosa and Pseudomonas putida, respectively.Figure\u00a0Pseudomonas putida with respect to the control sample that has no bacteria. The concentration of cells varied from 0.3 to 1.2\u00a0g DCW/L and the initial DBT concentration was 500\u00a0ppm. The results showed the effect of bacteria concentration on the biodesulfurization of DBT. It could be observed that biodesulfurization of DBT increased with an increase in the concentration of cells from 0.3 to 1.2 gDCW/L compared to when there were no bacteria in the control sample. This is an indication that the bacteria strain used the DBT for their metabolism as the only sulfur source. No DBT degradation was noticed when there were no bacteria in the medium. The result showed the highest desulfurization capability of DBT when the cell concentration was 1.2\u00a0g DCW/L with 80% degradation effect on DBT. This could be as a result of more Pseudomonas putida in the medium to degrade the DBT compound.Figure\u00a0Pseudomonas aeruginosa and Pseudomonas putida, respectively were achieved when cell concentration was 0.3\u00a0g DCW/L. In addition, 2-HBP produced for Pseudomonas aeruginosa and Pseudomonas putida at 0.3\u00a0g DCW/L was 117.34\u00a0ppm and 81.04\u00a0ppm, respectively, accounting for 23.47% and 16% Pseudomonas aeruginosa and Pseudomonas putida, respectively. The highest desulfurization performance was obtained when 1.2\u00a0g DCW/L was used. Enhancement in desulfurization of DBT and formation of 2-HBP at an increased concentration of bacteria could be attributed to the availability of more bacterial to feed on the DBT. It could be observed that formation of 2-HBP was lower than the degradation of DBT in all the cases. This could be attributed to the inhibition of bacteria growth as a result of the production of the 2-HBP31. It is a well-known fact that sulfate and 2-HBP, which are end products of DBT desulfurization, have direct adverse effects on the BDS. Therefore, enzymes of 4S pathway also undergo feedback inhibition exerted by\u00a02-HBP, hence, limiting the cell growth, resulting in low\u00a0desulfurization efficiency. These results obtained in this study agree with the findings reported by Mohebali and Ball31.Desulfurization of DBT and the production of 2-HBP is illustrated by the results in Fig.\u00a0Pseudomonas aeruginosa and Pseudomonas putida. The initial concentrations of DBT varied from 250 to 1000\u00a0ppm. The results showed that an increase in the initial concentration of DBT resulted in an increase in the growth of bacteria (Pseudomonas aeruginosa and Pseudomonas putida). This could be a result of enough availability of DBT for the metabolism of the cell in the medium, resulting thereby in enhanced growth. It was discovered that growth stopped before the final or complete desulfurization of the DBT. This could be attributed to the accumulation of the 2-HBP compound in the medium that inhibited the further growth of the cells at 7\u00a0h as a result of the inhibition effect. This agrees with the result of Maxwell et al.32. Other DBT metabolites of the 4S pathway such as DBTO, DBTO2, and HPBS were not detected by GC\u2013MS analysis, except 2-HBP. This could be attributed to the existence of an additional degradation pathway for DBT. Other authors also confirmed that other metabolites of the 4S pathway could not be detected in the experiment. However, they are indicated as postulated metabolites34.Figure\u00a0Pseudomonas aeruginosa was used as the biocatalyst. About 60.06%, 50.02%, 38.71%, and 30.21% desulfurization efficiencies were achieved when Pseudomonas putida was used as the biocatalyst for 250\u00a0ppm, 500\u00a0ppm, 750\u00a0ppm, and 1000\u00a0ppm, respectively.Figure\u00a0Pseudomonas aeruginosa and Pseudomonas putida, respectively. It could be observed from the result in Fig.\u00a0Pseudomonas aeruginosa and Pseudomonas putida in g DCW/L decreased with an increase in the percentage of oil to water at 50%. Results showed the best growth rate when a 20% oil phase was used compared to an aqueous medium without the oil phase and a 50% oil phase. The lower growth rate at 50% could be as a result of hydrophilicity of DBT, owing to the reduced concentration of DBT when the oil phase increased because the same initial concentration of DBT was used in all. It is assumed that the transfer of DBT from the oil phase to the aqueous phase is an important parameter especially when a biocatalyst that has lower capability to adhere at the interface is used30. Another reason this could be so, is that, there might be mass transfer limitation of DBT from the oil phase to the aqueous phase where the cells are present. It has however been discovered that the cells use DBT as its only sulfur source for it metabolic growth, since bacteria use the DBT in the model oil for its growth. Furthermore, it could mean that there was lower supply of oxygen as the oil phase increased. This result is consistent with Caro et al.35.Figure\u00a0Pseudomonas aeruginosa and Pseudomonas putida. The oil\u2013water ratio is an essential factor in defining the reactor productivity and hence the reactor volume. The volume ratio of oil to water (O/W) affects the bioavailability of DBT when biodesulfurization occurs in the interface between the organic and the aqueous phases. Results showed better DBT degradation of 2-HBP production in the biphasic medium than in the aqueous medium. This could be as a result of better growth achieved in the result as discussed in Fig.\u00a0Pseudomonas aeruginosa and Pseudomonas putida in biphasic media compared to aqueous media. Hence, the amount of 2-HBP production was more than in the aqueous phase for both bacteria. This might be attributed to substrate availability and reduced product inhibition since 2-HBP which causes feedback inhibition is in the organic phase. The inhibition effect of 2-HBP was therefore avoided by channeling the DBT compound to the organic phase allowing the desulfurization process to continue unhindered in the aqueous phase. These results support the reports of a few researchers37.Figure\u00a026 utilized resting cells of Lyophilized R-8 to degrade DBT in model oil with an initial concentration of 1807\u00a0ppm. The result showed 55.23% BDS performance at a BDS rate of 8.75\u00a0Mm (g DCW/L)\u22121\u00a0h\u22121. Comparing this with the result obtained in this study, when the 500\u00a0ppm initial DBT was in a model oil, a BDS performance and BDS rate of 67.53% at 21.25\u00a0mM (g DCW/L)\u22121\u00a0h\u22121, respectively by resting cells of Pseudomonas aeruginosa and 50.02\u00a0ppm and 13.90\u00a0mM (g DCW/L)\u22121\u00a0h\u22121 by resting cells of Pseudomonas putida were obtained. The BDS performance results obtained in this study are better than what was obtained by Mingfang et al.26. This could be due to a higher initial DBT concentration used in their study. Alcon et al.38 desulfurized DBT in a model diesel using Pseudomonas putida CECT 5259 for 10\u00a0h with an initial DBT concentration of 1.84\u00a0ppm. About 86% desulfurization efficiency was achieved by the bacteria. The result obtained in their study was higher than the result obtained in this study. This could be attributed to the higher initial DBT concentration of 120\u00a0ppm used in this study compared to the 1.84\u00a0ppm used by Alcon et al.38.Table Pseudomonas aeruginosa and Pseudomonas putida, respectively. It could be observed as well that 36% of 2-HBP was produced when Pseudomonas aeruginosa was used as biocatalyst and 33% of 2-HBP was formed for biodesulfurization of diesel by Pseudomonas putida. This result is low when compared to what was obtained in the biodesulfurization of model diesel. This could be because a lot of compounds are present in the real diesel which might have negatively affected the selectivity of DBT for biodegradation39.Results of biodesulfurization of real diesel samples obtained from a typical South African refinery are depicted in Fig.\u00a0Pseudomonas aeruginosa and Pseudomonas putida, with an initial DBT concentration of 5200\u00a0ppm. The results showed that about 36% and 33% desulfurization of diesel before HDS was achieved by Pseudomonas aeruginosa and Pseudomonas putida, respectively. After 8\u00a0h of biocatalyst activity at the resting stage, the diesel was shown to have reduced from its initial value (5200\u00a0ppm) to 3328\u00a0ppm for Pseudomonas aeruginosa and from 5200 to 3440\u00a0ppm for Pseudomonas putida. This low desulfurization performance could be attributed to the presence of other organosulfur compounds in the diesel (diesel sample obtained before HDS). In addition, high concentration of sulfur content could hinder the growth of bacteria (that is the concentration might be too toxic for the growth of the bacteria). This invariably affected the degradation efficiency of the DBT compound in the diesel. The formation of 2-HBP was also observed during the experiment as shown in Fig.\u00a0Pseudomonas aeruginosa and Pseudomonas putida, respectively39.Figure\u00a040.Figure\u00a017, desulfurized middle distillate diesel using cells Gordona CYKS1 for 10\u00a0h with an initial DBT concentration of 1500\u00a0ppm. The authors reported that the bacteria displayed a DBT degradation performance of 53%. The DBT biodegradation ability reported by the authors is higher than the DBT degradation ability of the Pseudomonas aeruginosa used in this study (about 30%). However, it should be noted that the initial concentration of 5200\u00a0ppm was used in this study as compared to 1500\u00a0pm used by Chang et al.17. The higher initial DBT concentration used in this study might have resulted in the lower DBT degradation performance of the bacteria as observed in this study. Higher DBT concentration could hamper the growth of the bacteria in the medium and subsequently result in lower consumption of DBT for growth. This speculation is evidently validated when a diesel sample obtained after HDS having initial DBT concentration of 120\u00a0ppm was used. With initial DBT concentration of 120\u00a0ppm, the biodegradation performance of the Pseudomonas aeruginosa was 70.54% and this result is higher than that of Chang and his co-authors17. Additionally, Chang et al.17 biodegraded the diesel sample for 10\u00a0h while 8\u00a0h was used in this study. In addition, their DBT in the middle distillate was degraded after 10\u00a0h, which is higher than 8\u00a0h, indicating that it could be possible that biodegradation performance of the Pseudomonas aeruginosa used in this study might be close to the value reported by Chang et al.17 if allowed for additional 2\u00a0h. Results reported in this article are comparable to the results of the investigation of Verma et al.41. The authors reported approximately DBT biodegradation performance of 88% with an isolate E1 bacteria when the initial DBT concentration was 368\u00a0ppm after 72\u00a0h biodegradation time. The shorter biodegradation time employed in this study (8\u00a0h) could have accounted for the lower biodegradation performance of Pseudomonas aeruginosa when compared to the results of Verma et al.41. Notwithstanding the disparities in the operating conditions of the reported literature when compared to the conditions employed in this study, results obtained in this study are comparable to the literature and could provide a platform for further research and development in this field. DBT metabolites such as DBTO, DBTO2 and HBPS were not detected in this study. Similarly, Silva et al.34 confirmed in their investigations that intermediate metabolites including DBTO, DBTO2, and HPBS were not discovered by GC\u2013MS analysis, which might be explained by the existence of another pathway for the breakdown of DBT. Therefore, further investigations are required to identify all the metabolites in the 4S pathway.Table Pseudomonas aeruginosa showed better BDS performance than Pseudomonas putida in all experiments. The results in this study showed that Pseudomonas aeruginosa and Pseudomonas putida can desulfurize DBT into less harmful compound, 2-HBP. However, further studies are required to determine their desulfurization efficiency for other sulfur organic compounds in real diesel.The Pseudomonas strains were successfully grown and utilized for desulfurization of synthetic and real diesel in this study. The final product, 2-HBP, detected shows that the specific activity of DBT desulfurization is 4S \u2013pathway. However, more investigations are still required in this field to detect all the various metabolites in the 4S pathway. In addition, the introduction of sufficient oxygen could improve the biodesulfurization performance of these bacteria cells.Pseudomonas aeruginosa and Pseudomonas putida could be better catalysts for the desulfurizing sulfur-containing compounds in South African diesel. The use of BDS has showcased in this study, in addition to HDS, could pave the way for the development of a hybrid process for the desulfurization of diesel. For future studies, a supply of oxygen into the bacteria medium may be required to enhance the growth of the bacteria, thereby enhancing the biodesulfurization efficiency. Furthermore, a thorough exploration into the understanding of the different microbial pathways that are involved in BDS may be required for the optimization and scale-up studies of the process.In a nutshell, the study has shown that"}
+{"text": "Bovine respiratory disease (BRD) has a significant impact on the health and welfare of dairy calves. It can result in increased antimicrobial usage, decreased growth rate and reduced future productivity. There is no gold standard antemortem diagnostic test for BRD in calves and no estimates of the prevalence of respiratory disease in seasonal calving dairy herds.To estimate BRD prevalence in seasonal calving dairy herds in Ireland, 40 dairy farms were recruited and each farm was visited once during one of two calving seasons (spring 2020 & spring 2021). At that visit the prevalence of BRD in 20 calves between 4 and 6 weeks of age was determined using thoracic ultrasound score (\u22653) and the Wisconsin respiratory scoring system (\u22655). Hierarchical Bayesian latent class analysis was used to estimate the calf-level true prevalence of BRD, and the within-herd prevalence distribution, accounting for the imperfect nature of both diagnostic tests.In total, 787 calves were examined, of which 58 (7.4%) had BRD as defined by a Wisconsin respiratory score \u22655 only, 37 (4.7%) had BRD as defined by a thoracic ultrasound score of \u22653 only and 14 (1.8%) calves had BRD based on both thoracic ultrasound and clinical scoring. The primary model assumed both tests were independent and used informed priors for test characteristics. Using this model the true prevalence of BRD was estimated as 4%, 95% Bayesian credible interval (BCI) . This prevalence estimate is lower or similar to those found in other dairy production systems. Median within herd prevalence varied from 0 to 22%. The prevalence estimate was not sensitive to whether the model was constructed with the tests considered conditionally dependent or independent. When the case definition for thoracic ultrasound was changed to a score \u22652, the prevalence estimate increased to 15% .The prevalence of calf respiratory disease, however defined, was low, but highly variable, in these seasonal calving dairy herds. Bovine respiratory disease (BRD) is one of the major challenges associated with rearing dairy calves internationally \u20133. BRD cEstablishing the prevalence of BRD is challenging as currently there is no gold standard ante mortem test for diagnosis of BRD . BayesiaTraditionally BRD was diagnosed by veterinarians using thoracic auscultation and clinical examination. It has been shown auscultation has moderate sensitivity (Se)  Se; 0.63\u20130.72) , 13 and \u20130.72 , 1Thoracic ultrasound (TUS) is a technique that has grown in popularity in recent years in research settings \u201320. It hA commonly used clinical scoring system is the Wisconsin respiratory scoring system , 25\u201329. Accepting these diagnostic limitations, numerous studies internationally have reported the calf-level apparent prevalence of BRD in dairy calves. Dubrovsky et al.  estimateSpring calving dairy herds are managed to synchronize the highest demand for feed (peak lactation) with the highest availability of grazed grass . This prThere are no studies that estimate the prevalence of BRD in seasonal systems and there are no studies that estimate the true prevalence of BRD when taking into account the lack of a gold standard test. Hence the aim of this study was to estimate the calf- and farm- level true prevalence of BRD using two imperfect diagnostic tests and a hierarchical Bayesian latent class model.This study was approved by University College Dublin, Animal Research Ethics Committee and the Health Products Regulatory Authority (V016/2020Q1).via a letter that had been sent to randomly selected spring calving dairy farmers in the Republic of Ireland that were in the HerdPlus database of the Irish Cattle Breeding Federation (ICBF). The dairy farmers who home-reared their heifers and enrolled in that study (n = 56) were sent a letter describing the current research project and asked if they were interested to contact a member of the research team. No geographical limit was put on recruitment, In total, 40 farms were recruited.Herds were recruited to this study using a database previously used in a national study of contract-reared vs. home-reared heifers . The conEach farm was visited once during either spring 2020 or spring 2021; the sampling period had to be split over 2 years due to the COVID 19 pandemic. In 2020 (February and March), 28 farms were visited and 547 calves were examined. In 2021 (February and March), 12 farms were visited and 240 calves were examined. In Ireland spring is considered to start in March 1; the maThe Wisconsin respiratory scoring system was used as described by McGuirk and Peek . The CRSth intercostal space on the left-hand side and the probe was moved in a dorso-ventral direction down each intercostal space to the 2nd on the left hand side. On the right hand side a similar technique was used but to allow for imaging of the most cranial portion of the lung lobe, the right fore-limb was drawn cranially by an assistant to allow ease of imaging of the 1st and 2nd intercostal space.All of the TUS was performed by a single operator (first author) using a portable linear rectal ultrasound scanner set at a depth of 7 cm and frequency of 7 MHz . A 70% isopropyl alcohol solution was sprayed onto the unclipped hair on both sides of the thorax. The technique described by Olivett and Buczinski  was usedThe entire lung field was examined and assigned a score from 0 to 5 as previously described by Ollivett and Buczinski : 0 = norThe BRD syndrome was classified into three separate presentations as defined by Ollivett and Buczinski  using thhttps://www.r-project.org/) for further data processing and statistical analysis. All data manipulation, visual presentation and statistical analysis was conducted using \u201ctidyverse\u201d  was modeled as a product of herd-level prevalence (hj) and the \u2018conditional' within-herd prevalence (\u03c8j). The herd-level prevalence was modeled using a Bernoulli distribution with mean \u03bc representing the proportion of herds that were disease free. The conditional within-herd prevalence was the within-herd true prevalence in affected herds only and was modeled using an intercept only random effect logistic regression  were obtained using JAGS called from R statistical software using the \u201crjags\u201d package (n = 2) chains from dispersed starting values . Initially autocorrelation was observed for some sample parameters, therefore chains were thinned by 10 for inference.Posterior inferences for each parameter , this was calculated using Excel (Microsoft). A gamma distribution of  was used as the prior for tau. The gamma distribution equates to the variance of the logit of normal distribution and considering our priors used, this allowed for the within-herd prevalence to vary from 0 to 100%.As part of the sensitivity analysis, several measures were undertaken: the final model was checked by changing the prior information of both TUS and clinical scoring Se and Sp to non-informative beta  distributions while retaining prior distributions for alpha, tau and mu. A case definition of TUS \u22653 was used for the primary models while a case definition of TUS \u22652 was used for subsequent models. A gamma distribution of  was used in the primary model with it being varied to  and . Finally, the analysis was also conducted assuming conditional dependence between detection methods. Assuming tests are conditionally independent implies that the association between TUS and CRS results are accounted for only by the latent class and no other variable. In contrast, assuming test dependency implies that test outcomes are influenced by other latent variables, other than the latent class of concern, that are common to both tests (TUS and CRS). In this case, dependency between diagnostic tests was modeled as described by Dendukuri and Joseph :The covariance between the two tests were estimated below:A uniform distribution was selected between these 2 bounds assuming an equivalent probability throughout this the range of scores.n = 29, 72.5%) with county Cork having the majority of farms within Munster (n = 15).The 40 dairy farms recruited all had spring calving herds ranging from 70 to 480 cows, with a median of 145. n = 38, 95%) with positive pressure ventilation in use on one farm (2.5%) and negative pressure ventilation in use on one farm (2.5%). Straw bedding was used in the majority of farms , one farm used rushes (2.5%), one farm used woodchip (2.5%). All calves were housed in group pens with group sizes varying between 5 and 45 calves. The median pen size in this work was 18 calves. The median number of pens that a calf moves through before weaning was 2 with a range of 1 to 4.All farms housed preweaned calves indoors, natural ventilation was utilized in the majority of farms (Eighteen farms (45.0%) fed colostrum from only the calves dam, 11 farms (27.5%) fed pooled colostrum without any exclusion criteria, 2 farms (5.0%) used pooled colostrum from selected cows. The remaining farms did not have clear colostrum management policies in place. The median volume of colostrum fed was 3 L. Fifteen farms fed only milk replacer (37.5%), 10 farms fed only whole saleable milk (25.0%), the remaining farms fed some combination of milk replacer and whole saleable milk, of those, 6 (15.0%) indicated that they fed some amount of non-saleable milk. Twenty-eight farms (70.0%) used teat feeders for feeding, 10 (25.0%) of farms used automatic milk feeders and two farms (5.0%) fed calves using buckets.n = 320, 40.7%) followed by Holstein x Friesian x Jersey , all other dairy breeds made up 26.7% (n = 210) and dairy beef crosses accounted for 11.7% (n = 92). The median calf body weight was 59 kg (range 31\u2013108 kg). Calves were not vaccinated against BRD on 34 farms, were vaccinated on 3 farms and on 3 farms the calves had unclear vaccination status as can be seen in In total, 787 calves were examined. Twenty calves were sampled on each farm with the exception of two farms where 6 and 21 calves were sampled. The median age of the calves was 34 days (range 10\u201358 days). The majority of calves examined were female  and 137 (17.4%) male with one calf's sex unrecorded. Holstein Friesian was the most common calf breed . The median TUS score of all calves was 1 (range 0\u20135). In 2020 thirty six of the 547 (6.6 %) calves had a TUS score \u22653 and fifty three of the 547 (9.7%) had CRS \u2265 5. In 2021 fifteen of the 240 (6.3%) calves had a TUS score \u22653 and nineteen of the 240 (7.9%) had a CRS \u2265 5.Upper respiratory tract disease was the most commonly identified type of BRD  in 58 calves, . Subclinical pneumonia  was identified in 37 calves (34.0% of BRD cases). Clinical pneumonia  was identified in 14 calves (12.8% of BRD cases). Convergence was assessed visually using autocorrelation trace plots, initially some auto correlation was detected so the model was thinned by 10. The PSRF values for all monitored variables were < 1.0038 indicating adequate convergence. The effective sample size for each variable ranged from 7,119 to 20,324.All the results of the final model, as well as the various other models, can be found in In the final model, the median herd prevalence within affected herds was 10% , the median prevalence within affected herds varied from 3% to 22% in models using TUS score \u22653 as case definition.In the final model the within-herd prevalence varied from 0% to 22%, the median within herd prevalence was 0% . The use of uninformed priors for the Se and Sp of each test resulted in an increase in the prevalence in all modeled scenarios, bar one. The change varied from a decrease of 2% prevalence to a maximum increase of 10% of median prevalence. However the 95% BCI in these scenarios remained relatively similar to that of the primary model [95% BCI ] with the 95% BCI increasing in width the most when uninformative priors were used for TUS [95% BCI ].Altering gamma from  to  resulted in an increase in prevalence from 15% [95% BCI ] to 18% [95% BCI ] in the model using TUS \u22652 as a cut off. In the primary model there was no change in the median prevalence estimate but a minor increase in the 95% BCI going from  to .Reducing the TUS score cut off from \u22653 to \u22652 as the case definition resulted in a 11% increase in the median prevalence of BRD in the models using independent tests and informed priors. It also resulted in the 95% BCI becoming wider increasing from  to .Test dependency had little effect on either of the primary models with a 1% decrease in conditionally dependent models using TUS \u22652 as the cut off, and a 1% increase in the conditionally dependent model using TUS \u22653 as the cut off. Test dependency had resulted in minor variation in 95% BCI.To the authors' knowledge, this is the first estimate of calf-level prevalence of BRD in in seasonal calving herds using hierarchical Bayesian latent class techniques. Bayesian latent class techniques have been used previously in the literature to estimate Se and Sp of TUS and CRS \u201325, thisAt the within-herd level the prevalence varied between 0% and 22%. Eleven of the farms visited were negative for BRD on TUS and CRS. This indicates that a low prevalence of BRD is achievable in commercial spring calving herds. Some herds did have a higher prevalence of BRD and a more detailed investigation of the management and environmental factors that influence the prevalence of BRD on those farms will follow on from this work.The farms used as part of this work were randomly recruited throughout Ireland, which should be considered as a strength of this work. The farms visited were seasonally calving and group-reared calves indoors, reflecting typical calf management in Ireland. The majority of farms were located in Munster, this was a reflection of the demographics of dairy farms in Ireland 2. The avThere was a 9% prevalence of CRS cases as defined by a score \u22655 or two scores \u22652 . This isIn this work 6.5% of calves had a TUS score \u22653 and 17.7% had a TUS \u22652. This level of lung consolidation is similar to other work where Buczinski et al.  found a Consistency in definition of TUS lesions has not been reached within the literature. Some authors, such as Rhodes et al. , have usvia veterinary practitioners this may bias the estimate higher, if recruitment uses farms with a pre-existing relationship with a university/research organization they may have better management that might bias for a lower BRD prevalence estimate. This makes it difficult to determine whether the prevalence of BRD as determined by CRS/TUS here is lower/similar to other studies with different farming systems. Based on the results from this study it appears that the broad differences in Irish management systems  does not have a negative impact or may even have a slightly positive impact on BRD prevalence. It is more likely that the individual farm management/facilities within a husbandry system impact BRD prevalence more than the over arching husbandry system itself.Caution must be taken when comparing the prevalence estimates from this work with others, as the method of farm recruitment is not consistent with several studies using convenience samples , 27\u201329. The data gathered here uniquely allowed assignment of BRD cases to one of three different subtypes. Upper respiratory tract disease (URTD) , lower respiratory tract disease (LTRD)  and clinical respiratory disease (CRD) . The breakdown of URTD, LRTD, and CRD is likely to be a reflection of the interaction between housing environment, calf immunity and pathogens present on a given farm. When the distribution of the different subtypes from this work was examined two major points emerged. Firstly, a substantial proportion of calves with pulmonary lesions, that may reduce calf performance, are likely to go undiagnosed if only examined for clinical signs using CRS. Of the fifty one calves that were identified with a TUS \u22653, only 14 (19.4%) were identified as cases to be treated using CRS. This means that the majority of calves with pulmonary lesions likely went untreated and so may have had reduced performance. Secondly, when TUS \u22653 was used as the case definition the majority of cases of BRD were classified as URTD. URTD cases were defined as calves that had CRS scores \u22655 or two scores \u22652 but did not have significant lung consolidation. This may have implications for antimicrobial usage as it is not clear currently whether it is of benefit to animals to treat URTD. Future work should investigate response to treatment, prognosis of different BRD subtypes and defining calves with active and non-active BRD.The epidemiology of the different BRD subtypes has implications for the use of both TUS as a screening method to assess farmer diagnostic Se and clinical scoring as a method by which to choose treatments. However, this was outside of the scope of this work. In the future integration of tools such as activity monitors and automatic feeders may allow for earlier detection of cases and detection of LRTD cases that may have otherwise have gone untreated .In previous publications TUS and CRS have been considered both independent and conditionally dependent , 25 whenMycoplasma bovis. In this case we chose to vary the TUS positive definition because there is still some uncertainty in the literature around the positivity threshold. Cramer and Ollivett  of BRD is impacted by environmental factors, the prevalence distributions might have been different across both years. To investigate, we repeated the analysis by modeling the prevalence distribution in each year separately. However, we found no difference in the median estimates across years and therefore decided to maintain the simpler model see . SecondlWe estimated the BRD prevalence to be 4% using a Bayesian approach to account for the lack of a gold standard antemortem test. The prevalence of BRD was lower or similar to that seen in other systems and regions. In future work investigating BRD prevalence, a Bayesian approach using two imperfect tests should be taken to allow for comparison of prevalence between different calf rearing systems. In addition, we found variation in the prevalence of different BRD subtypes with upper respiratory tract disease appearing to be more common. Future work should investigate the epidemiology of these different disease subtypes and what farm and pathogen-level risk factors influence them as well as treatment protocols.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Animal Research Ethics Committee University College Dublin. Written informed consent was obtained from the owners for the participation of their animals in this study.CM and JM contributed to conception and design of the study. JD collected and managed the data and wrote the first draft of the manuscript. JD and CM performed the statistical analysis. JM, CM, and JD wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version."
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