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+{"text": "While the availability of multiple high-efficiency SSRs would facilitate a wide array of genomic engineering possibilities, efficient recombination in mammalian cells has only been observed with Cre recombinase. Here we report the  Upon binding to their target recognition sequences, SSRs can induce the deletion, insertion, or inversion of DNA sequences leading to conditional gene inactivation or expression in vitro and in vivo. A second SSR from the \u03bb integrase family, FLP from Saccharomyces cerevisiae, has also been used in mammals Streptomyces lividans, also displays activity in mammalian cells de novo synthesis of mouse codon-optimized FLP (FLPo) and \u03a6C31 (\u03a6C31o) SSRs result in DNA recombination efficiencies similar to that of Cre.The use of site-specific recombinases (SSRs) both de novo according to the native amino acid sequence but with mouse codon usage (http://www.geneart.com/). During the optimization process a number of sequence motifs were avoided, including internal TATA-boxes, ribosomal entry sites, stretches of AT- and GC-rich sequence, repeat sequences, RNA secondary structure, and cryptic splice and polyadenylation sites .The use of multiple, highly efficient SSRs in ES cells and mice now opens possibilities for a broader range of molecular manipulations. These not only include the potential for additional genetic alterations during ES cell expansion  and performing multiple general or tissue-specific gene knockouts, but also for studying gene function in over-lapping domains of expression. Additionally, the use of multiple SSRs could potentially be used for controlling gene expression in a temporal \u201coff-on-off\u201d manner in single or multiple cell types or tissues. The optimized \u03a6C31o and FLPo expression constructs will be made available to academic researchers through the Addgene plasmid repository  based on the published FLPe and \u03a6C31 coding sequence The coding sequence of FLPo and \u03a6C31o was commercially synthesized in vivo. Similarly, the \u03a6C31o and \u03a6C31 coding sequences were blunt cloned into the pROSA26-1 vector to generate mouse strains that broadly express this SSR.ES cells harboring a Cre-inducible \u03b2-galactosidase or FLP-inducible PLAP reporter gene integrated at the broadly-expressed ROSA26 locus were used as previously described to monitor Cre or FLP activity 20 \u00b5g of each linearized SSR expression construct was co-electroporated in a 10\u22361 molar ratio with PGKHygromycin into corresponding ES reporter cell lines for Cre Alkaline phosphatase (FLP) and \u03b2-galactosidase (Cre and \u03c6C31) activity in ES cells and embryos was visualized as previously described Figure S1Nucleotide sequence of FLPo. A mouse codon-optimized FLP gene containing an N-terminal SV40 nuclear localization signal was generated de novo  according to previously described FLPe amino acid sequence (0.03 MB DOC)Click here for additional data file.Figure S2Nucleotide sequence of \u03a6C31o. A mouse codon-optimized \u03a6C31 gene with C-terminal SV40 nuclear localization signal was synthesized de novo according to the native \u03a6C31 amino acid sequence .(0.04 MB DOC)Click here for additional data file.Figure S3Establishment of ROSAattR reporter line. Diagram of \u03d5C31 reporter knock-in vector targeted to the ROSA26 locus. The stop cassette is flanked by 35 bp attB and 39 bp attP sites, as previously described (0.06 MB TIF)Click here for additional data file."}
+{"text": "TTDA/hTFB5 gene. One of these mutations completely inactivates the protein, whereas other TFIIH genes only tolerate point mutations that do not compromise the essential role in transcription. Nevertheless, the severe NER-deficiency in TTD-A suggests that the TTDA protein is critical for repair. Using a fluorescently tagged and biologically active version of TTDA, we have investigated the involvement of TTDA in repair and transcription in living cells. Under non-challenging conditions, TTDA is present in two distinct kinetic pools: one bound to TFIIH, and a free fraction that shuttles between the cytoplasm and nucleus. After induction of NER-specific DNA lesions, the equilibrium between these two pools dramatically shifts towards a more stable association of TTDA to TFIIH. Modulating transcriptional activity in cells did not induce a similar shift in this equilibrium. Surprisingly, DNA conformations that only provoke an abortive-type of NER reaction do not result into a more stable incorporation of TTDA into TFIIH. These findings identify TTDA as the first TFIIH subunit with a primarily NER-dedicated role in vivo and indicate that its interaction with TFIIH reflects productive NER.Transcription/repair factor IIH (TFIIH) is essential for RNA polymerase II transcription and nucleotide excision repair (NER). This multi-subunit complex consists of ten polypeptides, including the recently identified small 8-kDa trichothiodystrophy group A (TTDA)/ hTFB5 protein. Patients belonging to the rare neurodevelopmental repair syndrome TTD-A carry inactivating mutations in the Transcription/repair factor IIH (TFIIH) is a multi-subunit protein complex essential for RNA polymerase II transcription and nucleotide excision repair (NER). The authors show that the TTDA subunit is associated with TFIIH specifically during NER. DNA-damaging agents constantly challenge the integrity of DNA. A network of distinct DNA-repair systems collectively removes most injuries and safeguards the stability of the genome . NucleotBesides GG-NER and TC-NER, TFIIH is also engaged in RNA polymerase II transcription initiation, RNA polymerase I transcription, activated transcription, and cell cycle regulation \u201320. TFITTDA gene [TTDA gene of three non-related patients with TTD-A, lead either to the complete absence of the protein (mutation on the first ATG), or to non-functional truncated peptides, making TTDA the first TFIIH subunit for which a complete absence is compatible with life [Cells from patients with TTD-A have reduced steady-state levels of TFIIH, due to mutatedDA gene , suggestth life . DespiteIn order to investigate the participation of TTDA in DNA repair and transcription, we measured the differences in mobility of TTDA during repair and transcription in living cells by confocal imaging of a functional green fluorescent protein (GFP)-tagged TTDA (TTDA-GFP) expressed in TTDA-deficient transformed fibroblasts. To compare the mobility of TTDA with others TFIIH subunits, we also measured kinetic parameters of XPB-GFP and XPD-GFP in each of these processes.To investigate the localization, behavior and dynamic properties of TTDA in the most relevant biologically context\u2014the living cell, and under different conditions, such as transcriptional interference and introduction of DNA lesions, we have generated a GFP-tagged TTD A . To compImmunoprecipitation assays showed that the TFIIH core factor, XPB, co-precipitates with both fusion proteins, using anti-GFP cross-linked sepharose beads and extracts of TTDA-GFP D. In addHigh-resolution confocal imaging  of TTDA-The cytoplasmic localization of both XPD-GFP and TTDA-GFP argues for a possible assembly of TFIIH-related subcomplexes within the cytoplasm. In order to investigate this, we measured the mobility of TTDA-GFP, XPB-GFP, XPD-GFP, and GFP in both the cytoplasm and the nucleus by photo-bleaching experiments. We applied a tailor-made variant of fluorescence recovery after photo-bleaching (FRAP)  in whichWithin the cytoplasm A, the moIn order to eliminate the fluorescence of this non-incorporated fraction, we modified the FRAP procedure by pre-bleaching the cytoplasmic fraction see. AssuminTo investigate whether the presence of DNA damage influences the equilibrium between the various TTDA-GFP pools, we compared the dynamic behavior of TTDA-GFP before and after UV irradiation (measured between 5 to 30 min after exposure) . FRAP_abWe further explored the participation of the three TFIIH components in NER by determining the binding time of each of the components when actively engaged in NER. To that aim, we applied FRAP on locally UV-damaged cells C, as wasThis tighter association of TTDA-GFP to TFIIH during repair can be explained in two ways: TTDA is more tightly anchored to TFIIH when  bound to DNA or TTDA is indeed a NER-specific subunit of TFIIH docking with higher affinity to the complex when actively repairing. A standard not NER-related DNA interaction of TFIIH is of course its transient interaction with promoter sequences during transcription initiation. Varying the transcriptional competence of the cells would thus also affect the equilibrium of TFIIH-bound versus free TTDA. To verify this hypothesis, we measure the mobility of TTDA-GFP during inhibition of transcription by DRB , known to increase overall mobility of XPB-GFP by the reduced number of interactions with transcription initiation complexes . As showNext we tested the effect of actinomycin D (ActD), classically used as a transcription elongation inhibitor but also known to interfere with other DNA transactions \u201335. In TFIIH is implicated in a multitude of DNA-transactions, ranging from basal and activated RNA polymerase II transcription, to ribosomal RNA gene expression, participation in GG-NER and TC-NER, and possibly cell-cycle regulation. The complexity of this factor is also reflected by the dynamic composition of distinct subcomplexes, as well as its multiple enzymatic activities: DNA-dependent ATPases and helicases, kinase, and ubiquitin ligase. Participation in multiple reactions complicates the analysis of specific functions when studied in standard in vitro assays. Dynamic regulation of the activity in either one or another process and the existence of different kinetic pools usually escape scrutiny by test tube analysis. Moreover, thermodynamic parameters within mammalian cell nuclei, critical for regulating activity , cannot be fully mimicked in vitro. Recent developments in cell biology have opened new opportunities for studying complex processes in the most relevant context, i.e. the living cell.Prior to in depth live cell analysis of protein dynamics, the biological activity should be carefully checked. In this study we thoroughly analyzed the function of GFP-tagged TTDA. The most pertinent cellular phenotype of TTDA-insufficiency is the NER defect. Remarkably, despite the 4-fold larger size of the tag compared to TTDA itself and the fact that it has to fit in a complex with nine other subunits, that should have numerous interaction partners, TTDA-GFP appeared normally incorporated into TFIIH C, able tIn our live cell studies we have shown that TTDA-GFP and XPD-GFP reside both in the cytoplasm and in the nucleus, contrasting to the strictly nuclear localization of XPB. Photo-bleaching experiments indicated that both proteins do not interact in the cytoplasm. However, application of a newly developed variant of FRAP, FRAP_abc, revealed that TTDA and XPD do interact with TFIIH in the nucleus. In striking contrast to XPB, the association of TTDA to TFIIH  is much more dynamic. Previously we found by subcellular fractionation (unpublished data) and others found, by GFP-tagging , that enBoth TTDA-GFP and XPD-GFP accumulate in nucleolar foci (previously observed in fixed TTDA-HA-expressing fibroblasts ). NucleoReduced steady-state levels of TFIIH in TTD-A cultured fibroblasts appeared to be a critical determinant in NER efficiency , but thiThe in vitro NER studies  showed tThe complete absence of TTDA in patients, as deduced from the mutation abolishing the start codon, shows that cell viability is not critically depending on TTDA and on high amounts of TFIIH. This is in striking contrast with deletion mutants of each of the previously known TFIIH subunits, which are not viable in yeast and mammals. Incompatibility with live associated with deletions of TFIIH encoding genes was explained by the vital transcriptional role of TFIIH . Extrapo6 normal nucleotides) cannot be accomplished in one step [TTDA can be considered as integral component of TFIIH in biochemical terms  with 2-d intervals. Next, from these mass populations, stably expressing GFP-fusion protein clones were isolated after single cell sorting using the FACSVantage . Single-cell sorted clones were further analyzed for GFP expression by microscopic evaluation and immunoblot analysis.Stably expressing cells were isolated after subsequent rounds of selection; first Neomycin-dominant marker selection using G418, followed by selection on UV-resistance by exposing them three times to a dose of 6 J/m2 and 5% CO2.Cell strains used were SV40-immortalized human fibroblasts: TTD1BR-Sv (TTD-A), with and without stably expressing TTDA-GFP; XPCS2B-Sv (XP-B) stably expressing XPB-GFP ; XP6BE-S2 was used for total irradiation, and 40 J/m2 for local irradiation, through a micro-porous filter [Treatment with UV-C was performed using a Philips germicidal lamp. A dose of 8 J/m filter . Transcr3H-thymidine, as described previously [For UV-survival experiments, cells were exposed to different UV-C doses, 2 d after plating. Survival was determined 3 d after UV irradiation by incubation at 37 \u00b0C withviously .We prepared whole-cell extracts by isolating cells from six petri dishes (14 cm) per cell line. Cells were washed with phosphate-buffered saline (PBS) before lysing them by douncing  in 2 ml of buffer A , supplemented with anti-proteases. Cellular extracts were incubated overnight at 4 \u00b0C in buffer A with Rabbit polyclonal antibodies to GFP  and mouse monoclonal p44 (1H5), cross-linked to protein A-sepharose beads . Before immunoprecipitations, cross-linked beads were washed three times with buffer A. After incubation with the extracts, the cross-linked beads were washed extensively with buffer A. Subsequently, beads were boiled in Laemli SDS-PAGE sample buffer, separated on 11% SDS-PAGE, blotted onto nitrocellulose and analyzed using the following antibodies: anti-XPB (1B3) and monoclonal anti-GFP (Roche).To analyze expression levels of the fusion proteins we prepared whole-cell extracts by sonication. These were separated on an 11% SDS-PAGE and transferred to 0.45 \u03bcm nitrocellulose membranes (Millipore). Expression of the fusion proteins was analyzed by hybridizing the membranes with a monoclonal anti-GFP (Roche), followed by a secondary antibody (rabbit anti-mouse) conjugated with horseradish peroxidase  and detected using enhanced chemo-luminescence (ECL+ Detection Kit) (Amersham).+ (PBS containing 0.15 % glycine and 0.5 % BSA). Cells were incubated at room temperature with primary antibodies  for 2 h in a moist chamber. Subsequently, coverslips were washed three times with PBS/TritonX-100 and PBS+, incubated 1 h with secondary antibodies,  at room temperature and again washed three times in PBS/TritonX-100. Samples were embedded in Vectashield mounting medium  containing 0.1 mg/ml DAPI (4\u2032-6-diamino-2-phenylindole). Images were obtained by confocal laser scanning microscopy imaging, carried out with a LSM 510 microscope .Cells were grown on glass coverslips and fixed with 2% paraformaldehyde at 37 \u00b0C. Coverslips were washed with PBS containing 0.1% Triton X-100, three times for 5 min, and subsequently washed with PBS3 d prior to microscopy experiments, cells were seeded onto 24-mm-diameter coverslips. Imaging and FRAP were performed on a Zeiss LSM 510 meta confocal laser scanning microscope .FRAP analysis was performed at high time resolution . Briefly. After local irradiation of TTDA-GFP, XPB-GFP, and XPD-GFP fibroblasts with UV-C light, locally damaged areas were photo-bleached during 3 s with 100% laser intensity. Monitoring of the fluorescence recovery was followed by imaging the cell every 5 s for 200 s. A single image was taken prior to the photo-bleaching. Results are expressed by the ratio between damaged area and undamaged area. The first data point after photo-bleaching is set to 0, while final recovery is normalized to 1. Error bars, in the inset of the curves, represent the standard error of the mean. Statistical significance was checked, whenever two distinct curves were not easily dissociable, by using Student'st-test  within an appropriate time window: right after the photo-bleaching when evaluating mobility differences or after complete recovery when immobile fractions are being compared.To determine residence times of the different TFIIH components at locally damaged areas, we applied FRAP on local damageFigure S1Mobility of TTDA-GFP in the cytoplasm after bleaching the nuclear pool (red line) compared to the mobility of TTDA-GFP in the cytoplasm without bleaching the nuclear pool (green line). The relative fluorescence (fluorescence post-bleach divided by fluorescence pre-bleach) is plotted against time in seconds.(193 KB PPT)Click here for additional data file.Figure S2(A) Scheme of the plasmid used to transfect TTD-A-GFP expressing cells. The construct expresses small-interference RNA against XPC (sequence available upon request), under the control of an H1 (RNA polymerase III) promoter. The plasmid also contains a Mito DsRED_IRES_Hygro transcriptional unit, allowing the selection of transfected clones by the Hygromycin resistance and the mitochondrial DsRED signal.(B) Example of a TTDA-GFP expressing cell transfected with a mixture of two different XPC small-interference RNA-expressing constructs.(C) Strip-FRAP_abc of UV-irradiated TTDA-GFP expressing cells knocked-down for XPC (green curve), compared with untreated TTDA-GFP expressing cells (blue curve) and UV-treated TTDA-GFP expressing cells (red curve). Relative fluorescence (fluorescence post-bleach divided by fluorescence pre-bleach) plotted against time in seconds.(557 KB PPT)Click here for additional data file.Figure S3FRAP curve of TTDA-GFP expressing cells untreated (green line) and treated see with Act(48 KB PDF)Click here for additional data file."}
+{"text": "The development of non-small cell lung carcinoma proceeds through a series of well-defined pathological steps before the appearance of invasive lung carcinoma. The molecular changes that correspond with pathology changes are not well defined and identification of the molecular events may provide clues on the progression of intraepithelial neoplasia in the lung, as well as suggest potential targets for chemoprevention. The acquisition of anti-apoptotic signals is critical for the survival of cancer cells but the pathways involved are incompletely characterized in developing intra-epithelial neoplasia (IEN).We used immunohistochemistry to determine the presence, relative levels, and localization of proteins that mediate anti-apoptotic pathways in developing human bronchial neoplasia.Bronchial epithelial protein levels of the phosphorylated (active) form of AKT kinase and the caspase inhibitor cIAP-2 were increased in more advanced grades of bronchial IEN lesions than in normal bronchial epithelium. Additionally, the percentage of biopsies with nuclear localization of p65/RELA in epithelial cells increased with advancing pathology grade, suggesting that NF-\u03baB transcriptional activity was induced more frequently in advanced IEN lesions.Our results indicate that anti-apoptotic pathways are elevated in bronchial IEN lesions prior to the onset of invasive carcinoma and that targeting these pathways therapeutically may offer promise in prevention of non-small cell lung carcinoma. Lung cancer is the leading cause of cancer mortality in both men and women in the United States . Non-smaEvasion of apoptosis by tumor cells is a critical step during tumorigenesis. The serine/threonine kinase Akt is a critical mediator of anti-apoptotic signaling in eukaryotic cells and is activated in a signaling cascade downstream of Ras activation and phosphoinositide-3-kinase (PI3K) . AmplifiThe NF-\u03baB transcription factor family can stimulate both pro- and anti-apoptotic signals. Many studies have described a critical role for NF-\u03baB activity in promoting cell survival. Increased staining for NF-\u03baB subunits has been detected in breast  and cervL [Several NF-\u03baB-regulated genes that function in control of apoptosis have been described including cIAP-1, cIAP-2, A1/Bfl1, Traf1, Traf2 and Bcl-XL -33. cIAPL  and can L . ElevateL -38 and eL ,39, implWhile the expression of pro-survival genes has been examined in some detail in lung tumors, relatively few studies have examined the expression of these proteins in pre-neoplastic lesions. To determine the presence and abundance of pro-survival proteins in developing lung neoplasia, we examined human bronchial biopsies of various grades  for the presence of phospho-Akt, p65/RELA and cIAP-2/BIRC3 and determined their localization within cells. The results obtained provide new insight into the distribution of pro-survival genes in human lung neoplasia and pre-neoplasia. This information may be useful in designing studies to test the importance of these pathways experimentally and ultimately may lead to improved diagnostic or therapeutic strategies for human lung cancer.Bronchial biopsies were obtained during autofluorescence  bronchosRabbit polyclonal antibodies for phosphorylated  and total Akt  were purchased from Cell Signaling Technologies  and were both used at 1:100 dilution. The p65/RELA antibody  was purchased from Abcam America  and used at a 1:4000 dilution. The cIAP-2/BIRC3 antibody  was from R&D Systems  and used at a 1:500 dilution. Biotinylated goat anti-rabbit secondary antibody and normal goat serum was from Vector Laboratories .Paraffin embedded sections were deparaffinized through xylene and a graded series of ethanols. Endogenous peroxidase activity was quenched by incubation in 2% hydrogen peroxide in methanol for 15 minutes then cleared in PBS for 5 minutes. High temperature antigen unmasking in citrate buffer was used for all antibodies and carried out as described previously . Non-speBiopsies received a numerical score of 0 for negative staining, 1 for predominantly faint staining, 2 for predominantly moderate staining or 3 for predominantly strong staining. For the purpose of this study, the bronchial biopsies were divided into 5 categories for phosphorylated Akt and cIAP-2/BIRC3 analysis: normal, hyperplasia, mild dysplasia or moderate dysplasia, severe dysplasia or carcinoma in situ and carcinoma. For analysis of p65/RELA nuclear translocation, the mild and moderate dysplasia groups were analyzed separately as there was a large difference in the percentage of nuclear staining between these two groups. No significant difference in staining intensity between mild and moderate dysplasia was seen for phosphorylated Akt or cIAP-2/BIRC3. Statistical analysis was performed using SigmaStat software . Analysis of variance for each stain was calculated using a Kruskal-Wallis chi-squared test. Pairwise comparisons were calculated using a non-parametric Mann-Whitney rank-sum test. A p-value < 0.05 was considered significant.The serine-threonine kinase Akt is a central regulator of cell survival and apoptosis that is itself activated by phosphorylation. Antibodies that specifically recognize the phosphorylated form (Ser 473) were used to identify the phosphorylated, and thus activated, form of Akt. Intensity of staining was scored on a scale of 0 for negative, 1 for predominantly faint staining, 2 for predominantly moderate staining and 3 for predominantly strong staining. In normal bronchial epithelium, phospho-Akt staining typically consisted of faint cytoplasmic stain with occasional cells staining with a more intense nuclear pattern Figure . In someThe transcription factor NF-\u03baB is activated following Akt phosphorylation via several mechanisms and is in itself known to play a role in cell survival and protection from apoptosis. We thus characterized the immunohistochemical staining pattern of the NF-\u03baB subunit p65/RELA in pre-neoplastic bronchial biopsies. NF-\u03baB subunits are normally held in the cytoplasm, thus preventing them from activating transcription, and are translocated to the nucleus following various activation signals such as oxidative stress or growth factor/cytokine stimulation. We therefore determined the staining pattern of p65/RELA in the cytoplasmic or nuclear compartment of airway epithelial cells. Faint to moderate cytoplasmic staining for p65 was present in the cytoplasm of epithelial cells in normal biopsies with little staining in the underlying stroma Figure  and 4B. The inhibitor of apoptosis protein (IAP) family function to block apoptosis by caspase-dependent and caspase-independent means . SeveralWe compared staining intensity of phospho-Akt and cIAP-2 in biopsies where both stains were performed on serial sections (n = 53). There was a significant correlation between phospho-Akt and cIAP-2 staining intensities in these biopsies . This correlation was seen at all pathology grades.The cIAP-2 gene is a transcriptional target for NF\u03baB activation . To examWe also examined if there was a correlation between tumor stage and staining intensity or nuclear localization of p65/RELA in carcinomas. No correlation was seen between tumor stage and any of the stains examined.The pathways utilized by lung tumors to evade apoptosis, allowing the tumors continued survival and growth, are incompletely characterized. Equally important are the pathways that become activated in IEN lesions before they become invasive cancer. In this study, we examined the presence and cellular localization of the phosphorylated form of Akt kinase, the transcription factor p65/RELA and the cellular inhibitor of apoptosis protein cIAP-2/BIRC3 in human bronchial IEN lesions. In normal human bronchial epithelium, staining for phosphorylated Akt was present as a faint cytoplasmic stain. Staining intensity increased with increasing pathology grade and was present in a nuclear, perinuclear or plasma membrane pattern. The intensity of p65/RELA staining also increased with increasing pathology grade. Furthermore, more biopsies had nuclear localization of p65/RELA with advancing pathology grade, indicating nuclear translocation of the protein and the potential for transcriptional activation. The apoptosis inhibitor cIAP-2/BIRC3 also had increased staining intensity with increasing pathology grade. These results indicate that Akt, p65/RELA and cIAP-2/BIRC3, components of an anti-apoptotic pathway, are elevated in pre-neoplastic lung lesions.The serine/threonine kinase Akt is a central mediator of anti-apoptotic pathways in eukaryotic cells . ActivatThe staining for phospho-Akt observed in the biopsies in our study was diverse, consisting of cytoplasmic, nuclear or plasma membrane associated patterns. As PDK must associate with PI3K at the plasma membrane to be activated, localization of Akt has often also been localized to the cytoplasm or plasma membrane ,9. HowevThe NF-\u03baB transcription factor family is known to direct both apoptotic and anti-apoptotic signaling in eukaryotic cells . In unstWhile ample evidence has implicated NF-\u03baB activation as playing an important role in cell transformation, particularly in vitro, there is little data on the presence or localization of NF-\u03baB components in human lung tumors. Chemotherapeutic agents induced NF-\u03baB activity in NSCLC cell lines, increasing the cells resistance to these agents  suggestiIn our study, nuclear translocation of the p65/RELA subunit of NF-\u03baB was significantly increased in moderate dysplasias compared to lower grade lesions. In the carcinomas, there was a reduction of p65/RELA nuclear positivity similar to the decreased intensity of phospho-Akt staining observed in carcinomas compared to moderate and severe dysplasias. The percentage of cells with positive nuclear staining in carcinomas was also very low compared to moderate and severe dysplasias generally being restricted to isolated foci. In addition to the increased number of biopsies with nuclear staining at higher pathology grades, the overall intensity of cytoplasmic tended to be increased in moderate dysplasias in both the epithelium and stroma compared to normal epithelium . In a number of carcinomas, there was intense cytoplasmic staining for p65/RELA with an apparent paucity of staining in the nucleus. While these biopsies were scored negative for nuclear staining of p65/RELA, it was impossible to discount the possibility that nuclear p65/RELA was present in these cells but at levels lower than those observed in the cytoplasm. In the lung adenocarcinoma cell line NCI-H441 we have also observed higher levels of p65 immunoreactivity in the cytoplasm than the nucleus although these cells have very high levels of NF-\u03baB activity as measured by reporter gene assays . It should also be noted that activation of NF-\u03baB has been reported in lung cancer cells without an increase in nuclear localization ,54. In aTumor cells acquire the ability to escape apoptosis that is normally initiated in damaged cells. One mechanism for inhibiting apoptosis is to block the activity of the effector caspases that initiate apoptosis by degrading specific cellular targets . The celWhile increased staining for cIAP-2/BIRC3 in human lung adenocarcinomas has been reported , levels While nuclear staining localization of p65/RELA and increased staining for cIAP-2 did not correlate, we observed a positive correlation between staining intensity for cIAP-2 and phospho-Akt. While it is impossible to state whether this indicates that cIAP-2 is regulated by activation of Akt phosphorylation, this is a possibility. It should be noted, however, that staining intensity for phospho-Akt occurred one pathology grade prior to the observed increase in cIAP-2 staining intensity.Increased staining for phospho-Akt and cIAP-2/BIRC3 is present in pre-neoplastic human bronchial biopsy lesions compared to normal human bronchial epithelium. Additionally, the percentage of biopsies containing nuclear p65/RELA staining is increased in pre-neoplastic lesions compared to normal bronchial epithelium. The activation of genes involved in protecting cells from apoptosis in pre-neoplastic lesions suggests that the ability of lung epithelial cells to evade apoptosis is acquired prior to the cells becoming fully transformed. As pre-neoplastic lesions have not acquired all the genetic and epigenetic changes present in lung carcinoma, they may be more amenable to treatment with chemopreventive agents. Thus, identification of the pathways activated in human bronchial pre-neoplastic lesions is important in increasing our understanding of how these lesions develop into lung cancer with the eventual goal of targeting these pathways therapeutically.The author(s) declare that they have no competing interests.JWT reviewed histological staining, participated in design of the study and drafted the manuscript. YZ carried out the immunohistochemical analyses. JCL determined pathology grades of bronchial IEN lesions and reviewed histological staining. PWB determined pathology grades of lung carcinomas and helped draft the manuscript. SL participated in the design of the study, obtained IEN lesions and helped draft the manuscript. MWA conceived of the study, participated in study design and helped draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Epithelial to mesenchymal transition (EMT) in alveolar epithelial cells (AECs) has been widely observed in patients suffering interstitial pulmonary fibrosis. In vitro studies have also demonstrated that AECs could convert into myofibroblasts following exposure to TGF-\u03b21. In this study, we examined whether EMT occurs in bleomycin (BLM) induced pulmonary fibrosis, and the involvement of bronchial epithelial cells (BECs) in the EMT. Using an \u03b1-smooth muscle actin-Cre transgenic mouse (\u03b1-SMA-Cre/R26R) strain, we labelled myofibroblasts in vivo. We also performed a phenotypic analysis of human BEC lines during TGF-\u03b21 stimulation in vitro.We generated the \u03b1-SMA-Cre mouse strain by pronuclear microinjection with a Cre recombinase cDNA driven by the mouse \u03b1-smooth muscle actin (\u03b1-SMA) promoter. \u03b1-SMA-Cre mice were crossed with the Cre-dependent LacZ expressing strain R26R to produce the double transgenic strain \u03b1-SMA-Cre/R26R. \u03b2-galactosidase  staining, \u03b1-SMA and smooth muscle myosin heavy chains immunostaining were carried out simultaneously to confirm the specificity of expression of the transgenic reporter within smooth muscle cells (SMCs) under physiological conditions. BLM-induced peribronchial fibrosis in \u03b1-SMA-Cre/R26R mice was examined by pulmonary \u03b2gal staining and \u03b1-SMA immunofluorescence staining. To confirm in vivo observations of BECs undergoing EMT, we stimulated human BEC line 16HBE with TGF-\u03b21 and examined the localization of the myofibroblast markers \u03b1-SMA and F-actin, and the epithelial marker E-cadherin by immunofluorescence.\u03b2gal staining in organs of healthy \u03b1-SMA-Cre/R26R mice corresponded with the distribution of SMCs, as confirmed by \u03b1-SMA and SM-MHC immunostaining. BLM-treated mice showed significantly enhanced \u03b2gal staining in subepithelial areas in bronchi, terminal bronchioles and walls of pulmonary vessels. Some AECs in certain peribronchial areas or even a small subset of BECs were also positively stained, as confirmed by \u03b1-SMA immunostaining. In vitro, addition of TGF-\u03b21 to 16HBE cells could also stimulate the expression of \u03b1-SMA and F-actin, while E-cadherin was decreased, consistent with an EMT.We observed airway EMT in BLM-induced peribronchial fibrosis mice. BECs, like AECs, have the capacity to undergo EMT and to contribute to mesenchymal expansion in pulmonary fibrosis. Myofibroblast cells, an intermediate cell type between fibroblasts and smooth muscle cells (SMCs), have been suggested to play an important role in the development of interstitial pulmonary fibrosis (IPF), which produces excessive amounts of extracellular matrix (ECM), leading to formation of fibroblastic foci -3. HowevIn the present study, we employed the Cre/LoxP recombinase system, using the \u03b1-smooth muscle actin (\u03b1-SMA) promoter to drive Cre-dependent recombination in presumptive myofibroblast cells as well as SMCs. We then generated an \u03b1-SMA-Cre/R26R transgenic mouse strain that allows permanent \u03b2-galactosidase  labeling in airway SMCs and the other structural cells undergoing transdifferentiation into myofibroblasts. Since the recombination is achieved by Cre-dependent removal of the transcriptional stop sequence between the two LoxP sites upstream of the lacZ gene in R26R mice, lacZ expression will permanently label Cre-expressing cells ,14. As eFor histological immunofluorescent staining, anti-\u03b1-SMA monoclonal antibody (mAb) was purchased from Sigma ; anti-bovine smooth muscle myosin heavy chains (SM-MHC) polyclonal antibody (pAb) was kindly provided by Professor Mary Anne ; rabbit anti-human E-cadherin pAb was purchased from Santa Cruz Biotechnology (Cat sc-7870), rabbit anti-mouse/human E-cadherin pAb was purchased from Boster Company (Cat BA0475). GAPDH mAb was purchased from Chemicon (Cat CB1001). Secondary antibodies of goat anti-rabbit pAb conjugated with FITC and goat anti-mouse pAb conjugated with TRITC were purchased from Bethyl(Cat A120-201F) and Open Biosystems (Cat SAB1428), respectively. Goat anti-mouse pAb conjugated to HRP was purchased from Santa Cruz Biotechnology (Cat sc-2005). Bleomycin (BLM) used for establishing the peribronchial fibrosis model was purchased from Nipponkayaku . Primers were synthesized in Sangon . All chemicals for \u03b2gal staining were purchased from Jingmei Company .U63129 and M57409). The \u03b1-SMA promoter-Cre fragment was released from the construct using Sphl and EcoRI for transgenic microinjection  using the following primers: forward 5'-GAAGATCTATGCCCAAGAAGAAGAGGAAGGTGTCCAATTTACTGAC-3' and reverse 5'-CGGAATTCTGAACAAACGACCCAAC-3'. The PCR product was then sub-cloned into the BamHI-EcoRI site of the VSMP8 plasmid  which contains the mouse \u03b1SMA promoter fragment -1070~+2582, including the first exon and part of first intron  or with 80 \u03bcl PBS (n = 4 for each group). These mice were sacrificed 20 days later for western blot analysis, \u03b2gal and immunofluorescent staining.Organs were dissected from BLM or PBS treated transgenic mice and subjected to \u03b2gal staining. Briefly, organs were fixed in 0.1 M PBS, pH7.3 containing 0.25% glutaraldehyde, 2 mM MgCl2, 5 mM EGTA at 4\u00b0C for 1\u20132 hrs. Left lung lobes were perfused with 1 ml fixing solution by endotracheal injection and right lobes were ligated and removed. Tissues were then incubated in wash buffer  3 times for 30 min each, and then in staining buffer 6, 6 mM K4Fe(CN)6, 0.02% NP-40) at 37\u00b0C overnight. Following staining, wholemount tissues were observed under XTL-3400 Zoom Stereo Microscope  or processed by dehydrating, wax embedding, sectioning at 8 \u03bcm intervals and counterstaining with Carmine Alum. Microscopic analyses were performed with a Leica DM LB2 microscope equipped with a digital camera.After sacrificing \u03b1-SMA-Cre/R26R mice, right lung lobes (upper and middle lobes) were dissected and fixed in formalin and processed by conventional histological procedures. After sectioning at 4 \u03bcm intervals, sections were dewaxed, rehydrated, blocked with 10% goat serum for 60 min at room temperature and immunofluorescently stained with \u03b1-SMA, SM-MHC or E-cadherin. Sections were incubated with anti-\u03b1-SMA mAb (1:400), anti-bovine SM-MHC pAb (1:400) or co-incubated with E-cadherin pAb (1:100) overnight at 4\u00b0C and subsequently incubated with goat anti-mouse IgG-TRITC (1:800) pAb or goat anti-rabbit IgG-FITC (1:400) pAb for 1 hour. DAPI was used to stain nuclei (500 ng/ml in 95% ethanol) for 20 sec, and coverslips were mounted with 80% glycerol. Slides were examined using a Leica DC 500-fluorescence microscope equipped with a digital camera.Alternatively, lung sections were processed for Masson's trichrome staining to detect collagen and elastin. The staining was carried out using Masson trichrome staining Kit  according to the manufacturer's instruction.\u03b1-SMA protein levels in lungs were evaluated by western blot as previously described . After cThe human bronchial epithelial cell line 16HBE-14o (16HBE), a generous gift from Professor S. Holgate  was routinely maintained in growth medium consisting of MEM  and 10% FCS . Cells were seeded into sterile round coverslips placed inside 12-well plates. On reaching 70% confluence, medium was changed to FCS-free MEM, and rhTGF-\u03b21  was added to a subset of wells to a final concentration of 10 \u03bcg/L. 72 hours later, all wells were washed twice with cold PBS and a subset of wells were fixed with cold methanol:acetone (1:1) at -20\u00b0C for 10 min. Coverslips were removed from the wells and placed on glass slides, blocked with 10% goat serum for 60 min. Cells on coverslips were incubated with anti \u03b1-SMA mAb (1:400) or rabbit anti human E-cadherin (1:50) overnight at 4\u00b0C and subsequently incubated with goat anti-mouse IgG-TRITC (1:800) or goat anti-rabbit IgG-FITC (1:400) for 1 hour. Another subset of wells were fixed with PFA at RT for 20 min and treated with 0.1% TritonX-100 for 5 min. Coverslips were removed from wells, placed on slides, blocked with 10% goat serum for 30 min and incubated with 100 \u03bcl Alex 594 phalloidin (1:500) for 20 min at RT. DAPI was used to stain nuclei (500 ng/ml in 95% ethanol) for 20 sec, and coverslips were mounted with 80% glycerol. Slides were examined using a Leica DC 500-fluorescence microscope equipped with a digital camera.To permanently label myofibroblasts, we firstly generated transgenic mice bearing an \u03b1-SMA promoter driven Cre. Ten pseudopregnant mice were implanted oviductally with fertilized eggs injected with the construct, yielding 19 offspring, 4 of which were identified to carry the randomly integrated transgene. Two founder mice were selected and used to produce inbred strains. We then crossed an \u03b1-SMA-Cre transgenic strain to reporter strain R26R+/+ whereby Cre-specific recombination at the ROSA26 locus allows expression of \u03b2-galactosidase in smooth muscle cells and myofibroblasts.\u03b2gal staining was performed on offspring of the \u03b1-SMA-Cre/R26R and the R26R mice respectively. Positive \u03b2gal staining was observed in the trachea of the \u03b1-SMA-Cre/R26R strain Fig. , but notAs expected, \u03b2gal staining was highly restricted to SMCs in smooth muscle-rich organs isolated from the \u03b1-SMA-Cre/R26R mice (data not shown). In pulmonary arteries and veins, \u03b2gal staining was consistent with the natural distribution of smooth muscle tissue at these sites Fig. . In the The double transgenic mouse strain described above provides a simple means to follow expression of \u03b1-SMA, and thus the regulation of myofibroblast development during pulmonary fibrosis. We next endotracheally administrated BLM in the transgenic mice for induction of lung injury and fibrosis. As shown in figure Histological observations reveal a significantly increased number of \u03b2gal staining- positive cells located at subepithelial areas of bronchioles, terminal bronchioles Fig.  and tuniCorrespondingly, western blot analysis revealed an overall increase in lung \u03b1-SMA protein in the BLM treated transgenic mice, compared with the control mice Fig. As shown in Fig. In vitro immunofluorescent staining of 16HBE cells  demonstrates that exposure to TGF-\u03b21 results in an apparent reduction of E-cadherin staining, an epithelial marker, concomitant with its redistribution from intercellular junction areas into the cytoplasm Fig. . In contUsing the Cre/Loxp system, we generated a transgenic mouse strain that expressed lacZ specifically in SMCs and myofibroblasts containing tissues. The \u03b2gal expression pattern in the \u03b1-SMA-Cre/R26R transgenic model closely resembled the expression of endogenous \u03b1-SMA in the airways, and that in the gastrointestinal channel, vessels and genitourinary tract under normal physiological conditions. These data suggest that the SMP8 promoter region of the \u03b1-SMA gene, including the first exon and part of the first intron (-1070 to +2582 of the mouse \u03b1-SMA promoter), is sufficient to recapitulate endogenous \u03b1-SMA expression patterns, in concordance with previous studies -19.Smooth muscle-targeted Cre recombinase mice that have previously been generated by others for study of diseases, including SM22-CreER and SMMHC-Cre strains in which Cre is driven by the promoter of SM22 gene or SM-MHC gene, respectively. Feil and colleagues have generated the SM22-CreER transgenic mice to the effect that the expression of the transgene is confined to smooth muscle cells for studying vascular and gastrointestinal diseases . HoweverAdditionally, for the reason that in vivo recombination in Cre/Loxp system is irreversible, \u03b2gal staining in the lung of our transgenic strain could reflect past and present myofibroblast transition events post BLM treatment. This may assist discovery of the cellular source of the active myofibroblasts in the development of pulmonary fibrosis. In the chronic progression of fibrosis, multiple cycles of injury and repair may occur repeatedly with a broad time period and range of sites. Activation of the \u03b1-SMA promoter may be a transient event and limited to a subgroup of cells at a given time point . So the In the BLM-treated \u03b1-SMA-Cre/R26R mice, we observed a number of \u03b2gal staining positive cells emerging in the subepithelial areas of bronchioles and terminal bronchioles and in the ectoblast of vessels, concomitant with extensive Masson Trichrome-stained extracellular matrix. In comparison, in the PBS-treated \u03b1-SMA-Cre/R26R mice, \u03b2gal positive cells were seldom seen, suggesting that not only can the reporter mice demonstrate the inherent distribution of pulmonary SMCs under physiologic condition, but also have the capability to sensitively record the trail of myofibroblast transition in the lung of the mice following pathologic stimulation. We demonstrate herein that increased \u03b2gal expression in the lungs of the BLM-treated mice is mainly to be due to the appearance of myofibroblasts in the subepithelial areas of bronchiole and terminal bronchiole. Previous studies had shown that airway BLM administration does not result in remarkable morphological changes in the SMC layer [There have been prior suggestions that EMT occurs in the lung during fibrogenesis, but these suggestions derive largely from studies of transformed cells or primary AECs cultured on plastic, the in vivo significance of which is unclear ,9. It haAdditionally, \u03b2gal was stained positively in a few basal epithelial cells and columnar epithelial cells lining the bronchiole during bleomycin-induced lung fibrosis in the reporter mice. The immunofluorescent co-staining of the both E-cadherin and \u03b1-SMA confirmed further that the BECs were undergoing EMT. When we focused on the BEC cell line 16HBE in vitro, we found that exposure to TGF-\u03b21 led to a remarkable myofibroblast cell-like phenotype, marked by expression of \u03b1-SMA and F-actin and the reduction of the epithelial-specific junction localization of E-cadherin. Taken together, these observations suggest that BECs might also be capable of undergoing EMT and thereby provide another cellular source for the parenchymal aggregation of myofibroblasts during fibrosis.As mentioned above, however, the present histological observations in the reporter mice do not support the rationale that EMT exerts a critical influence on the progression of pulmonary fibrosis, because EMT indicated by \u03b2gal staining and \u03b1-SMA immunostaining was rarely detected in BECs and AECs of BLM-treated mice. For the predominant activation of sub-epithelial myofibroblasts in the development of BLM-treated lung fibrosis, fibroblasts or other sources of progenitors may play more essential roles which contribute to the pool of expanded myofibroblasts after lung injury. To what extent does EMT contribute to the aggravation of fibrosis, whether similar EMT in BECs occur in IPF patients are all interesting questions for future studies.Additionally, the \u03b1-SMA-Cre single transgenic strain bearing the \u03b1-SMA driven Cre is sufficiently sensitive to test the function of a candidate gene in SMCs or myofibroblasts on the development of pulmonary fibrosis. Tissue-specific gene knockout or knock-in can be accomplished via crossing the \u03b1-SMA-Cre mouse to a strain containing a loxP site flanked sequence of interest.In conclusion, we have developed a double transgenic reporter mouse strain to map the natural distribution of \u03b1-SMA-expressing cells in vivo under basal physiological condition. Moreover, lung cells that do not express \u03b1-SMA under normal conditions may permanently express \u03b2gal via \u03b1-SMA activation in response to pathologic stimulation, thus allowing tracking of the cellular source of myofibroblasts and to definitively test whether EMT occurs in vivo.The author(s) declare that they have no competing interests.ZW carried out the transgene construction, transgenic screening and breeding, histological works and drafted the manuscript. LLY carried out the microinjections and transgenic screening and breeding. LC participated in the histological work and mice screening and breeding. MZ participated in the in vitro immunostaining. XC participated in the microinjections. XY guided the microinjection and animal breeding. JX design the study, technical support the research, revise the manuscript and give final approval of the version to be published. All authors read and approved the final manuscript."}
+{"text": "The human genome lies deep within the cell interior, sequestered behind the double-walled membrane of the nucleus, yet still remains vulnerable to a wide variety of hazardous materials. Two of DNA's worst enemies, ultraviolet light and chemical carcinogens, can wreak havoc on the molecule by mutating individual nucleotides or changing its physical structure. In most cases, genomic integrity is restored by specialized suites of proteins dedicated to repairing specific types of injuries.XPB, XPD, andTTDA\u2014have been implicated in the photosensitive form of a rare inherited premature aging syndrome called trichothiodystrophy (TTD), which is characterized by brittle hair and nails, scaly skin, and neurological degeneration.One mending mechanism, called nucleotide excision repair (NER), recruits and coordinates the services of roughly 25 proteins to recognize and remove structure-impairing lesions, including those induced by ultraviolet light. At the center of this effort is the ten-subunit transcription/repair factor IIH (TFIIH) complex. As its name suggests, in addition to its role in DNA repair, it also regulates transcription; how TFIIH coordinates these very different activities is still under investigation. Regardless, three TFIIH genes\u2014In a new study, Giuseppina Giglia-Mari, Jan Hoeijmakers, Catherine Miquel, Wim Vermeulen, and colleagues created a fluorescently tagged version of trichothiodystrophy group A (TTDA) to investigate its role in repair and transcription. By experimentally modifying the transcription function and by triggering DNA repair in human cell lines expressing the fluorescent TTDA protein, the researchers show that TTDA first of all dynamically associates with TFIIH, and that TTDAs become stably incorporated only while the complex is engaged in NER.TTDA mutations that produce either truncated, nonfunctional versions of the protein or no protein at all, resulting in reduced TFIIH levels. TTD-A cells also have NER defects and are hypersensitive to ultraviolet light.NER steps in when damage interferes with the elongation of a newly transcribed gene, promoting cell survival. It also plays an anti-cancer role by surveying the genome for damage. In ultraviolet-induced NER, TFIIH unzips the double helix to access the injury, and then recruits four proteins to stabilize the open strand, evaluate the damage, and cleave the DNA on either side of the lesion. Other proteins produce new nucleotides to fill the gap. Cells collected from patients with TTD-A haveTo study the role of the TTDA subunit in the TFIIH complex's functions, the researchers tagged TTDA and the XPD subunit with green fluorescent protein (GFP) to monitor and compare their movement and behavior using high-resolution confocal microscopy. Both TTDA-GFP and XPD-GFP could stably incorporate into TFIIH and function in DNA repair, proving reliable tools for studying TFIIH's spatial and temporal distribution and activity during transcription and DNA repair.Both TTDA-GFP and XPD-GFP were observed in the cytoplasm and nucleus, in contrast to the XPB subunit, which is known to localize only in the nucleus. To determine if TTDA and XPD assemble with TFIIH in the cytoplasm, the researchers used a customized version of a motility-monitoring technique called fluorescence recovery after photobleaching (FRAP). Applying a high-intensity laser to a specific region in a cell destroys fluorescence in that area; as unaffected GFP-tagged molecules from other regions replace the zapped molecules, fluorescence is recovered. Recovery is much faster for free-moving GFP-tagged proteins than for those caught up in interactions with other cellular proteins.In the cytoplasm, TTDA-GFP moved much faster than XPD-GFP, but did not interact with each other. Both proteins, however, were less motile in the nucleus, where they interacted with TFIIH. These results indicated that TTDA exists in two pools in non-irradiated cells, one free and one bound to TFIIH. After irradiation, however, fluorescence was not completely recovered for either TTDA-GFP or XPD-GFP, suggesting a lack of motility one would expect by participating in DNA repair. When the researchers inhibited a protein essential for NER initiation in the irradiated cells, they observed a reduced fraction of immobilized TTDA-GFP, confirming that TFIIH must incorporate TTDA-GFP  for NER. By interfering with the transcription machinery, they go on to show that TFIIH does not stably incorporate TTDA when TFIIH is simply bound to DNA; TFIIH only enhances association with TTDA during NER.Altogether, these results show that NER-specific lesions induce TTDA to form a stable association with the TFIIH in living cells. Giglia-Mari et al. propose that once TTDA nestles into place after the core TFIIH complex attaches to a lesion, it triggers a conformational change that recruits the other subunits required for repair. TTDA may also help TFIIH fold properly, preventing it from being degraded and allowing it to accumulate to the levels necessary for NER function. TFIIH molecules spend far longer at NER sites (four to five minutes) than they do at transcription sites (two to ten seconds), suggesting why TFIIH concentrations are so important to NER\u2014and why people that can't produce TTDA experience such debilitating symptoms."}
+{"text": "Lxx)4 (LYPLTSLRSLFG). Intriguingly, the deletion of the C-terminal proline-rich region of ALIX prevented detectable binding to p6. In contrast, a four-amino acid deletion in the central hinge region of p6 increased its association with ALIX as shown by its ability to bind to ALIX lacking the proline rich domain. Finally, by using a random screening approach, the minimal ALIX391\u2013510 fragment was found to specifically interact with this p6 deletion mutant. A parallel analysis of ALIX binding to the late domain p9 from EIAV revealed that p6 and p9, which exhibit distinct ALIX binding motives, likely bind differently to ALIX. Altogether, our data support a model where the C-terminal proline-rich domain of ALIX allows the access of its binding site to p6 by alleviating a conformational constraint resulting from the presence of the central p6 hinge.The interaction between the HIV-1 p6 late budding domain and ALIX, a class E vacuolar protein sorting factor, was explored by using the yeast two-hybrid approach. We refined the ALIX binding site of p6 as being the leucine triplet repeat sequence ( For HIVene 101) -6, and Aotein X) ,6.In this context, the reduction of Tsg101 levels by siRNA or the introduction of a dominant-negative Tsg101 mutant severely blocks viral budding ,8, whileMechanistically, the interaction between p6 and Tsg101 is well-characterized: Tsg101 interacts with the p6 PTAP motif via its N-terminal UEV domain in a process that appears to be up-regulated when p6 becomes monoubiquitinylated at conserved Lys residues in positions 27 and 33 ,10. The in vivo with p6 and p9 late domains [The other p6-interacting partner, ALIX, which consists of 868 amino acids, is organized in three domains: (i) a N-terminal BroI domain responsible for CHMP4 recruitment in the endosomal pathway ,14, (ii) domains , and  [By using an , 2003)  have not, (2003) . However03  have The following work revealed that specific p6-ALIX association could be achieved through contacts between a minimal ALIX fragment containing amino acids residues 391\u2013510 in the long arm of the V domain and a p6 late domain which has been mutated in its central hinge region. This mutant which displayed intermediary affinity for ALIX compared to HIV-1 p6 wild type and EIAV p9, suggests that in physiological conditions the constrained conformation of the HIV-1 late domain weakens its association with ALIX.in vivo interaction between EIAV p9 and ALIX were previously designed using the Y2H assay [Several studies concerning the 2H assay ,21. For 2H assay . These Gn L consensus sequence as well as the leucine triplet repeat sequence (Lxx)4 are crucial for HIV-1 p6 to interact with ALIX. This motif overlaps completely with the helix-2 in p6 as identified by NMR analysis [in vivo closely depends on the complete integrity of the secondary structure of helix-2. In details, the alanine substitution has variable effect from complete abolition of binding for Y36A and L38A, severe reduction in binding for E34A, L35A, P37A, L41A, R42A and a moderate but significant reduction for L44A, while the substitution of all other residues were well tolerated. Collectively, the binding data of our p6 mutants, are in full agreement with experimental data obtained in vitro with p6-derived peptides [in vitro binding assay measured by SPR [As shown in Figure analysis , thus inpeptides , except d by SPR -18. Howe\u0394PRR) from amino acids 716 to 868 impaired ALIX binding to p6 in vivo, while p9 still bound to the ALIX mutant  [In subsequent experiments, we tested in the Y2H system the potential interaction between the p6 domain and different ALIX mutant constructs. Side-by-side comparison was carried out in the presence of the EIAV p9 domain. Unexpectedly, a truncation of the proline-rich region in ALIX  and cou582 Fig.  providesin vitro [From structural studies, the ALIX viral late domain-binding site has been mapped to a large hydrophobic pocket on the long arm of the V domain. Mutational experiments targeting amino acid residues, which form the surrounding walls, revealed in particular that substitutions V509A in \u03b15 and F676D in \u03b111 caused a dramatic effect on the ability of the protein to bind a p6-derived peptide in vitro .in vivo using the Y2H assay. As expected, both the V509A and F676D mutations prevented the yeast cell growth on selective medium when tested against the bait-p6 protein  and the EIAV p9-derived peptide (QTQNLYPDLSEIKKE) have reported that both peptides interacted d values , while fd values . A possid values   enhanced binding to ALIX, and partially allowed the rescue of HIV-1 PTAP/LIRL upon ALIX overexpression. This limited enhancing effect of this deletion on HIV-1 PTAP/LIRL p6 upon ALIX overexpression is indicative of a negative modulation played by the hinge region of HIV-1 p6. This negative modulation would be part of the highly complex process that optimises the HIV-1 budding.The overexpression of wild type ALIX has been shown to partially rescue budding defects of HIV-1 particles with a p6 domain containing mutations in the PTAP motif , i.e. unable to recruit the ESCRT I component Tgs101 ,21. We t\u0394SQKQ we used a previously described Y2H assay called Y2H-TPCR [\u0394SQKQ bait. After selection on selective SD/-Trp/-Leu/-His/-Ade medium, one ALIX fragment encompassing residues 391\u2013510 was found to bind to p6\u0394SQKQ. This fragment also interacted with p9, but not with p6 or with the double mutant p6\u0394SQKQ Y36A unable to bind to ALIX as reported above, thus demonstrating that the interaction was specific . It is worth noticing that ALIX391\u2013510 fragment partially rebuilds the arm 2 of the V-shape domain and encompasses the great majority of the hydrophobic surface residues which presumably contact the late domains [391\u2013510 that we identified are present in the truncated ALIX409\u2013715[364\u2013716, and ALIX1\u2013503 [To isolate a minimal region in ALIX that was still able to bind to p6Y2H-TPCR  Fig. 3)\u0394SQKQ we  domains . RemarkaLIX1\u2013503  fragment\u0394SQKQ mutant, which still binds to ALIX\u0394PRR, sheds some light on a particular structural aspect of p6. Indeed, analyzing HIV-1 subtypes sequenced until now, the p6 domain appears by far the most variable domain in the Gag polyprotein precursor and natural deletions or insertions are frequently observed in the central region of p6 between S14 and I31 [in vivo analysis, a tightly interaction between late domain inhibited partially rescue of particle production upon ALIX over-expression. We can speculate that the ALIX-binding site is not necessarily optimized for high-affinity particularly in the context of HIV-1 which employs two late domains. Taken together, these observations point out that the negative activity of the p6 hinge may provide an additional ALIX-dependent regulatory process in the mechanisms that control HIV-1 budding, the complexity of which is far from being fully understood as shown by the recent finding of nucleocapsid binding to ALIX [\u0394SQKQ and p9 binding site down to the ALIX 391\u2013510 fragment. Furthermore, from our data, both HIV-1 p6 and EIAV p9 bind to an overlapping site on ALIX but in a quite different way. If the interaction between ALIX and p9 is direct, that of p6 to ALIX occurs in two steps. We propose that the PRR domain of ALIX could first contact p6 so as to alleviate the conformational constraints of the p6 hinge region and enable the subsequent binding of the HIV-1 late domain to the ALIX V domain within the 391\u2013510 fragment.If HIV-1 budding process is dependent on the presence of both Tsg101 and ALIX proteins, ALIX recruitment by the p6 late domain occurs at relatively low levels. On an evolutionary point of view, it has been proposed  that a s and I31 . Interes and I31 , it is uDuring the submission of this work the crystal structures of ALIX V domain in complex with short peptides spanning the HIV-1 and EIAV late-domain motifs was reported . BecauseAa: amino acid; AD: activation domain; Bp: bp; DBD: DNA binding domain; EIAV: equine infectious anemia virus; HIV-1: human immunodeficiency virus type-1; PMSF: phenylmethanesulphonylfluoride; SD: synthetic dropout; SPR: surface plasmon resonance; X-\u03b1-Gal: 5-Bromo-4-Chloro-3-indolyl \u03b1-D-galactopyranoside; Y2H: yeast two-hybrid.The authors declare that they have no competing interests.DG, NC, LB and J-CC have conceived the study and analyzed data. J-CC, NC and CL performed the laboratory work and wrote the manuscript. CL and NC equally contributed to this work. All the authors have read and approved the manuscript"}
+{"text": "A decrease in postural stability may increase the risk of falling in the older adult. The relationship between athletic footwear and postural stability has been reported in previous studies with conflicting results. The aim of the current study is to evaluate differences between two different types of athletic footwear and barefeet, in relation to postural stability in asymptomatic older adults.\u2122; shoe 2: ASICS Cardio Velcro\u2122) and barefoot. Participants gave informed consent and attended a laboratory setting where they carried out standard tests of quiet standing balance of 30 seconds duration on a Tekscan Matscan\u00ae pressure mat. Each participant performed three repetitions of bipedal standing with eyes open and eyes closed under three randomised conditions. Two-way, repeated measures, within-groups Analysis of Variance (ANOVA) examined significant differences between the three footwear conditions and two vision conditions in terms of postural sway in quiet standing. Postural sway was measured as centre of pressure excursions in an anterior-posterior (AP) and medio-lateral (ML) direction (cm).Twenty-one older adults (mean: 74 SD: 5 years) were recruited from a University-based clinic. The cross-sectional study evaluated two different walking shoes (shoe 1: ASICS Gel OdysseyThe results demonstrated a significant difference (p<0.05) in AP postural direction with eyes open between barefoot  and shoe 1 ; and shoe 2 . The results also demonstrated a significant difference in AP postural direction (p<0.05) with eyes closed between barefoot  and shoe 1 ; and shoe 2 . No significant difference in mean ML postural direction between the two footwear conditions and barefoot, with eyes open and eyes closed was found (p>0.05).In both of the walking shoes, when standing with eyes open and eyes closed, AP sway range was significantly increased when compared to barefeet. The results suggest that older adults demonstrate an initial destabilisation effect, which could possibly be of benefit to functional ability over a longer duration. The potential of athletic footwear to enhance postural stability requires further long-term investigation."}
+{"text": "We used first-pass contrast-enhanced MRI to quantatively measure the myocardial Ktrans, a parameter indicating myocardial perfusion and vascular permeability, in mice with or without vasodilation. We measured a significant increase in myocardial Ktrans with vasodilation. We believe this may be the first report showing that first-pass imaging can quantify increased myocardial perfusion in mice relative to baseline.First-pass contrast-enhanced MRI is a well-established technique for quantifying myocardial perfusion in humans and large animals and has recently emerged as a viable tool for quantifying myocardial perfusion in mice -3. AppliImaging was performed on a 7T Clinscan MR system equipped with a gradient system having a full strength of 650mT/m and a slew rate of 6666 mT/m/ms, and using a 30mm diameter birdcage RF coil. A saturation-recovery spiral sequence was employed, with TE = 0.36 ms, TR = 3.9ms, interleaves = 14, FOV = 25.6 x 25.6mm, matrix = 128x128, saturation delay = 40 ms, alpha = 20\u00b0, and slice thickness = 1mm. Data acquisition required 55 ms/image, approximately 40% of the murine R-R interval, and was placed in the latter part of the cardiac cycle. C57Bl6/J mice were imaged with (n=5) and without (n=5) an intraperitoneal bolus injection of the vasodilator ATL313 . First-pass images were acquired for one mid-ventricular short-axis slice. A dual-bolus gadolinium injection technique was used, acquiring the arterial input and tissue functions (AIF and TF) in separate scans. Myocardial Ktrans, the product of myocardial perfusion and the first-pass extraction fraction of gadolinium, was quantified using a standard Kety model deconvolution method.Administration of ATL313 significantly increased the heart rate in all mice. First-pass images displayed uniform tissue enhancement. Example Gd concentration vs. time curves for the TF and AIF are shown in Figure These findings indicate that first-pass MRI in mice can quantitatively measure increased myocardial Ktrans with a vasodilator. Taken together with our previous studies quantifying perfusion after myocardial infarction1, these results indicate that first-pass imaging can accurately measure myocardial perfusion in mice in a variety of flow conditions.Funding was provided by NIH R01 EB 001763."}
+{"text": "Antibodies against mutated citrullinated vimentin (AMCV) represent a useful diagnostic marker with correlation to disease activity in patients with rheumatoid arthritis (RA). Since seropositivity for citrullinated autoantibodies was predictive for response to B-cell depleting therapy (BCDT) with rituximab (RTX), we investigated whether differences in antibody fine reactivity and immunoglobulin (Ig) isotype kinetics among AMCV-positive patients could provide additional information about outcome.A total of 50 AMCV IgG-positive RA patients (RTX responders (RRs) n = 37 and non-responders (NRRs) n = 13) were analyzed for reactivity against MCV epitopes and co-existent AMCV isotypes IgM and IgA. Antibody titers were determined by enzyme-linked immunosorbent assay at baseline and 24 weeks after the first cycle of RTX, and compared to kinetics of rheumatoid factor (RF) and antibodies against cyclic citrullinated peptide (ACCP).Recognized MCV epitopes by AMCV IgG of RRs and NRRs showed similar baseline patterns, with reducing reactivity in RRs and unchanged or even expanding reactivity in NRRs upon RTX treatment. At baseline, RRs were more frequently negative for AMCV subtypes, especially for IgA (68 %), compared to NRRs (31 %). Being AMCV IgA-negative at baseline indicated a good treatment response to RTX . Co-existence of AMCV IgA and IgG with stable titers upon treatment were associated with poorer responses to RTX. Furthermore, reductions of AMCV IgA levels upon RTX correlated with the improvement of 28-joint Disease Activity Score (DAS28). In comparison, subtypes of RF and ACCP were not of additional value for prediction of RTX response.Restrictive IgG seropositivity against MCV with treatment-associated decline in fine reactivity and titers was predictive for response to RTX. Double-positivity for AMCV IgG and IgA was associated with failure to respond to BCDT, suggesting a pathogenetic and less sensitive IgA-producing B-cell subset in NRRs.The online version of this article (doi:10.1186/s13075-015-0717-z) contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is one of the most common systemic autoimmune diseases worldwide, characterized by chronic and erosive arthritis, as well as by an increased mortality, mainly due to infections, cardiovascular events and malignant lymphoma . Early dResponse to a B-cell targeted therapy with RTX is usually evaluated by clinical and laboratory signs  \u20138. Due tAn objective of this study was to differentiate subgroups of seropositive patients for response to RTX. For this purpose, we investigated the epitope recognition patterns against MCV and the AMCV isotypes in AMCV IgG-positive patients with RA, in relation to their therapeutic outcome to RTX. The aim was to determine a predictive and monitoring parameter for RTX treatment, and to gain further insights into the differential behavior of humoral autoimmune responses under such targeted therapies.Our cohort was comprised of AMCV IgG-seropositive RA patients (n = 50) fulfilling the new ACR/EULAR classification criteria , who werAnti-MCV reactivities in RRs and NRRs were investigated by an MCV epitope mapping of AMCV IgG, and by determination of AMCV isotype profiles in both groups at baseline and after 24 weeks of RTX-treatment.From the patients in our cohort, the antibody reactivities of 34 AMCV IgG-positive RA patients (23 RRs compared with 11 NRRs) were tested against 88 epitopes of MCV using enzyme-linked immunosorbent assay (ELISA). Referring to the MCV sequence, 18-mer peptides with 12 overlapping amino acid residues to the adjacent peptide were generated . Mutations (from glycerine to citrulline and serine to histidine) and citrullinations (replacement of each arginine by citrulline) were inserted. The resulting peptides were applied in ascending order corresponding to the wells of a microtiter plate (A1-H1 (peptide 1\u20138), A2-H2 (peptide 9\u201316), A3-H3 (peptide 17\u201324), A4-H4 (peptide 25\u201332), A5-H5 (peptide 33\u201340), A6-H6 (peptide 41\u201348), A7-H7 (peptide 49\u201356), A8-H8 (peptide 57\u201364), A9-H9 (peptide 65\u201372), A10-H10 (peptide 73\u201380), A11-H11 (peptide 81\u201388))  with a cut-off level of 20 U/ml . The Wilcoxon signed rank test was applied to compare the autoantibody titer difference from baseline to week 24 for RRs and NRRs. In order to compare the titers of RRs with NRRs for each autoantibody subtype, for differences at baseline and at week 24, we used the Mann\u2013Whitney U test. Furthermore, the frequency distribution of seropositivity of antibody subtypes in RRs and NRRs was tested by the chi-squared and Fisher\u2019s exact test. In order to clarify whether AMCV IgG-positive RRs n = 23) and NRRs n = 11) to RTX differ in their AMCV reactivities, we performed an epitope mapping using overlapping MCV peptides that were recognized by AMCV IgG of the patients. An illustration of the recognition pattern is shown for RRs . Being AMCV IgA-negative at baseline with an isolated IgG response could indicate a better response to RTX, . In return, baseline double-positivity of IgA and IgG was predictive for a poor response to RTX .In order to clarify whether the response outcome among seropositive patients is associated with Ig isotypes, we analyzed the qualitative distribution of IgM and IgA in AMCV IgG-positive patients and compared them with isotypes of RF and ACCP IgG. At baseline, a lower prevalence of AMCV subtypes was observed in RRs compared to NRRs, with significant differences for AMCV IgA Table\u00a0. In deta2P = 0.105). In contrast, IgG, IgM and IgA subclasses of RF, as well as ACCP IgG, showed nearly similar distributions in RRs and NRRs without apparent differences at baseline , whereas the titer of NRRs was twice as high at baseline and rather remained stable, with a non-significant increase of 19.76 % during the follow-up period  , but increased slightly in NRRs by 20 % . As a result, the change of AMCV IgA in RRs was significantly different compared to NRRs at the end of the follow-up period (P = 0.007).For quantitative analysis, we determined the mean titer courses of AMCV isotypes upon RTX treatment in RRs and NRRs and compared them with RF subtypes and ACCP IgG Tables\u00a0, Fig.\u00a04.P <0.0001) and by 62.5 % in NRRs (P = 0.03), with a substantial difference between both groups at week 24 (P = 0.0003) , the difference did not reach statistical significance at week 24 (Mann\u2013Whitney U P >0.05) . The greater the difference and reduction of AMCV IgA titers were, the greater the difference and improvement in DAS28 were. This was in agreement with the above-mentioned AMCV IgA reductions in relation to their DAS28 response. In contrast, no significant correlation was observed for the other autoantibody subtypes and DAS28 values (data not shown).In line with these findings, a moderate correlation of AMCV IgA and DAS28 could be described Fig.\u00a0. In ordeP = 0.012). Furthermore, only the subtype IgA of RF showed a significant coincidence with AMCV IgA. ACCP IgG and RF IgM were not significantly associated with AMCV IgA (P <0.05)  but negative for RF IgG (n = 47 out of 50) Tables\u00a0 and 2. A05) Fig.\u00a0.Table 4CThe objective of this study was to identify a predictive biomarker among RA patients undergoing BCDT with RTX. Since seropositivity for RA-associated autoantibodies are accepted indicators for response to RTX, we investigated whether differences in fine reactivity and Ig isotype kinetics among AMCV-positive patients could provide additional information. Since the pathogenetic role of these immunoglobulin isotypes is also of interest, we compared the profile of AMCV antibodies to established RF subtypes and ACCP IgG.Analysis of AMCV IgG-positive patients revealed differences with respect to MCV epitope recognition patterns, serological baseline status and follow-up changes in relation to response outcome. Patients with response to RTX were characterized by a restrictive antibody response of AMCV IgG, decreasing mean titers and reduction of initially recognized epitope pattern upon treatment. In contrast, double-seropositivity for AMCV IgG and IgA , stable antibody mean titers and an unchanged or even expanded epitope recognition pattern during the follow-up period were associated with non-response to RTX. These findings were confirmed by a moderate correlation of AMCV IgA reductions with improved DAS28 response.In general, non-response to RTX could be due to resistant B-cell repertoire or/and pharmacokinetic differences in individual cases, like different tissue penetration of RTX to lymphoid niches . In thisA close link of RTX response to CD20-positive memory B-cell compartment has been shown by previous studies. In most of these investigations, NRRs had an incomplete depletion and higher repopulation numbers of memory B-cells \u201326. ThesThe association of non-response to RTX with the appearance of IgA anti-MCV points to the significance of this Ig subset in RA. At early stages of RA investigations, the presence of IgA RF was shown to be associated with increased disease activity and severity . FurtherOur study is limited by the relatively small sample size and inclusion of patients from two cohorts, of whom one part received RTX as a first-line biologic. Thus, different pretreatment with biologicals and concomitant medication could have influenced our results. Furthermore, by preselecting for AMCV IgG-positive patients, we could have missed different subtype kinetics in other serologic subgroups. Although this study design limits the comparability to existing studies, we were able to perform detailed analyses of the autoantibody kinetics in RTX-treated patients. In summary, our results confirm a correlation of AMCV subtypes with disease activity, and provide evidence for their potential prediction and monitoring value.Restrictive baseline seropositivity for anti-MCV IgG was identified as a predictor for response to RTX. Co-expression of IgA was associated with treatment failure to RTX. Reduction of AMCV IgA titers under RTX showed a correlation with EULAR response in RA. Furthermore, rigid IgA isotype under such targeted therapies could be related to refractory B-cell subsets, giving evidence of greater heterogeneity within the so called seropositive RA cohort."}
+{"text": "The digital revolution is changing the dental profession.Intraoral, desktop and face scanners, Cone Beam Computed Tomography (CBCT), software for Computer Assisted Design Computer Assisted Manufacturing (CAD/CAM) and guided surgery, new aesthetic materials, milling machines and 3D printers are radically transforming the dental profession.The modern digital workflow consists of four phases that are closely dependent on each other: the acquisition of information, processing that information in a project, producing the necessary devices and the clinical application on the patient. These phases integrate into the traditional workflow based on anamnesis, clinical examination, 2D radiology, treatment plan formulation, and execution of the therapies, but all based on 3D perspective, leading us to the virtual patient.via low dosage radiation Cone Beam Computed Tomography (CBCT). It is therefore possible to plan the optimal positioning of implants with software to guide the surgery. Planning data are transferred to a surgical template that can be physically fabricated in various ways and different materials. This guide will help the surgeon correctly position the implants, without the need to raise a flap.Now we can, through an intraoral scan of one or more prosthetic preparations with powerful intraoral scanners, acquire 3D information for the realization of a prosthetic Computer Assisted Design (CAD) project; within the modeling software We can design our restoration that is then milled in a highly aesthetic material  and applied on the patient. The same goes for implants, without having to take conventional physical impressions with impression trays, which our patients have never appreciated. At the same time, information related to teeth and gingiva, received from an intraoral scan, can be superimposed on the bone-related information acquired However, fixed prosthesis and implant surgery are not the only disciplines affected by the digital revolution; aesthetic dentistry, orthodontics and regenerative and maxillofacial surgery have also experienced this change. In aesthetic dentistry, the so-called digital smile design techniques, which can design and realize the patient's smile through 2D  3D  tools are very successful. Similarly, the application of digital techniques opens up new horizons in orthodontics, which is due to greater possibilities for diagnosis and planning (through the \u201csafe bone\u201d set-up), but above all, due to possibility of clinically implementing and employing a whole range of customized devices such as aligners. Regenerative bone surgery can apply personalized bone synthetic grafts on a patient's defect, because they are drawn in 3D from CBCT. The benefits for the clinician are many, such as the greater simplicity of surgery and reduction of surgical time. In the future, these bone grafts will be printed in 3D and loaded with growth factors or the patient's stem cells to accelerate and enhance healing processes. This is the perspective of Bone Tissue Engineering. Lastly, in maxillofacial surgery, it is now possible to draw and realize, by means of rapid prototyping techniques such as laser sintering and laser melting, personalized metal devices and custom-made implants, even in titanium.All these changes represent a true revolution for our profession, comparable to what was, over 30 years ago, the introduction of dental implants.The digital revolution opens up interesting scenarios and possibilities, but it also represents a challenge for the dentist and his/her team. In fact, it is necessary to learn about new devices, software and machines and to understand how to integrate them efficiently into the workflow.In this special issue, entirely dedicated to the world of digital dentistry, we have gathered several scientific and clinical papers that deal with digital topics. We hope you will find them interesting and useful."}
+{"text": "Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2\u0394. We find, in addition, that the viability of rif1\u0394 cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1\u0394 cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the  Rif1 is a conserved eukaryotic protein implicated in regulation of both the temporal pattern of DNA replication initiation and the DNA damage response (DDR). We found that in budding yeast several of Rif1\u2019s DDR-related phenotypes stem from its ability to interact with the Glc7/PP1 phosphatase and inhibit DNA replication initiation at the highly repetitive and highly transcribed rDNA locus. Each rDNA copy contains a potential replication origin flanked on one side by a proteinaceous replication fork barrier, a crucial player in rDNA array size maintenance. Additionally, the rDNA RFB ensures that replication proceeds in the same direction as transcription, thus presumably minimizing collisions between the replication and transcription machineries. Our results show that inhibition of rDNA origin firing by Rif1-Glc7/PP1 prevents the buildup of an excess of stalled forks within the rDNA locus, which can lead to genome instability and cell death. These findings highlight the challenges posed by the replication of repetitive loci, and in particular the need to limit DNA replication initiation events at such vulnerable regions. Our study may have important implications for metazoan genomes, which contain a much higher fraction of repetitive sequences than budding yeast. Finally, since tumor cells already exhibit elevated levels of replication stress, our results suggest that inhibition of systems that limit DNA replication initiation may jeopardize the viability of these cells and thus prove to be a useful therapeutic strategy. In eukaryotes DNA replication initiates from multiple sites (origins) in a characteristic sequential pattern referred to as \u2018replication timing\u2019 . ReplicaSaccharomyces cerevisiae about one-third of all potential replication origins  are located within the rDNA repeat array on chromosome XII [In the budding yeast some XII . The rDNsome XII , 7, and some XII , 9.S. cerevisiae, is essential for fork arrest at RFBs both within and outside of the rDNA [rrm3\u0394 cells leads to fork collapse and breakage, and to loss of viability in combination with mutation of DNA repair genes such as MRE11, SGS1, and SRS2 [mre11\u0394 mutants [The rDNA repeat RFB, which is generated by sequence-specific binding of the Fob1 protein , is belithe rDNA , 14 and and SRS2 , 16. Sim mutants . Apart f mutants  the MRX  mutants ) and in  mutants .Repair of broken replication forks at the yeast rDNA RFB leads to repeat array instability due to recombination-driven gain or loss of copies . AccordiRIF1 in budding yeast leads to advancement of the replication timing of most late origins [Rif1, a budding yeast Rap1-interacting factor, was initially described as an inhibitor of telomerase-dependent lengthening of telomeres in yeast . Rif1 is origins . Importa origins , 34. Modrif1\u0394 accounts for the majority of its DNA damage response (DDR)-related phenotypes, suggesting that the rDNA is a key target of Rif1 action. These findings offer a new perspective on the relationship between replication timing, repeated DNA sequences and genome stability.Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus and thus promotes the stability of rDNA repeat array. Moreover, the increase of rDNA instability in RIF1 leads to an increase in the number of active replication forks during S phase [rif1 mutants. Consistent with our previous observations [RIF1 or cells mutated in its Glc7-binding RVxF/SILK motifs (see ARS305 at the rif1 mutants Pol2 is recruited at 45 minutes after the release into S phase). On the other hand, Pol2 was detected over a shorter time interval at the late replicating HMR locus in both rif1 mutants, which might reflect its earlier and/or faster replication. Given the fact that many dormant replication origins are located within the repetitive rDNA locus [RIF1 or mutation of its Glc7 (PP1)-interacting RVxF/SILK motifs advanced Pol2 binding by ~15 minutes, to a time similar to that of the early ARS305 origin. Importantly, acute depletion of Rif1 from the nucleus in G1 phase by the anchor-away method  was not affected  is responsible for inhibition of rDNA replication, defining the rDNA locus as a novel Rif1-Glc7 target.Origins that fire early in S phase, but not late-replicating regions, recruit the pre-replicative complex (pre-RC) component Sld3 in G1, prior to DNA replication initiation . Signifi1\u0394 cells , whereas ARS607,  in rif1\u0394rif1\u0394 at late origins , whereas levels of BrdU incorporation at early origins  were not affected  incorporated BrdU very similarly in rif1\u0394, rif1-RVxF/SILK and wild type cells. Analysis of the source data from the Peace et al. study [rif1\u0394 compared to wild type.To further investigate the role of Rif1 in rDNA replication, we used bromo-deoxyuridine (BrdU) incorporation followed by anti-BrdU immunoprecipitation (IP) and quantitative PCR (qPCR) as a more direct method to measure newly synthesized DNA. We released G2/M arrested (nocodazole-treated) cells into S phase in the presence of 0.2 M hydroxyurea (HU) and BrdU   . To addrsir2\u0394 cultures, similar to that in rif1\u0394  leads to the accumulation of a red pigment when adenine in the medium is limiting, and the appearance of red sectors in colonies [rif1\u0394 cells compared to wild type  nor the double mutation rif1\u0394 rrm3\u0394 affected cell growth, either under normal conditions or in the presence of DNA damaging agents , we first assessed rDNA instability in the rif1-RBM mutant, which, like rif1\u0394, leads to an increase in telomeric silencing and telomere TG-tract length [rif1-RBM has no effect on rDNA stability  using a semi-quantitative ChIP assay, we found no difference in Sir2 binding there by ChIP-qPCR, nor at three other sites along the rDNA locus: at rARS (which is located in IGS2), at an adjacent region at the 35S rRNA gene promoter, and at a site within the 35S rRNA gene coding sequence  [rif1\u0394-dependent elevation in rDNA instability is not a consequence of DDC activation.Martina et al.  recentlyby rif1\u0394 . We thernt (DDC) , these rArrested replication forks need to be stabilized and/or restarted to avoid formation of DSBs and/or inappropriate recombination events . IncreasRIF1 also severely compromises growth of mre11\u0394 cells, both in untreated cells and upon exposure to phleomycin, which generates DSBs  would be expected to rescue this synthetic sickness. Indeed, fob1\u0394, tof1\u0394, or csm3\u0394 deletions completely rescued rif1\u0394 mre11\u0394 synthetic sickness, both in normal conditions and upon treatment with genotoxic agents  [rif1\u0394 mre11\u0394 synthetic sickness, nor any effect of FOB1 deletion  . Survivadeletion .rif1\u0394 . It woreplisome \u201360 and peplisome . The synrif1\u0394 cells might lead to higher DRC activation  activation due to accumulation of single-stranded DNA at stalled replication forks, and is accompanied by Rad53 phosphorylation .rif1\u0394, deletion of SIR2 did not increase the level of Rad53-p upon HU  phosphatase through mutation of the conserved RVxF/SILK motifs led to elevated instability of the rDNA array as detected by the frequency of marker loss from within the array, popping-out of the repeats in the form of ERCs and increased smearing of the chromosome XII on PFGE gels , don\u2019t accumulate ERCs, but still exhibit an increase in rDNA replication. These data show that chromosomal (rDNA array) instability and not exclusively that of episomal (ERC) rDNA repeats, are repressed by Rif1-Glc7 inhibition.Deletion of els Figs  and S3. els Figs  and S3A els Figs  and S3A.sir2\u0394 and rif1\u0394 deletions lead to more intensive rARS firing, we note an important difference in their effects on the firing of late origins outside of the rDNA. While sir2\u0394 leads to a decrease in the activity of non-rDNA origins, in accordance with models proposing the re-localization of limiting replication factors during S phase [rif1\u0394 instead leads to increased firing of non-rDNA origins  Fig 7AFig 7A. A1\u0394 cells . We imags , or of slower fork progression speed. The increase in Y arcs in sir2\u0394 would fit with previously observed clustering of the activated origins in groups of ca. 2\u20133 adjacent repeats [SIR2 was shown to increase the sheer number of clusters without disrupting cluster formation. The clustering would lead to faster merging of the forks from adjacent repeats and loss of the bubble arc signal from the 2D gels. We imagine that the clustering of origins might be also beneficial for faster rescue of the forks arrested at the RFBs. Further studies using methods that give spatial resolution, such as DNA combing or electron microscopy would be necessary to investigate the effects of Rif1 and Sir2 on the spacing of origin firing and fork rates at the rDNA array.Although both  repeats . Deletiosir2\u0394 and rif1\u0394 show a similar increase in rDNA replication initiation, it is interesting to note that Sir2 has a quantitatively larger influence on rDNA stability compared to Rif1. This phenotype has been ascribed to the repressive effect of Sir2 on a bi-directional non-coding promoter (E-pro) located between the 5S rRNA gene and the RFB [rif1 mutants. However, our data do not exclude the possibility that other Rif1-Glc7 dephosphorylation targets exist at the rDNA locus, which could mediate the observed phenotypes. The deacetylation targets of Sir2 relevant for the biology of the rDNA locus also remain poorly explored. Further studies aimed at characterizing known and identifying additional Rif1-Glc7 and Sir2 target proteins should help to shed light on this important question. Another interpretation of our data is that Sir2 is required to recruit Rif1 to its site of action within the rDNA. At present, though, we have no evidence for Rif1 binding within the rDNA from ChIP assays, where we did not detect enrichment above background levels.Although both  the RFB , and it  the RFB  is instrrif1\u0394 and sir2\u0394 cells might be able to efficiently \u2018\u2018rescue\u201d the ones blocked at RFBs, thus neutralizing the effect of the latter, or perhaps even increasing rDNA stability. However, it may also be the case that the proximity of the RFB to the rARS , compared to the nearest possible non-blocked fork (~ 8 kb from rARS in the adjacent rDNA repeat), will mean that forks blocked at RFBs will have to persist for an extended period of time before they can be rescued by a non-blocked fork approaching from the other side. Furthermore, it was shown that both elevated and decreased DNA replication initiation rates at rARS increase rDNA instability [The detrimental effect of enhanced replication in a uni-directionally replicated locus may seem paradoxical. Indeed, one can imagine that the elevated number of unimpeded forks at the rDNA in tability  may act rif1\u0394 mutants themselves are not overtly sensitive to DNA damaging agents, and those mutations that display synthetic growth defects in combination with rif1\u0394  so far point to a specific role of Rif1 within the rDNA. However, our data do not rule out a subtle role for Rif1 in repair at all DSBs, and this is a subject worth further investigation. Regarding Rif1\u2019s role within the rDNA, our data point to a specific role for Rif1 Glc7 (PP1) recruitment, and by inference in rDNA replication control. Nevertheless we cannot rule out an additional downstream role of Rif1 in processing breaks generated at the RFB. Finally, we note that the fob1\u0394 background, which would appear to bypass the role of Rif1 in the rDNA stability, may be a valuable tool to study rDNA independent functions of Rif1 in DNA replication and DNA damage response.Since Rif1 is recruited to DNA DSBs generated by induction of the HO endonuclease  it is poOur finding that the effect of budding yeast Rif1 on replication timing is of most consequence at the repetitive rDNA locus may have important implications in more complex eukaryotes where repetitive DNA sequences are much more prevalent. We imagine that there is a strong selection for replication origins within extensive repetitive sequences, and as a consequence mechanisms that help to assure that not all of these identical elements fire within any given cell cycle.All yeast strains described in this study are listed in the RIF1-FRB) was constructed on the basis of the starting strain HHY168, which contains RPL13A-2XFKB12, tor1-1, and fpr1\u0394::NAT alleles [RIF1 gene. The depletion of Rif1 from the nucleus was achieved by addition of rapamycin (1 \u03bcg/ml) to the yeast culture.The Rif1 anchor away strain  at 50V x 18 hrs. Lanes were excised and run on 2nd dimension gels  at 175V for 13.5 hrs. The resolved DNA was transferred onto nylon membranes, UV cross-linked and hybridized with rDNA probes as described in [The neutral-neutral 2D agarose gel electrophoreses were performed according to  with minribed in . The rDNUndigested genomic DNA was resolved on 0.9% agarose gels, transferred onto nylon membranes and hybridized with rDNA probe.XhoI restriction fragments was performed essentially as described in [Telomere southern blot to measure the length of the telomere terminal ribed in .Protein extraction by the TCA method and western blotting were performed essentially as described . Rad53 pChromatin immunoprecipitation of TAP-tagged Fob1 and Sir2  and quanH. wingei, 170\u20133667). The gel was stained with ethidium bromide, photographed under UV, transferred to a nylon membrane and hybridized with an rDNA probe.PFGE was performed as previously described  using BiADE2 marker, were excluded from the calculations.rDNA instability was measured by the marker loss assay , 82. SatThe significance of the difference of the mean values obtained in BrdU IP-qPCR and rDNA instability assays was assessed with two-tailed paired Student\u2019s t-test. The mean and standard error of the mean (SEM) are reported on the graphs.S1 Figrif1\u0394 cells. (B) Representative FACS profiles from the G2/M arrest and 0.2M HU release experiments. (C) BrdU incorporation at the indicated loci in WT, rif1\u0394 mutant and negative control strains . Cultures were released from nocodazole (G2/M) arrest into 0.2 M HU for 2 hrs. Data are presented as mean +/- SEM and a t-test was used to compare the means of WT and mutant cultures. (*) P < 0.05. (D) The schematic representation of the restriction fragments of the rDNA repeats analysed in the 2D agarose gel electrophoreses. (E) 2D gels of NheI digested genomic DNA from asynchronous cultures probed with rARS probe. Representative images (left panel) and quantification of n = 4 experiments (right panel); the abbreviations are as on the BglII digested genomic DNA from asynchronous cultures probed with rDNA RFB probe.(A) Mcm4-13xMyc ChIP at indicated loci in G1-arrested WT and (TIF)Click here for additional data file.S2 FigADE2 marker-loss assay) at mre11\u0394 mutant serves as a positive control for PHL, MMS and CPT plates. (D) rDNA instability for the indicated WT and mutant strains was measured by the ADE2 marker-loss assay.(A-B) Representative pictures of plates with colonies of strains with indicated genotypes for the rDNA instability assays ((TIF)Click here for additional data file.S3 Figrrm3\u0394 mutant . (D) Deletion of FOB1 does not alleviate the rif1\u0394 -dependent increase in rDNA replication (2D gels of NheI-digested genomic DNA from G2/M arrested cultures released in 0.2M HU for 2 hrs).(A) The chromosomes (chr) from indicated strains were resolved using pulsed-field gel electrophoresis (PFGE) and stained with ethidium bromide (top panel). The same gel was transferred by Southern blot and hybridized with an rDNA probed to detect chr XII (bottom panel). The asterisk marks the position of chr XII in the 20x rDNA strain. (B) ERC accumulation in the indicated strains see also . (C) PFG(TIF)Click here for additional data file.S4 FigXhoI-digested genomic DNA in the indicated strains. (B) rDNA instability measured by ADE2 loss in the indicated strains.(A) Telomere length assayed by Southern blot of (TIF)Click here for additional data file.S5 FigMRE11/mre11\u0394 in combination with (left to right): RIF1/rif1-RBM; RIF1/rif1-RVxF/SILK; RIF1/rif1\u0394.Part I. (A\u2013B) Exponentially growing cultures of the indicated genotypes were serially diluted 1:10 and spotted onto solid YPAD media at the indicated temperature supplemented or not with indicated chemicals . (C) Tetrad dissection plates of the heterozygous diploids (TIF)Click here for additional data file.S6 FigRIF1/rif1\u0394, RRM3/rrm3\u0394, MRE11/mre11\u0394, and FOB1/fob1\u0394 (left panel) and serial dilution spot assays with some of the derived strains (right panel). (B) Serial dilution spot assay of strains harboring combinations of RIF1, SIR2 and MRE11 gene deletions. (C) Tetrad dissection of a diploid strain with the genotype: RIF1/rif1\u0394, SIR4/sir4\u0394, MRE11/mre11\u0394, FOB1/fob1\u0394.Part II. (A) Tetrad dissection plate of a diploid strain heterozygous for 4 gene deletions: (TIF)Click here for additional data file.S7 FigRIF1-FRB anchor-away strains . (B) Rad53 phosphorylation upon HU treatment detected by Western blot in cells harboring rif1\u0394, combined with rad9\u0394, mrc1\u0394, and sml1\u0394 mec1\u0394 mutations. (C) Asynchronous cell cultures of the indicated genotypes were treated with HU for 2 hours. Subsequently, cells were pelleted, washed and released in fresh media lacking HU, to monitor recovery from the DNA replication checkpoint. Proteins were extracted and Western blotting was performed with antibodies against total Rad53 (Rad53) or the activated (autophosphorylated) protein (Rad53-p), and total Mcm4-13xMyc (which serves as a control).(A) Rad53 phosphorylation upon HU treatment in asynchronous cultures in S288C and JC482 backgrounds (upper panel) and additional rapamycin treatment of WT and (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file."}
+{"text": "The fact that piezoelectric ceramic transducer (PZT) precision drive systems in 3D printing are faced with nonlinear problems with respect to positioning, such as hysteresis and creep, has had an extremely negative impact on the precision of laser focusing systems. To eliminate the impact of PZT nonlinearity during precision drive movement, mathematical modeling and theoretical analyses of each module comprising the system were carried out in this study, a micro-displacement measurement circuit based on Position Sensitive Detector (PSD) is constructed, followed by the establishment of system closed-loop control and creep control models. An XL-80 laser interferometer  was used to measure the performance of the precision drive system, showing that system modeling and control algorithms were correct, with the requirements for precision positioning of the drive system satisfied. As technology advances with time, studies in various disciplines are also making progress, during which people have turned attention from macro-machinery to micro-machines. When developing micro systems, people are faced with various problems caused by high-precision instruments to realize high-precision operation. Currently, it is required that operations in the micro technique field reach nanoscale positioning that features positioning, driving, and control capacity with 0.1\u2013100 nm precision. With vast potential for future development, nanoscale positioning systems can be used in various industrial fields, including robotics, aerospace engineering, precision machinery, rapid prototyping, automation, and ultra-precision machining.Piezoelectric ceramic transducer (PZT) delivers no heat, noiselessness, and high positioning precision in nanoscale positioning; thus, it has been widely applied in precision manufacturing and aerospace engineering . To overRichter et al. shortened the regulation time, improved system performance, and shortened the time when the whole platform reached stability by 3\u20134 ms by improving the rise time of the system step response .Domestic researchers have also made outstanding contributions to PZT-based precision drive systems. Zhang Yulin, a professor from Shandong University, established an online identification model based on an artificial neural network and constructed a PZT control console based on neural network algorithms . ResearcThe PZT precision drive system in this study mainly consisted of a piezoelectric ceramic transducer, drive unit, an STM32F103 single-chip computer , an analog/digital (A/D) conversion module, a digital/analog (D/A) conversion module, a voltage amplification circuit, and a Position Sensitive Detector(PSD) photoelectric displacement sensor, as shown in In PZT driving power, high-precision D/A conversion is used for output; a microcontroller is used to output digital signals that undergo D/A conversion; the converted voltage goes through power amplification to output the control voltage of the PZT, as shown in The PSD detection system mainly consists of a micro-displacement detection device, i.e., PSD, and the following I-V conversion circuit, filter circuit, and digital operational circuit, as shown in In this study, a light source was fixed on the micro-control panel and moved with the worktable, which was driven by the PZT actuator. As the position of the light source changed, the voltage output by the PSD also varied accordingly, and the electrical signal was then sent to the control system through the A/D. In this study, an optical lever principle was used to amplify the micro-displacement of the PZT to improve system resolution to enable PSD positioning, ensuring its ability to reach a satisfactory level. In the center area of the PSD is approximately linear. When the light spot is away from the center, the PSD becomes nonlinear. Therefore, the center should be used as far as possible when the range is satisfied.x\u2032 is the actual displacement of PZT; U1 and U2 are the output voltages by the PSD; 2L is the length of PSD; and \u03b2 is the magnification of the optical system.Here, To better analyze the system, mathematical modeling was required for the precision elements, PZT, and driving power of the control system.x is the variation of displacement (\u03bcm); \u0394U1 is the voltage (V) variation; mk is the displacement-voltage conversion coefficient; mT is the time constant; and mT = CRC.The PZT micro-actuator can be considered equivalent to a capacitor in the circuit. With a stack structure, the equivalent capacitance of a PZT can reach as high as several microfarads. When a step voltage is applied to both ends of the PZT, it shows some features that are similar to capacitance charge-discharge. Therefore, a transient process is needed for the PZT to reach a stable state. The voltage applied is linearly correlated with the output displacement of the PZT to some degree. The PZT and power-driven equivalent resistance constitute an RC loop that is a first-order inertial loop in automatic control, as shown in Measured by the precision capacitance meter, the equivalent capacitance of the PZT is The PZT precision worktable could be deemed as a mass-spring-damper second-order system based on its own structure and features, as shown in m is the mass of the platform, \u03bcD is the damper, and BK is the elastic coefficient of the spring. According to the reference manual of the platform and relevant tests, m = 0.5 kg, BK = 0.83 \u00d7 106 N/m, and \u03bcD = 249.2 Ns/m. Force analysis of the spring-mass-damping system suggested that the displacement output by the platform is y(t) in the presence of force f(t). According to Newton\u2019s second law, there is an acceleration speed on the platform in the presence of force. The force on the platform is given as follows:Here, After Laplace transformation of this formula, we obtain:When the formula above was converted into a standard second-order oscillation loop, its step response output could be described by Digital signals that needed to be converted were sent to the D/A converter through the interface circuit by the single chip computer and then converted into corresponding DC voltage analog signals. In the high voltage amplifier, low voltage output by D/A was converted into a high voltage available for PZT operation in order to control the PZT\u2019s displacement and, thus, realize the platform positioning. The DC high voltage amplification circuit was able to amplify the analog voltage signal output by a D/A converter, which was simplified into a proportional amplifier loop under automatic control. As a result, its transfer function could be determined: The amplification coefficient The proportional amplifier loop, the first-order RC inertial loop, and the second-order oscillation loop in the PZT precision worktable were connected serially, i.e., the output of the last loop served as the input of the next loop. The transfer function could be established by multiplication of the transfer functions of the various loops. The transfer coefficients of the first-order and the second-order systems suggested that the transfer function of the system is:Here, The system response curve depicting the effect of a step signal is shown in t is the creep time, and t.The impact of creep, one of the inherent characteristics of PZTs, on PZT positioning precision could not be neglected. When a step voltage was applied to the PZT actuator, an instantaneous step response that was generally several milliseconds was generated within the time scale determined by its mechanical resonance, followed by a slow creep response. It was generally assumed that the creep process of PZT took on a logarithmic form: When voltage was applied t, It could be assumed that it was the input voltage that led to a certain creep displacement of PZT according to the contravariant relationship between electric energy and mechanical energy, whereas constant strain could also result in the voltage creep of the PZT. Therefore, voltage creep could be analyzed by the law of displacement creep. The voltage creep model is given as:A creep model-based feed-forward control based on a proportional\u2013integral\u2013derivative controller (PID) closed-loop control was able to further reduce the impact of creep on displacement. In this study, a PSD feedback-based PID algorithm implementation was designed. Deviation Controller tuning, also known as \u201coptimal tuning\u201d, is essentially matching its characteristics and controlled characteristics by adjusting the controller\u2019s parameters, so that the PID controller is able to perform effectively. The controller parameters obtained with this method are designated \u201coptimal tuning parameters\u201d. Of all tuning methods, Ziegler-Nichols tuning and the performance index setting are the most widely used. Manual tuning of PID parameters using the add-up method was implemented in this test. Given that PK = 7.8, iK = 0.0029 and DK = 0.000725. PID parameters obtained by Ziegler-Nichols tuning method were The comparison between In this study PZT is the stacked type, with a size of 5 \u00d7 5 \u00d7 18 mm. The power supply of PZT is a 0\u2013100 V adjustable DC stabilized voltage supply. The displacement was measured with the XL-80 laser interferometer from Renishaw, Wotton-under-Edge, UK. The linear measurement displacement resolution of this laser interferometer is 1 nm.Owing to the fact that the PZT showed excellent linearity after 40 V, 40 V was set to be the initial voltage. The mechanical amplification factor of the PSD photoelectric displacement sensor detection device was set to 10. The experimental system is shown in x-axis and the displacement of the closed-loop precision worktable with creep on the y-axis.The laser interferometer was used to measure the precision worktable 20 times based on preset displacement, with the mean value taken. y = 1.0001x \u2212 10.966, whose linearity was 0.197% and maximum error was 19.65 nm, which was calibrated as 20 nm in the test. With creep-based feed-forward modeling, the fitted linear equation of the measured data was y = 0.9999x + 3.3668, whose linearity was 0.073% and maximum error was 7.25 nm, which was calibrated as 10 nm, given in Closed-loop system-based positioning data was fitted to the curve When closed-loop control was used in the system, the maximum displacement error of the precision drive system could reach 19.65 nm, which was attributed to three factors. First, the overall performance of the PZT drive power, including stability and precision, would make a difference. Second, errors might also occur in the control system as a whole during installation and debugging. Most importantly, the PZT itself was not able to eliminate the impact of nonlinear features, such as creep and hysteresis, without a creep compensation algorithm. When the system was connected to a creep compensation algorithm-based closed-loop control system, the maximum error of the PZT displacement was 7.25 nm; i.e., the error caused by creep was reduced. The circuit was provided with a circuit module for quick PZT discharge, which was used to reduce the response time of the PZT when the voltage at both ends changed, thus weakening the impact of the creep features and hysteresis and improving overall system performance. Data analysis indicated that the system error with only closed-loop control was much larger than that based on the creep compensation algorithm, which possessed an absolute advantage in system performance.In this study, a PID closed-loop control algorithm was the core of precision drive system, and a creep-forward compensation model based on a PSD photoelectric position feedback system was introduced. This effectively eliminates the effect of nonlinear hysteresis on PZT control accuracy and system error in control systems. The PSD detection system is compact and simple, which can be used in a limited space.Through theoretical analysis and experiment, the algorithm is verified, and a precision drive system was designed. The maximum error of this drive system was less than 10 nm, which satisfied the requirements for precise positioning, with a high practical value in high-precision positioning systems."}
+{"text": "Eliminating the excess energetic driving force in organic solar cells leads to a smaller energy loss and higher device performance; hence, it is vital to understand the relation between the interfacial energetics and the photoelectric conversion efficiency. In this study, we systematically investigate 16 combinations of four donor polymers and four acceptors in planar heterojunction. The charge generation efficiency and its electric field dependence correlate with the energy difference between the singlet excited state and the interfacial charge transfer state. The threshold energy difference is 0.2\u00a0to 0.3\u2009eV, below which the efficiency starts dropping and the charge generation becomes electric field-dependent. In contrast, the charge generation efficiency does not correlate with the energy difference between the charge transfer and the charge-separated states, indicating that the binding of the charge pairs in the charge transfer state is not the determining factor for the charge generation. Understanding the energetic driving force is important for optimizing the performance of organic solar cells. Here Nakano et al. suggest that the dominant driving force is the energy difference between the singlet excited state and the charge transfer state after assessing 16 material combinations. The enthalpy difference between the two states (Egopt\u2009\u2212\u2009ECS) is the primary energetic driving force for the overall charge generation. In high-performance OSCs, internal quantum efficiencies (IQEs) as high as 100%2 and fill factors (FFs) of upto 80%4 have been reported, comparable with inorganic and perovskite solar cells5. The high FF is associated with flat current-voltage curves around the short-circuit condition, which indicates that the charge generation efficiency is independent of the electric field at the donor/acceptor (D/A) interfaces. These observations have proved that efficient, electric field-independent photoelectric conversion using organic semiconductors is possible with a sufficiently large driving force of Egopt\u2009\u2212\u2009ECS. However, this excess energy (Egopt\u2009\u2212\u2009ECS) is wasted as heat, resulting in large overall energy loss in the form of low open-circuit voltage (VOC). This fundamental trade-off between the energetic driving force for charge generation and the cell voltage is a reason for the power conversion efficiency (PCE) of OSCs being limited to 15% to date6. Therefore, the essential question in realizing efficient OSCs beyond the current limit is how much the excess energy can be reduced to increase VOC while maintaining efficient charge generation.In a single-particle state picture, the photoelectric conversion process in organic solar cells (OSCs) involves the transition from an initial singlet S excited 9. The overall energy loss during the charge generation can, therefore, be separated into two components: the exothermic transition from the S1 to CT states and the subsequent endothermic transition from the CT to CS states, written asECT is the lowest energy of the CT state. Egopt\u2009\u2212\u2009ECT represents the energetic loss to form the CT state and ECS\u2009\u2212\u2009ECT corresponds to the binding energy of the charge pairs in the CT state.Several experimental studies have shown that the relaxed charge transfer (CT) state at the D/A interface is the main precursor for the charge separation processEgopt\u2009\u2212\u2009ECT has been used as an empirical indicator to show the low energy loss for mixed bulk heterojunction (BHJ) OSCs. High photoelectric conversion efficiency with Egopt\u2009\u2212\u2009ECT of 0.1\u2009eV or even smaller has been proposed12. However, there is a fundamental problem in estimating the interfacial energetics of BHJs due to their microscopic inhomogeneity; the disorder and the molecular intermixing can change the molecular properties at the D/A interface substantially compared with those in the bulk15. Therefore, for BHJs, using the bulk Egopt, EHOMOD, and ELUMOA values measured for the pristine materials to calculate the overall energy loss is questionable. Moreover, large deviations in molecular properties near the D/A interface in BHJs make it difficult to discuss the data reported from different groups for various material combinations. In contrast, planar heterojunctions (PHJs) are a suitable structure for investigating the direct correlation between the interfacial properties and the device performance23 because of their well-defined interfacial structure. Even with PHJs, however, reliable discussion of the interfacial energetics has suffered from the uncertainty of ELUMOA due to the lack of accurate measurement methods, leading to a lack of quantitative studies of the intrinsic link between the interfacial energetics and the photoelectric conversion process.Recently, EHOMOD and ELUMOA, which are directly measured by ultraviolet photoemission spectroscopy (UPS) and low-energy inverse photoemission spectroscopy (LEIPS)25 for each pristine film. The correlations of the charge generation efficiency and its electric field dependence with the energetic difference of Egopt\u2212ECT and ECS\u2212ECT are investigated to explore the minimum requirement of the energetic driving force in efficient OSCs.In this study, we systematically investigate the charge generation in OSCs with 16 combinations of four donor polymers and four acceptors using PHJ structure. A well-defined interface of PHJs allows us to eliminate the effects of the complicated mixed interfaces and the inhomogeneity of the structures. We also use reliable electronic PHJ properties, such as 29. One fullerene derivative (PCBM) and three non-fullerene materials (BTAs) were used as the acceptors. BTAs have similar core \u03c0-conjugated structures but different energy levels due to their different end functional groups. Using BTAs as the acceptor in BHJ-type OSCs gave a high VOC (1.15\u20131.30\u2009eV) with a PCE of upto 8.25%32.Figure\u00a0EHOMOD and ELUMOA values were measured by UPS  because of the equilibrated charge distributions when the materials are in contact. The energy levels with respect to the vacuum level are shown in Supplementary Fig.\u00a01 state . The device structure was indium tin oxide (ITO)/polyethylenimine ethoxylated (PEIE)/acceptor/donor/MoOx/Ag. Current density-voltage (J-V) characteristics were recorded under AM1.5 100\u2009mW\u2009cm\u22122 simulated sunlight irradiation. The J-V characteristics and EQEs are shown in Supplementary Figs.\u00a0JSC), VOC, FF, and PCE of all the OSCs are summarized in Supplementary Table\u00a0The PHJ devices with the combinations of the four donor polymers and the four acceptors were prepared. Supplementary Table\u00a0ECT at the D/A interface of OSCs. First, we measured the temperature (T) dependence of the J-V characteristics under light irradiation and evaluated ECT by linearly extrapolating the qVOC-T plot to 0\u2009K according to the literature36. This method relies on the assumptions that the non-geminate charge recombination occurs exclusively through the single manifold of the interfacial CT state and that the non-radiative component of the charge recombination does not occur at 0\u2009K. We observed that the qVOC-T plots for all the combinations of materials showed linear relationships in the range of 300 to 210\u2009K  measurements to evaluate the absorption and emission of CT states for all the systems  is the light absorption efficiency at position z (with the origin at ITO/acceptor interface), \u03b7ED(z) is the efficiency for the excitons generated at position z to diffuse to the D/A interface, \u03b7gen is the interfacial charge generation efficiency from the exciton, and \u03b7col is the charge collection efficiency , we calculated light absorption profiles in the multilayered films by using the optical transfer matrix formalism following a reported procedure39. The optical constants of the eight materials and the other layers were evaluated separately by using a spectroscopic ellipsometer , which indicate how much light at each wavelength is absorbed at each position in the multilayered films.To evaluate \u03b7gen as the unknown parameter, in principle, the diffusion equation can be solved numerically in PHJs to obtain \u03b7ED from the diffusion constants and the exciton lifetimes. However, accurate evaluation of these parameters is difficult due to many factors, such as the dimensionality of the diffusion process. Indeed, the reported diffusion constants and diffusion length vary greatly in the literature depending on the measurement method40. Therefore, we used a simple model assuming the exciton collection length (ECL) near the D/A interface for each material and that all the generated excitons reach the D/A interface  to estimate the possible error range of \u03b7gen. The uncertainty of the ECL is shown as the error bars for calculated \u03b7gen. The EQE spectra calculated with these ECLs and \u03b7gen reproduced the experimental EQE spectra of most of the systems well  and the acceptor sides (\u03b7genA), respectively. The error bars indicate the variations of the assumed ECL. There was a weak trend with scattering in which a smaller driving force produced lower \u03b7gen. Interestingly, when the logarithms of \u03b7gen were plotted against Egopt\u2009\u2212\u2009ECT, the trend was much clearer with a threshold behavior  quenching43. The clear correlation between \u03b7gen and Egopt\u2009\u2212\u2009ECT suggests that the charge transfer process from the S1 to CT states can be described by Marcus theory and that this process has a large effect on the overall charge generation process of organic solar cells. When we used the ECT values determined by EQE/EL measurements, the trend in the dependence of \u03b7gen on Egopt\u2009\u2212\u2009ECT discussed above remained unchanged  is not apparent on the charge generation of OSCs. In addition, there were large differences in \u03b7Dgen and \u03b7Agen for each PHJ  in the disordered density of states of the acceptor (donor)48. It has also been proposed that the entropic gain plays a significant role in the CT states splitting into CS states50. Either way, we conclude that the energetic  driving force is important for the S1 to CT transition, whereas factors other than the energetics are more important for CT states splitting into CS states.The charge transfer from the CT to CS state is endothermic due to the Coulombic binding of the charge pairs; hence, ECT Fig.\u00a0. This ini.e., short circuit) to \u22121.0\u2009V asNext, we investigated the electric field dependence of the charge generation. For quantitative discussion, we defined the degree of the electric field dependence as the ratio of the charge generation efficiency with an applied bias of 0\u2009V  in this system. The S1 state of J61 generated within the ECL was efficiently converted to the CT states regardless of the internal electric field. The S1 state generated outside of the ECL (far from the D/A interface) decayed to the ground state, and the emission was mainly detected as PL. In contrast, in BTA3/J61 the PL from the S1 state of BTA3 decreased substantially with an applied bias of \u22121.5\u2009V. This is consistent with the relatively large field dependence of \u03b7genA (50%) in this system; the larger internal electric field generated by the applied bias promoted the CT state formation and decreased the population of the S1 state in BTA3, which decreased the PL signal intensity. We conducted the same experiments for BTA2/J61 , PCBM/PTB7 (electric field dependence of \u03b7genD was 24%), and BTA2/P3HT (electric field dependence of \u03b7genD was 10%) and found that the electric field dependence of the S1 emission appeared in only the BTA2/J61 PHJ 12. For these efficient cells, the charge generation does not depend on the electric field and the FFs are larger than 0.6. In this study, however, we observed a stricter requirement, that Egopt\u2009\u2212\u2009ECT larger than 0.2\u00a0to\u00a00.3\u2009eV is necessary for efficient electric field-independent charge generation. To explain this discrepancy, we hypothesize that Egopt\u2009\u2212\u2009ECT of BHJ is underestimated. In the random mixture of the donor and acceptor material, the aggregation and crystallinity near the D/A interface differ from those of the bulk domain15 because of the interfacial disorder or molecular intermixing56. In this situation, Egopt at the interface becomes larger than that of the bulk due to the disorder, and ECT also increases due to a deeper EHOMOD in the disordered donor or a shallower ELUMOA in the disordered acceptor  could be due to the difference in the interfacial structures; there could be key features in the interfacial structures of BHJs that universally reduce the apparent thresholds for the charge separation. Revealing these factors will lead to find the methodology for reducing the energy loss while maintaining efficient charge generation.In 16 organic PHJ systems, the charge generation efficiency and the electric field dependence of the charge generation clearly correlated with the energy offset, Mw: 36,000, Rieke Metals), polyoxy]benzodithiophene-2,6-diyl}{3-fluoro-2-[(2-ethylhexyl)carbonyl]thienothiophenediyl}) , and -phenyl C61 butyric acid methyl ester  were used as received. Poly , J61, and the BTA series were synthesized following literature methods30.Regioregular poly   was spin-coated onto a pre-cleaned glass substrate at 3000\u2009rpm for 30\u2009s. A donor polymer was spin-coated onto the glass/PSS substrates. Details of the spin-coating conditions and film thicknesses are summarized in Supplementary\u00a0Table\u00a034. A MoO3 hole-transporting layer (7.5\u2009nm) and Ag electrodes (100\u2009nm) were deposited by thermal evaporation through a metal mask under high vacuum (~10\u22124\u2009Pa). All samples were encapsulated with a glass cap and UV-curable resin in a dry N2-filled gloveboxA glass substrate with a patterned ITO electrode was cleaned by sequential ultrasonication in detergent solution, water, 2-propanol, and acetone, followed by UV-O61. The samples were irradiated with an electron beam in a vacuum chamber with pressure below 1\u2009\u00d7\u200910\u22127\u2009Pa. The kinetic energies of the incident electrons were 0\u20134\u2009eV to avoid sample damage, and the electron current densities were between 10\u22126 and 10\u22125\u2009A\u2009cm\u22122. The emitted photons were collected by a photon detector equipped with an optical band-pass filter and a photomultiplier tube. The LEIPS spectrum was obtained by scanning the electron kinetic energy. To minimize the uncertainty in the electron affinity, the spectra were taken at different center wavelengths of the band-pass filter of 260, 285, and 335\u2009nm.ITO substrates were used for the UPS and LEIPS measurements. UPS was performed with a photoelectron spectroscopy system  with He I excitation (21.2\u2009eV). For all UPS measurements, a \u22125.0\u2009V bias was applied to the samples. Detail of the LEIPS setup has been described elsewhereJ-V characteristics of the devices were measured under simulated solar illumination  from a solar simulator with a 150\u2009W Xe lamp . The light intensity was calibrated with a standard silicon solar cell . The active area of each device was defined by using a 0.12\u2009cm2 metal mask.The The EQE of each device was measured with monochromatic light . The light intensity was calibrated with a standard Si and InGaAs photodetector. The photocurrent was recorded using a lock-in amplifier  with a low-noise current amplifier . The lock-in frequency was 85\u2009Hz. For measurements with a reverse bias, the DC voltage output of the current amplifier was used.\u03bb\u2009=\u20090.154\u2009nm) was generated at 45\u2009kV and 200\u2009mA. Films were prepared on a Si/SiO2 substrate using the same spin-coating conditions as for the photovoltaic devices.X-ray reflectivity was performed with an X-ray diffractometer , and the reflection patterns were fitted by using GlobalFit software (Rigaku). Monochromatized Cu K\u03b1 radiation  from 400 to 900\u2009nm. Reflectance mode with an integrating sphere was used to measure the reflectance and transmittance of the devices, and the device absorptance was calculated by subtracting the measured reflectance and transmittance from 1.2 (Kitano Seiki). The light source was a 5\u2009W warm white light-emitting diode (LED)  with a homemade condensing lens system. The LED output power was controlled so that irradiated devices exhibited nearly identical performance to that under AM1.5, 100\u2009mW\u2009cm\u22122 solar light. Light intensity was altered by a combination of two neutral-density filters. The intensity was calibrated using a standard silicon photodiode. Temperatures at both the sample stage and the device surface were measured by thermocouples.Devices were placed in a stainless-steel chamber filled with dry N2-cooled InGaAs detector for the infrared region . A bias voltage was applied to the devices and the current was recorded using a source measurement unit .PL and EL were measured by using a spectrofluorometer equipped with a photomultiplier for the visible region and a liquid N39. The film thickness of each layer was measured by X-ray reflectivity. The electric field distributions and energy dissipations of monochromatic light were calculated for all the locations in each layer. The unit of the layer thickness and the wavelength were set to 1\u2009nm. The simulations were performed by using lab-made code run on MATLAB.Ellipsometry measurements were conducted on an ellipsometer  in the wavelength range of 210 to 1690\u2009nm with incident angles of 45\u00b0 to 75\u00b0. The thin films of the organic materials were prepared on a fused silica substrate by spin-coating. The transmittance of each film was measured at the same time and was used to determine the optical constants. The isotropic optical model was used for the PCBM thin film, whereas out-of-plane anisotropic models were used to analyze the data for the other organic materials. A gradient component model in the vertical direction was used for the ITO. Optical simulations of the bilayer OSC devices were performed based on the previously reported transfer matrix model2-dye pulse laser  with an excitation wavelength, repetition rate, and pulse duration of 532\u2009nm, 100\u2009Hz, and 0.4\u2009ns, respectively. Resistance (50\u2009\u03a9) was put parallel to the input of a digital oscilloscope , and the transient current was calculated using Ohm\u2019s law. The integral of the transient current over time provided the amount of transient charge.An LED bias light with neutral-density filters was used. Perturbation was performed with an NSupplementary InformationPeer Review FileSolar Cells Reporting SummarySource Data"}
+{"text": "The necessity for diagnosis and treatment of this infection is emphasized by the high number of complications and death rate. Pasteurella spp. infections can cause various diseases in wild and domestic animals; in humans, most infections are associated with cat or dog bites, licks, and scratches   during 2002\u20132015. For culture, we used Columbia agar with 5% sheep\u2019s blood and chocolate agar with PolyViteX  and incubated the cultures at 37\u00b0C for 24 h in a 5% CO2 incubator. We performed identification using the VITEK 2 GN ID and API NH Systems (bioM\u00e9rieux) before 2012; after 2012, we used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry . We obtained patients\u2019 medical history from the local medical database. We identified local pasteurellosis if the patient had a superficial wound or tenosynovitis or if small joints were involved. Invasive pasteurellosis was present if large joints, including shoulder, hip, knee, or prostheses, were inflamed or if the patient had neurologic, pulmonary, cardiovascular, abdominal, or pelvic involvements or had septic shock or bacteremia. We included patients in this study if they had microbiological investigation with positive results for This retrospective study was approved by the University Research Ethics Committee, Faculty of Medicine, University of Szeged, Hungary. Collection of data about participating patients was in accordance with ethical standards at the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments. https://www.microsoft.com). We used Mann-Whitney U-test or \u03c72 tests with Yates\u2019 correction to compare groups. A p value <0.05 was considered statistically significant. We analyzed the data using GraphPad Prism version 5.03 (https://www.graphpad.com/scientific-software/prism).We collected the following data from the medical database: age, sex, concurrent medical conditions, animal exposure history, outcomes, and laboratory findings at hospital admission. For statistical analysis, we collected and analyzed demographic and clinical data about the patients. We used descriptive statistics including means or medians with ranges and percentages to characterize data; in this case, we used Microsoft Excel 2013  and women , and we observed increasing rates of pasteurellosis cases by advanced age in both sexes   isolated from the lower respiratory specimens were grouped into the invasive infection cluster.We determined the distribution of  fluids) . Most st fluids) , but somPasteurella sp. was the only isolate, whereas polymicrobial infections were detected in 83 patients (51.2%). In these cases, the most frequent anaerobic bacteria isolated from clinical specimens were Fusobacterium nucleatum (n = 18), Peptostreptococcus anaerobius (n = 11), Prevotella oralis (n = 10), Prevotella melaninogenica (n = 7), Prevotella loescheii (n = 7), and Bacteroides pyogenes (n = 7). Among facultative anaerobic bacteria, Staphylococcus aureus (n = 12) and Escherichia coli (n = 5) were the most common species.In 79 patients, 2\u00a0=\u00a08.345, df\u00a0=\u00a01, p = 0.004). The difference in the age distribution among those with localized (median age 53.5 [range 0\u201397] years) and invasive (median age 63 [range 0\u201387] years) infection was also statistically significant . In the invasive-infection group, the largest number of patients  had various abscesses ; 8 (16.7%) patients had pneumonia, and 8 (16.7%) had bacteremia. Respiratory failure was diagnosed in 6 (12.5%) patients, osteomyelitis was in 4 (8.3%) patients, pleurisy in 2 (4.2%) patients, and arthritis in 2 (4.2%) patients. Central nervous system infection, adnexitis associated with pelvic inflammatory disease, peritonitis, pacemaker infection, cirrhosis, and gangrene were also recorded.Localized infections secondary to bites, scratches, or licking were the most prevalent type during the study period . Most patients with localized infections were female . For invasive infections (n = 48), however, male predominance  was observed, and that difference was statistically significant . Some patients with invasive infections had multiple underlying disorders. Cardiovascular disease, diabetes, and malignancy were the most frequent underlying diseases.In 8 (7%) patients with localized infections and 37 (77.1%) patients with invasive infections, underlying diseases were recorded in the medical history ; the difOf 114 patients with localized infection, 94 (82.5%) had contact with a dog  or cat . Ten patients had no animal contact, and no data were available for 10 patients. Animal contacts were recorded in 10 (20.8%) of 48 cases of invasive infection; no information about animal contacts was available in 16 (33.3%) cases. Twenty-two (45.8%) of 48 patients with invasive pasteurellosis had no animal contact.Pasteurella spp. between localized and invasive infections could not be detected (Most injuries (74.6%) in the localized-infection group were attributed to animal bites. Scratch wounds caused by cats were recorded in 12 patients; however, some of them had simultaneous bite injuries. Six cases of cat-associated injury were observed in women 21\u201330 years of age; in the same age group of male patients (3 patients), none had animal contact . Differedetected .2\u00a0=\u00a05.999, df\u00a0=\u00a01, p = 0.014), and the average length of stay among patients with invasive pasteurellosis was also longer. Hospital admission was almost the same in patients with dog bites (68.3%) and in patients with cat-induced injury (64.2%). The average length of hospital stay was longer (11 [range 1\u201360] days) in cases of dog bites than in cases of cat bites or scratches (8.3 [range 3\u201341] days). In patients with localized infections, injuries affected mostly the upper extremities ; in 23 patients, lower extremities were affected, mainly shins; in 6 patients, the face; in 1 patient, the eye; and in 1 patient, the genitals.When we compared data about hospitalization in patients with localized and invasive infections, we found 71 (62.3%) of 114 patients with localized infections had hospitalizations; the average length of stay was 8 (range 1\u201330) days. In the invasive-infection group, 40 of 48 patients (83.3%) were hospitalized, and the average length of hospital stay was 12.7 (range 1\u201360) days. Patients with invasive infections were more frequently hospitalized than patients with localized infections ; functional disability, mainly in fingers, developed in 10 patients ; and lymphangitis was observed in 7 of 53 (13.2%) patients. Following bites and scratches, 3 patients had amputations , affecting mainly the fingers; 3 patients had osteomyelitis (associated with dog bites) as a consequence of pasteurellosis. The high number of complications emphasizes the importance of localized and invasive pasteurella infections.After localized Of the 162 patients with pasteurellosis, 143 (88.2%) patients recovered; in 6 cases, no data were available about the outcome of pasteurellosis . Thirteen of the 162 patients died; all these patients had invasive pasteurellosis infections. Seven of these 13 patients had pneumonia and respiratory failure, 5 patients had bacteremia, and 1 patient had panencephalitis after traumatic brain injury. The median age of patients who died was 69.5 (range 39\u201384) years; most of them had multiple underlying diseases. Laboratory investigations revealed elevated leukocytes, C-reactive protein levels, and liver enzymes in all patients who died from pasteurellosis. In 4 cases, animal contacts, including dogs, cats, and other animals, were recorded in the patient\u2019s medical history. No information about animal contact was available in 5 cases, and in 4 cases, no animal contact could be found. In cases of animal contacts, no bite or scratch was mentioned by the patients or relatives. P. multocida was isolated from 12 patients, and P. canis from 1 patient. All 13 of these patients were hospitalized; the average of length of hospital stay was 10.3 (range 1\u201330) days. Pasteurella sp. was isolated from respiratory specimens or pleural fluid from 6 patients, from blood culture from 4 patients, from 2 wound specimens, and from 1 cerebrospinal fluid specimen.From clinical specimens , Pasteurella sp. infection have been increasing in the United States , and third was P. pneumotropica (5.2%). Differences between species isolated from localized and invasive infections could be detected in our study, a finding also confirmed by Nollet et al. , which may also cause underestimation of the number of animal contacts. The number of infections caused by Pasteurella infections was higher than it was in earlier studies. We observed that Pasteurella patients with cat- and dog-associated injuries were frequently hospitalized. In cases of invasive infections, the source of Pasteurella infection was frequently unknown. In spite of the adequate treatment on the basis of medical chart review and antimicrobial drug susceptibility of the isolated strain, the death rate from these infections was 27.1% in our study. We also found that complications after localized infections were detected frequently, and certain complications, such as lymphangitis, are associated only with injuries caused by cat bites; this connection was not described in earlier publications. We think that the rate of pasteurellosis is much higher than estimated because many patients with smaller injuries do not seek medical advice, and in many cases, general practice physicians try to treat smaller injuries and do not perform sample collection or order microbiological investigations. Our results, as well as international results, show that education about the possible health hazards associated with pet ownership should be provided, and the increased risks for infection in elderly and immunocompromised patients should be emphasized.Pets, including dogs and cats, are frequently recommended to patients with chronic illness because animal therapy might provide a potential health benefit. Elderly patients may also own companion animals to combat loneliness; however, pets may be the potential source of various infections or injuries ("}
+{"text": "Biological safety and antiviral properties of the cluster are as yet unknown. Here, we show the effect of acute exposition of human cells and red blood cells to the molybdenum cluster and its interaction with proteins and antiviral activity in vitro. We measured cell viability of HepG2 and EA.hy926 cell lines exposed to increasing concentrations of the cluster (0.1 to 250 \u00b5M), by 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Hemolysis and morphological alterations of red blood cells, obtained from healthy donors, exposed to the cluster (10 to 200 \u00b5M) at 37 \u00b0C were analyzed. Furthermore, quenching of tryptophan residues of albumin was performed. Finally, plaque formation by rotavirus SA11 in MA104 cells treated with the cluster (100 to 300 \u00b5M) were analyzed. We found that all doses of the cluster showed similar cell viability, hemolysis, and morphology values, compared to control. Quenching of tryptophan residues of albumin suggests a protein-cluster complex formation. Finally, the cluster showed antiviral activity at 300 \u00b5M. These results indicate that the cluster [Mo6Cl14]2\u2212 could be intravenously administered in animals at therapeutic doses for further in vivo studies and might be studied as an antiviral agent.The molybdenum cluster [Mo In tTo establish whether cluster administration can induce toxic effects and/or hemolysis, HepG2, EA.hy926, and human red blood cells were exposed to different doses of the cluster in vitro and evaluated for possible effects on cell viability, hemolysis, and morphological alterations . FurtherAcute exposition to the cluster induces low effects in both cell viability and hemolysis, and the cluster apparently forms a complex with albumin in solution. The anticancer activity of the cluster were tested in HepG2 and EA.hy926 cells. The targeting efficacy of the cluster showed higher toxicity and accumulation in both lines cells. The HepG2 was employed in the assays because it retains better hepatic characteristics, has been widely used for cytotoxicity studies ,29, and,Cell uptake and morphological changes were observed in HepG2 and EA.hy926 human cells after 24, 48 and 72 h of cluster exposition (50 and 100 \u03bcM). HepG2 and EA.hy926 cells treated for 24 h did not show accumulation of the cluster at 50 and 100 \u03bcM A,B, whilThe cytotoxicity of molybdenum cluster was evaluated on HepG2 and EA.hy926 cell lines using the 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. All doses tested (0.1 to 250 \u00b5M) showed no toxicity on both HepG2 and EA.hy926 cells following 48 h of incubation . HoweverSeveral reports have described the antitumor effect of compounds synthesized from molybdenum. These clusters can be stimulated to generate the death of tumor cells  by inducAcute exposition of human red blood cells to therapeutic doses of cluster did not induce acute B or delaWe examined the hemolytic properties of increasing doses of the cluster in human red blood cells in vitro, finding a minimal hemolysis level at therapeutic and higher concentrations. This in vitro study needs to be corroborated by in vivo analysis of hemolytic properties, thus ensuring that administration in animals does not induce hemolytic anemia .0 versus cluster concentration . This inhibitory effect was Mo-cluster dose-dependent A,B. SurpBiological properties of hexanuclear clusters have been previously described ,17,43,44A final issue in biological safety is the potential of metal based drugs to induce a damaging immune response. Some metal based drugs have been reported as immunogenic in vitro, showing mainly innate immune response, evaluated on monocyte culture and proteomics analysis , and theA biomarker is an indicator of a biological state of disease. It is characteristic of a specific state and, therefore, can be used as a marker for a target disease . These b6Cl14]2\u2212 was previously reported (OH2)x .Preparation of (OH2)x  2\u2212 is a promising fluorescent component and could be studied in animal models at therapeutic doses for potential use in tracking cancer cells in vivo and as an antiviral agent.Thus, the cluster [Mo"}
+{"text": "Exosomes contain different functional bimolecular characteristics related to physiological or pathological processes and are now recognized as new biomarkers in different human cancers. Rapid detection and classification of cancer-related exosomes might be helpful in the rapid screening of patients that may have cancer. Here, we report a surface enhanced Raman scattering technology for rapid and label-free exosomal detection (Exo-SERS) to aid in the discrimination of different cancer cells based on specific Raman phenotypes and multivariate statistical analysis. The results demonstrated that exosomes derived from both tumor cells and normal cells exhibit special, unique Raman phenotypes. Using the Exo-SERS method, the cancer cells were accurately discriminated from normal cells, and subtle molecular changes between the different cell types could be detected with high sensitive. This research provides a rapid, label-free and non-destructive manner for detecting and discriminating between cancer types. Exosomes are nano-sized phospholipid bilayer-enclosed vesicles that are secreted by all cells into the extracellular milieu . ReleaseTo date, the rapid detection of exosomes is still a challenging task. Current methods include electron microscopy (EM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), resistive pulse sensing (RPS), western blot, flow cytometry, and mass spectrometry ,10,11. NSurface enhanced Raman scattering (SERS) technology provides a promising perspective on fast, sensitive, nondestructive, and label free detection. The enhanced Raman signals belong to test samples absorbed on the rough metal surfaces used, which are typically the Ag or Au nanoparticles in the colloid. As a laser beam penetrates through the suspension colloid, the collected SERS signals are a reflection of the whole detecting systems; therefore, SERS may be used to gain the whole fingerprint spectrum of the analytes. Theoretically, exosomes from a variety of different origins have specific functional bio-macromolecules. This means that exosomes from variety of origins have specific genotypes and molecular phenotypes, which in turn can reflect different SERS phenotypes. Therefore, combining SERS technology with exosomal detection (Exo-SERS) to discriminate cell origin is likely feasible according to specific Raman phenotypes and multivariate statistical analysis. Stremersch et al. reported, for the first time, the capability of applying SERS for distinguishing melanoma cell B16F10-derived exosome-like vesicles from red blood cells . SubsequBased on the above studies, it is clear that the cancerous exosomes can be easily distinguished with normal exosomes due to the presence of various bio-functional components of the SERS technology. However, as one of the potential biomarkers in liquid biopsy techniques, the question of whether different types of cancerous exosomes can be rapidly discriminated still needs to be addressed. Therefore, the overall goal of this work is to rapid discriminate exosomes collected from different cancer types in a label-free manner which has the potential of facilitating the nondestructive cancer diagnosis. The exosomes collected from eight cell lines  were rapidly detected and classified based on the label-free SERS method. Because the Raman signals can be collected in several seconds, the Raman fingerprints could reliably reflect the different combinations of cargoes in vesicles. It is therefore easy to compare and distinguish the Raman phenotypes of cancerous exosomes from those of normal cells. In conjunction, a multivariate statistical analysis was subsequently applied to further discriminate between the different types of exosomes. The characterization of various types of exosomes would be benefit for nondestructively monitoring maternal neoplastic tissue types through liquid biopsy. Apart from diagnostic applications, this method is potentially useful for deepening insight into the molecular composition/diversity of vesicles secreted by different cancer types. Consequently, these results will pave the way for the development of liquid biopsy for cancer detection and diagnosis.Exosomes were isolated via differential centrifugation combined with ultracentrifugation of the supernatants of eight cultured cell lines that contained several types of shed membrane fragments and vesicles a.Therefore, it was critical to ensure that the extracted exosomes were purified vesicles with no or very little contaminating material before performing detection analysis. To avoid the presence of large vehicles, differential centrifugation methods were used in addition to a 0.22 \u03bcm micron filter before performing ultracentrifugation to ensure that the obtained exosomes were as pure as possible. Traditional methods were applied to identify the extracted exosomes. The dynamic light scattering (DLS) data showed that the particle size distribution of exosomes collected from MCF7 cells was between 60 nm and 130 nm, with an average diameter of 110 \u00b1 17 nm b. The otThe normal Raman signal of exosomes suspended in PBS is rarely detected because the Raman scattering of small exosomes is an extremely inefficient process. Increasing the laser power and extending the exposure time can enhance the faint Raman signals, but since the laser power is above 10 mW, it is easy to burn the biological specimens . This we\u22121, which corresponding to deformation vibration of adenine ring [3CH2 wagging  [2CH3 deformation vibration, respectively [\u22121 to 1600 cm\u22121, which is the so-called distinctive exosomal SERS phenotype (abbr. Exo-SERS phenotype). From the Exo-SERS profiles, esophageal cancer cell EC109-, EC9706- and Kyse150-derived exosomes showed a higher Raman peak intensity at lower vibrational region of 700\u2013750 cm\u22121 and a relatively lower peak intensity from 1200 cm\u22121 to 1600 cm\u22121  [\u22121, i.e., 727 and 735 cm\u22121 in HepG2-exosomes, 735 and 755 cm\u22121 in L02-exosomes. The vibrations at this region were mainly assigned to the nucleotide peak stretching. While in other Exo-SERS, it was only showed up one main Raman peak, which was 735 cm\u22121 in mammary exosomes, and near 725 cm\u22121 in oesphago-exosomes. ine ring , adenineine ring ,23, adenine ring , symmetrine ring ,22, CH dollagen) , nucleotollagen) , and CH2600 cm\u22121 a. In the\u22121 to process the PC-LDA, all of the samples precisely fell into two categories, one group was the esophageal cancer cell-derived exosomes, and another was the remaining cell-derived exosomes  and C-C vibration  [\u22121  [\u22121 and 1000 cm\u22121. By applying the Raman data from 940 cm\u22121 to 1100 cm\u22121 to process the PC-LDA, the results indicated that the breast cell-derived exosomes could be correctly distinguished from other exosomes  . Aside fbration) , while Eexosomes c. The acexosomes . Based o\u22121 and 998 cm\u22121 were sizable in the M10A-exosome, while the peak intensity of 735 cm\u22121 was indistinct . It is reported that 1221 cm\u22121, 998 cm\u22121 and 735 cm\u22121 are assigned to amide III attribution [\u22121), carbohydrates  and lipids  on the membranes were stronger in M10A-exosomes than that of M231- and MCF7-exosomes. Thus it can be seen that changes in the relative amounts of these molecular species, or even the species ratios, can refer a measurable and useful amount of overall spectral variance. However, there still exist unknowns that require further study of the Raman information. For example, in normal exosome of L02 and M10A, the intensity of 1374 cm\u22121  was stronger than 1318 cm\u22121 (amide III); however, the cancerous exosomes were the opposite. The meaning of this result requires in-depth study.To further highlight the difference between normal cell-derived exosomes and cancerous exosomes, peak intensity ratio, and differential spectrogram were analyzed. It can clearly be seen that the Raman peaks at 1221 cmdistinct a. Additi below 1 a,b. In near to 1 b. This cribution , phenylaribution  and adenribution , respectribution . The disribution . In this\u22121 were significantly higher than that of the cancerous exosomes, while the intensity of 735 cm\u22121 was lower , freshly prepared DMEM containing 10% exosome-depleted FBS (Gibco) was added, and cells were cultured for 48 h. Once the cells reached 80\u201390% confluence, the culture media was collected for exosome isolation as depicted in g at 4 \u00b0C for 80 min. After carefully removing the supernatant, the sediment was suspended in PBS and ultra-centrifuged again at 100,000\u00d7 g for 80 min to pellet the small vesicles that correspond to exosomes [Human breast cancer cells MDA-MB-231 (abbr. M231) and MCF7, human hepatoma cells HepG2, human esophageal cancer cells EC109, Kyse150 and EC9706 were used to produce exosomes from different cancer types. Normal human breast cells MCF-10A (abbr. M10A) and normal human liver cells L02 were used to collect normal exosomes. All of the cells were cultured in DMEM media  with 10% fetal bovine serum  and 1% penicillin/streptomycin (HyClone) at 37 \u00b0C under 5% COexosomes . The isoExosome size distribution and morphology were tracked by dynamic light scattering  and transmission electron microscope . The acquisition time of DLS was set at 10 s. The exosomes were measured via negative staining and visualization with the TEM. Then, 10 \u00b5L of sample was dripped onto a 2 mm copper mesh and precipitated for 1 min. Then, 10 \u00b5L of 3% phosphotungstic acid (pH 7.0) was added to stain for 5 min and the samples were put under an incandescent lamp to dry at room temperature. The acceleration voltage was 40 kV and the resolution was 0.14 nm. The expression of exosomal biomarkers was determined by western blotting. A primary antibody for TSG101 , a secondary antibody to IgG-HRP , and the control \u03b2-actin (Solarbio) was used. Protein concentration was determined by using a micro BCA protein assay kit .The Au nanoparticle colloid was synthesized according to the primary work published by Wei et al. . The plaAfter mixing 10 \u03bcL of exosome solution with 10 \u03bcL of Au colloid in the PCR tubes, the mixture was immediately pipetted onto a quartz slide to collect the Raman signals. The Exo-SERS data were collected using a laser confocal micro-Raman spectrometer . The excitation was provided by a 785 nm laser with 0.5 mW power for the exosome solution. A 50\u00d7 long objective lens was applied with an exposure time of 20 s and integrated once. Approximately 35 SERS spectra from each samples were acquired at different positions. The experiments were repeated three times. Before testing, calibration was carried out through the built-in silicon wafer of the instrument to ensure that the experimental conditions were consistent.p-value \u2264 0.01 was considered to be significantly different, and p \u2264 0.05 indicated a difference. Sensitivity and specificity is calculated according to Equation (1):The original data were preprocessed using Wire 4.1 software . The cosmic rays were removed from the original spectra and baseline correction was applied to reduce the interferences from the fluorescent background and instrument noise. The spectral regions that reflected the Raman phenotype species were cut and selected for further data analysis, including principal components analysis (PCA) and linear discriminant analysis (LDA). PCA was performed to reduce the dimensionality of the SERS data. Two or three PC scores were applied to LDA and the PC scatter plot, which displays the classification results of the different types of exosomes. A leave-one-out cross validation was used to verify the PCA-LDA results. Multivariate statistical analysis was calculated and analyzed by using SPSS software (version 19.0). One-way ANOVA was applied to compare the Raman peak intensities across different Exo-SERS. A In summary, we have reported a surface enhanced Raman scattering technology with rapid and label-free exosomal detection (Exo-SERS) for discriminating different cancer types based on specific Raman phenotypes and multivariate statistical analysis. Different types of exosomes containing different functional biomolecules resulted in specific SERS phenotypes, thus the cancer derived exosomes could be distinguished from the normal cell-derived exosomes. Furthermore, different cancer type derived exosomes varied in their SERS spectral patterns. By applying PCA, LDA, and relative Raman peak intensity analysis, the subtypes of exosomes could be accurately distinguished. The sensitivity and specificity were greater than 95% and the accuracy was 96.7% for classifying the eight exosome subtypes. This proof-of-concept study provides a rapid, label-free and non-destructive manner for detecting and discriminating between cancer types."}
+{"text": "Hematopoietic stem cell transplantation (HCT), also referred to as blood and marrow transplantation (BMT), is a high-risk, but potentially curative therapy for a number of cancer and noncancer conditions. BMT Roadmap (Roadmap 1.0) is a mobile health app that was developed as a family caregiver\u2013facing tool to provide informational needs about the health status of patients undergoing inpatient HCT.This study explored the views and perceptions of family caregivers of patients undergoing HCT and their input regarding further technology development and expansion of BMT Roadmap into the outpatient setting (referred to as Roadmap 2.0).Semistructured qualitative interviews were conducted among 24 family caregivers. Questions were developed from existing literature coupled with prior in-depth observations and interviews in hospital-based settings to explore the study objectives. Participants were recruited during routine outpatient clinic appointments of HCT patients, and all interviews were conducted in the participants\u2019 homes, the setting in which Roadmap 2.0 is intended for use. A thematic analysis was performed using a consistent set of codes derived from our prior research. New emerging codes were also included, and the coding structure was refined with iterative cycles of coding and data collection.Four major themes emerged through our qualitative analysis: (1) stress related to balancing caregiving duties; (2) learning and adapting to new routines (resilience); (3) balancing one\u2019s own needs with the patient\u2019s needs (insight); and (4) benefits of caregiving. When caregivers were further probed about their views on engagement with positive activity interventions , they preferred a \u201cmenu\u201d of positive activities to help support caregiver health and well-being.This study involved family caregivers as participants in the development of new components for Roadmap 2.0. Our research provided a further understanding of the many priorities that hematopoietic stem cell transplant family caregivers face while maintaining balance in their lives. Their schedules can often be unpredictable, even more so once the patient is discharged from the hospital. Our findings suggest that expanding Roadmap 2.0 into the outpatient setting may provide critical caregiver support and that HCT caregivers are interested in and willing to engage in positive activities that may enhance well-being and attenuate the stress associated with caregiving.RR2-10.2196/resprot.4918 Hematopoietic stem cell transplantation, also commonly referred to as blood and marrow transplantation (BMT), is a high-risk, but potentially curative therapy for a number of cancer and noncancer conditions . Given tReceiving education on patient illness has been identified as an approach for reducing stress and anxiety ,10. CompBMT Roadmap (Roadmap 1.0) was developed as a caregiver-facing mobile health app to provide information, education, and skills building to meet informational needs during the inpatient transplant process  14-19].-19.14-19Roadmap 1.0 was designed as a multicomponent app that included the following modules: an overview of the criteria needed for discharge along with training for self-management, which was required for discharge ; real-time laboratory results graphed so that visual trends could be tracked longitudinally across time; personalized medication lists with common indications, dosing and schedule, and side effects; health care provider \u201cFacebook\u201d photos; details about clinical trials that patients were enrolled in; and educational materials defining commonly used medical terms and concepts that the medical team discusses with families. Caregivers were instructed to use it freely throughout their hospital stay.Roadmap 1.0 was rated as highly useful and easy to use ,23; careThis study aimed to explore the views and perceptions of HCT caregivers for expanding Roadmap 1.0 into the home environment, outside of the hospital, where the app would be most used (the expanded version of the app will be referred to as Roadmap 2.0 henceforth). We were also interested in examining the life of a caregiver in her/his home environment to inform Roadmap 2.0.The field sites in this study included participant\u2019s homes, defined as permanent residence, rental apartment, and local extended-stay accommodations . The homes were all within approximately 90 minutes of travel distance from the University of Michigan, a large, tertiary academic medical center, as mandated by the transplant program.Semistructured interviews were conducted among 24 family caregivers. Eligibility for study participation included the following: primary family caregiver who had already experienced the transplant procedure with their loved one (patient) and was in the posttransplant phase of care; age\u226518 years; comfortable with reading and speaking English; willing to participate in a face-to-face interview in a home setting; and able to provide informed consent. Participants were recruited through referrals from the hematopoietic stem cell transplant clinical team and included caregivers of transplant patients visiting the hospital for routine outpatient clinic appointments. The hematopoietic stem cell transplant clinical team notified the research team if the caregiver agreed to learn more about the study. All caregivers approached for the study agreed to participate. One caregiver was approached by the study coordinator, but was deemed ineligible because English was not her primary language and she was not comfortable with reading and speaking English. Our primary dataset included semistructured interviews with the primary caregivers of hematopoietic stem cell transplant patients . Saturation is defined in qualitative research as a criterion for discontinuing data collection and/or analysis . RecruitEthical approval for this study was obtained by the Institutional Review Board. All participants signed an informed consent prior to the research investigators traveling to their residence. Once the informed consent document was signed, the research team worked with the participant to arrange for a date and time that worked well for both. All interviews were conducted in the participants\u2019 homes. A minimum of two research assistants traveled to the homes. The home semistructured interview questions were informed by existing literature as well as prior observations and interviews conducted in hospital-based settings (inpatient and outpatient transplant units). The questions were subsequently optimized by the study team. Approximately 250 hours of hospital-based observations and interviews were conducted by a minimum of two research assistants . Observations were recorded as field notes, and interviews were audio-recorded, with permission, and subsequently professionally transcribed . The home interviews explored several domains including demographic information, general caregiver duties after transplant, posttransplant life experiences, use of technology and social media platforms to support caregiver self-management and provide informational resources, and positive activity interventions to promote caregiver health and well-being .Based on prior in-depth observations and interviews conducted in the hospital of Roadmap 1.0, we found that caregivers were interested in and willing to engage in positive activity interventions. It was acknowledged that caregiving can be stressful and personal well-being needed to be attended to in order to best support the patient. Thus, in this study, positive activity interventions were defined to caregivers as simple pleasant activities that were intentional and could be developed into routine practices such as expressing gratitude or scheduling activities that promoted positive thoughts, emotions, or behaviors -30. ArtiThe median age of the study participants was 57 years . The majority were married or in a domestic partnership (23/24 [96%]), white (23/24 [96%]), and female (20/24 [83%]). In addition, 20/24 (83%) received at least a 2-year college degree and 12 participants (50%) received at least a 4-year college degree. The interviews took place in permanent residences (11/24 [46%]), rental apartments (10/24 [42%]), or local accommodations  stress related to balancing caregiving duties, (2) learning and adapting to new routines (resilience), (3) balancing one\u2019s own needs with the patient\u2019s needs (insight), and (4) benefits of caregiving.Once patients were discharged home, caregivers reported heightened anxiety of the new duties and tasks as well as being in the small living conditions of temporary housing . A representative participant quote is shown in Despite the burden of these new duties and routines, some caregivers voiced optimism and strength. Having beautiful flowers or Get Well cards in the room provided encouragement and support . For exaCaregivers gained insight by recognizing the need to care for their own health while also serving their loved ones. In addition to developing new routines on behalf of the patient, many were also adjusting to living in new, temporary accommodations in order to be closer to the hospital, as mandated by the transplant team. More than half of the study participants (52%) were not living in their permanent residence. As such, caregivers developed new strategies over the transplant trajectory that focused on themselves as well as the patients. Many of these strategies included self-care or management of their own stress that enabled them to be \u201cbetter\u201d caregivers. Some examples that were provided included taking a quiet moment for themselves , focusing on sleep, maintaining pre-existing relationships or friendships, and engaging in sports or physical activity . One participant noted that simply getting out of the house was useful .Caregivers made considerable adjustments in their daily lives. Some caregivers took time off work and had not yet returned to work at the time of interviews. Nonetheless, caregivers found meaning in performing their medically related duties on behalf of the patient or simply from spending time with the patient in \u201cisolated\u201d environments . Caregivers noticed that they found benefits in caregiving, such as deeper compassion and empathy toward the patient; they identified the positive aspects of caregiving. For example, while one caregiver shared that she now had to rely on other family members, this allowed deeper relationships to form . AnotherCaregivers were asked to rank a particular positive activity on a scale of 1-10, with the higher number indicating that the activity could be very beneficial . PleasanIn this study, we explored the views and perceptions of HCT caregivers to expand Roadmap 2.0 into the home environment. Through our qualitative findings, four major themes emerged: (1) stress related to balancing caregiving duties; (2) learning and adapting to new routines (resilience); (3) balancing one\u2019s own needs with the patient\u2019s needs (insight); and (4) benefits of caregiving. These findings support the importance of linking caregivers to resources throughout the transplant trajectory, beyond the hospital course, because in many instances, caregivers are not even aware of the support services available. The artifacts collected  and semistructured interviews in caregivers\u2019 home environment provided content and design considerations for the outpatient version of Roadmap 2.0; suggested topics or categories are provided in Caregivers in this study gained new insights and recognized the importance of maintaining their own health and well-being, while also caring for the patient. Accordingly, they indicated the willingness to engage in positive activity exercises. The research team asked the caregivers to rank each activity on a Likert scale. Caregivers reflected on their own subjective values and experiences in determining the ranking. In general, they preferred having a \u201cmenu\u201d or choice of a variety of positive activity exercises. The majority favored Pleasant Activity Scheduling, where caregivers would set aside time each day for a positive activity. This was followed by Gratitude journaling and Savoring the Moment, a mindfulness-based exercise. Interestingly, these activities correlated with the examples caregivers provided when discussing strategies employed while caring for the patient .Increasing amounts of data suggest that simple strategies aimed at enhancing positive thoughts, emotions, and behaviors are effective and highly scalable -30. PosiStudies have shown that positive psychology interventions enhance subjective and psychological well-being and help reduce depressive symptoms in a cost-effective and targeted manner, particularly when combined with other evidence-based interventions . FurtherHCT caregiving is intense and complex with unexpected fluctuations throughout the transplant trajectory. This paper has specifically focused on descriptive qualitative approaches conducted in the home setting of HCT caregivers. We focused on the home environment, which was informed from prior observational and qualitative work in the hospital. Since the early prototype of Roadmap 1.0 ,44, we hOur work supports caregivers as key stakeholders in the development of mobile health technology for the provision of patient care. Although we recognize the limitations of this study, such as single-center design, and relatively homogeneous participant population  with bias toward willingness to participate, the strengths of the study include rigorous data collection and analyses and novel study site (home environment). Based on our findings, we have constructed a Caregiver Health Survey to quantify the views and perspectives of HCT caregivers nationally, which will capture a larger, more diverse population. We hope that our collective qualitative and quantitative findings will inform the design and development of a positive psychology intervention to provide caregiver support in the outpatient setting. The multicomponent intervention will include a \u201cmenu\u201d of positive activities that will be tested in a randomized controlled trial design."}
+{"text": "Panophrys within genus Megophrys from southern and eastern China. This study demonstrates that the Panophrys specimens from the hilly areas among Guangdong, Guangxi and Hunan can be morphologically distinguished from all recognized congeners, thereby providing additional supports for the recognitions of four new species of Panophrys, namely Megophrys (Panophrys) mirabilis Lyu, Wang & Zhao, sp. nov. from northeastern Guangxi, Megophrys (Panophrys) shimentaina Lyu, Liu & Wang, sp. nov. from northern Guangdong, and Megophrys (Panophrys) xiangnanensis Lyu, Zeng & Wang, sp. nov. and Megophrys (Panophrys) yangmingensis Lyu, Zeng & Wang, sp. nov. from southern Hunan. The descriptions of these species take the number of Megophrys species to 101, 46 of which belong to the subgenus Panophrys.Recent phylogenetic analysis encompassing multilocus nuclear-gene and matrilineal mtDNA genealogy has revealed a series of cryptic species of the subgenus  Megophrys Kuhl & Van Hasselt, 1822 within the family Megophryidae Bonaparte, 1850, is a typical representative for Oriental fauna, spreading throughout southern China, southern and eastern Himalayas, across Indochina to islands of the Sunda Shelf and the Philippines  and partial cytochrome C oxidase 1 gene (CO1), were used for phylogenetic analysis. All sequences were attained from GenBank, encompassing 17 samples of the unnamed species  of CO1 and 541 bp of16S, were concatenated seriatim into a 1173-bp sequence, and were further tested in jmodeltest v2.1.2 with Akaike and Bayesian information criteria, all resulting the best-fitting nucleotide substitution models of GTR+I+G. Sequenced data was analyzed using Bayesian inference (BI) in MrBayes 3.2.4 . Two independent runs were conducted in a BI analysis, each of which was performed for 10,000,000 generations and sampled every 1000 generations with the first 25% samples were discarded as burn-in, resulting a potential scale reduction factor (PSRF) of < 0.005. Mean genetic distances of 16S gene between and within species were calculated in MEGA 6 using the uncorrected p-distance model.DNA sequences were aligned by the Clustal W algorithm with default parameters  and trimmed with the gaps partially deleted in MEGA 6 . Two gene segments, 632 base pairs  was used to output the spectrograms and to measure interrelated parameters with Fast Fourier transform of 256 points and a 50% overlap. The following measurements were performed: call/note duration , notes per call, inter-note intervals (the difference between end time for a selected note and begin time for the next selected note), peak frequency , high frequency , low frequency , bandwidth 90% .Morphology. Thirty-six unnamed specimens from the hilly areas among Guangdong, Guangxi and Hunan, southern China were examined, 17 of which have been used in the phylogenetic analysis. All examined specimens were fixed in 10% buffered formalin and later transferred to 70% ethanol. All studied specimens are deposited in The Museum of Biology, Sun Yat-sen University (SYS), and Chengdu Institute of Biology, Chinese Academy of Sciences (CIB), China.External measurements were made for the unnamed specimens with digital calipers  to the nearest 0.1 mm. Mean and standard deviation (SD) were calculated in R 3.3.2 (R Core Team 2016). These measurements were as follows:ED eye diameter (from the anterior corner of the eye to posterior corner of the eye);FTL foot length ;HDL head length (from tip of snout to the articulation of the jaw);HDW head width (head width at the commissure of the jaws);HND hand length ;IND internasal distance (distance between nares);IOD interorbital distance (minimum distance between upper eyelids);RAD radio-ulna length ;SNT snout length (from tip of snout to the anterior corner of the eye);SVL snout-vent length (from tip of snout to posterior margin of vent);TD tympanum diameter ;TED tympanum-eye distance (from anterior edge of tympanum to posterior corner of the eye);TIB tibial length (from the outer surface of the flexed knee to the heel).Sex was determined by secondary sexual characters, i.e., the presence of vocal sac, nuptial pads/spines in males .Panophrys for comparisons were based on the examination of museum specimens listed in Appendix I and on information available in the literature  for major nodes > 0.90. The mean p-distances of 16S gene among all in-group and out-group species used in this study are given in Table Panophrys are given in Table The BPP 1.00) and almost have no molecular divergences (p-distances 0.0), which was defined as a cryptic species Megophrys sp25 in Megophrys (Panophrys) mirabilis sp. nov.The unnamed samples from Huaping Nature Reserve, Guangxi (samples ID 1\u20134 in Table BPP 1.00) and almost have no molecular divergences (p-distances 0.0), which was defined as a cryptic species Megophrys sp29 in BPP 1.00) and have small molecular divergences (p-distances 0.3), which was defined as a cryptic species M. sp28 in p-distances 4.1), and can be distinguished from all congeners by a combination of distinctive morphological characters (see Taxonomic accounts below). Therefore, the populations from Shimentai Nature Reserve and Mt Yangming represent two separately evolving lineage, and are described as new species, Megophrys (Panophrys) shimentaina sp. nov. and Megophrys (Panophrys) yangmingensis sp. nov., respectively.The samples from Shimentai Nature Reserve, Guangxi (samples ID 5\u20138 in Table BPP 1.00) and almost have no molecular divergences (p-distances 0.0), which was defined as a cryptic species Megophrys sp2 in Megophrys (Panophrys) yangmingensis sp. nov. in phylogeny. Furthermore, this population can be distinguished from all congener species by a combination of distinctive morphological characters (see Taxonomic accounts below). Therefore, this population from Mt Yangming represents a separately evolving lineage, and is described as a new species, Megophrys (Panophrys) xiangnanensis sp. nov.The other samples from Mt Yangming, Hunan , Lingui District, Guilin City, Guangxi Zhuang Autonomous Region, PR China.917 Figs , 4A, aduParatypes. Three adult specimens from the same locality as the holotype: male SYS a002192 and female SYS a002193 collected on 10 July 2013 by Jian Zhao and Yu-Long Li; female SYS a002289 collected on 9 September 2013 by Zu-Yao Liu.mirabilis means marvelous, referring to its distinctive habitus and color pattern of this species within the subgenus Panophrys.The specific epithet Huaping Horned Toad (in English) / Hu\u0101 P\u00edng Ji\u0103o Ch\u00e1n (\u82b1\u576a\u89d2\u87fe in Chinese)SVL 55.8\u201361.4 mm (N = 2) in adult males and SVL 68.5\u201374.8 (N = 2) mm in adult females; (2) snout rounded in dorsal view; (3) internasal distance smaller than interorbital distance; (4) tympanum clear, moderate size, TD/ED 0.49\u20130.63; (5) absence of vomerine ridge and vomerine teeth; (6) tongue small, majorly attached to the mandible, free margin small and rounded, not notched behind; (7) hindlimbs slender, heels overlapping and tibio-tarsal articulation reaching forward at the central eye; (8) fingers with distinct lateral fringes, presence of indistinct subarticular tubercles at the bases; (9) toes with distinct lateral fringes and rudiment of webs, presence of indistinct subarticular tubercles at the bases; (10) presence of slightly large horn-like tubercle at the edge of upper eyelid; (11) dorsal skin smooth with granules, (12) skin on flanks flabby, with spiny tubercles; (13) supratympanic fold distinct, with dense tubercles, forming an extremely swollen large shoulder gland above insertion of arm; (14) grayish brown above, tinged with blue in males, but dorsum of head and body reddish brown in females; (15) ventral surface of throat and chest with grayish blue latticed patches and black spots in males, but with orange latticed patches and black spots in females; (16) presence of underdeveloped nuptial pads on the dorsal surface of the first finger in adult males.(1) Body size relatively large, Megophrys (Panophrys) mirabilis sp. nov. can be easily distinguished from all recognized congeners, by having a small tongue, majorly attached to the mandible, flank skin flabby with spiny tubercles, and supratympanic fold with dense tubercles forming an extremely swollen large shoulder gland above insertion of arm.Megophrys (Panophrys) mirabilis sp. nov. with 42 recognized congeners of Panophrys are given in Table Further, detailed comparative data of Panophrys species were previously recorded from the hilly areas among Guangdong, Guangxi, and Hunan, namely Megophrys (Panophrys) acuta, M. (Pa.) brachykolos, M. (Pa.) nanlingensis, M. (Pa.) obesa, and M. (Pa.) shunhuangensis. M. (Pa.) mirabilis sp. nov. differs from M. (Pa.) acuta by the larger body size, SVL 55.8\u201361.4 mm in males and 68.5\u201374.8 mm in females , snout rounded in dorsal view (vs. strongly remarkably pointed), fingers with distinct lateral fringes (vs. absent), and overlapping heels (vs. not meeting). M. (Pa.) mirabilis sp. nov. differs from M. (Pa.) brachykolos by the larger body size, SVL 55.8\u201361.4 mm in males and 68.5\u201374.8 mm in females , slightly large horn-like tubercle at upper eyelid , fingers and toes with distinct lateral fringes , overlapping heels (vs. not meeting). M. (Pa.) mirabilis sp. nov. differs from M. (Pa.) nanlingensis by the larger body size, SVL 55.8\u201361.4 mm in males (vs. 30.5\u201337.3 mm), slightly large horn-like tubercle at upper eyelid , absence of vomerine ridge and vomerine teeth (vs. both present), tongue not notched behind (vs. notched), and fingers with distinct lateral fringes (vs. absent). M. (Pa.) mirabilis sp. nov. differs from M. (Pa.) obesa by larger body size, SVL 55.8\u201361.4 mm in males and 68.5\u201374.8 mm in females , slightly large horn-like tubercle at upper eyelid , absence of vomerine ridge (vs. present), fingers and toes with distinct lateral fringes , and overlapping heels (vs. not meeting). M. (Pa.) mirabilis sp. nov. differs from M. (Pa.) shunhuangensis by larger body size, SVL 55.8\u201361.4 mm in males and 68.5\u201374.8 mm in females , slightly large horn-like tubercle at upper eyelid , and fingers and toes with lateral fringes .Five SVL 55.8\u201361.4 mm in adult males and 68.5\u201374.8 mm in adult females, Megophrys (Panophrys) mirabilis sp. nov. is significantly different from 30 congeners whose SVL < 50 mm in males or < 60 mm in females, namely M. (Pa.) baolongensis, M. (Pa.) binchuanensis, M. (Pa.) boettgeri, M. (Pa.) cheni, M. (Pa.) daweimontis, M. (Pa.) dongguanensis, M. (Pa.) fansipanensis, M. (Pa.) hoanglienensis, M. (Pa.) huangshanensis, M. (Pa.) insularis, M. (Pa.) jiangi, M. (Pa.) jinggangensis, M. (Pa.) jiulianensis, M. (Pa.) kuatunensis, M. (Pa.) latidactyla, M. (Pa.) leishanensis, M. (Pa.) lini, M. (Pa.) lishuiensis, M. (Pa.) minor, M. (Pa.) mufumontana, M. (Pa.) nankunensis, M. (Pa.) ombrophila, M. (Pa.) palpebralespinosa, M. (Pa.) rubrimera, M. (Pa.) spinata, M. (Pa.) tuberogranulatus, M. (Pa.) wugongensis, M. (Pa.) wuliangshanensis, M. (Pa.) wushanensis, and M. (Pa.) xianjuensis.With a large body size, Megophrys (Panophrys) mirabilis sp. nov. can be further distinguished from the remaining seven congeners by the following characteristics: SVL 55.8\u201361.4 mm in adult males and 68.5\u201374.8 mm in adult females ; slightly large horn-like tubercle at upper eyelid ; vomerine teeth absent ; tongue not notched behind ; lateral fringes on toes narrow ; rudimentary webs on toes [vs. more than one-fourth webs in M. (Pa.) jingdongensis and M. (Pa.) shuichengensis].SVL 61.4 mm; head width slightly larger than head length, HDW/HDL 1.02; snout rounded in dorsal view, projecting, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eyes large, ED 0.31 of HDL, pupil vertical; nostril oblique-ovoid; canthus rostralis well developed; loreal region slightly oblique; internasal distance smaller than interorbital distance; tympanum clear, TD/ED 0.49; large ovoid choanae at the base of the maxilla; absence of vomerine ridge and vomerine teeth; tongue small, majority attached at the mouth, margin rounded, not notched behind; absence of vocal sac.Adult male. Body size large, SVL and hand 0.28 of SVL; hand without webs, fingers with distinct lateral fringes, relative finger length II < I < IV < III; tips of fingers slightly dilated, round; one indistinct subarticular tubercle at the bases of each finger; metacarpal tubercles indistinct, the inner one observably enlarged and the outer one smaller; presence of underdeveloped nuptial pad on the dorsal surface of the first finger, without nuptial spines. Hindlimbs slender, tibio-tarsal articulation reaching forward at the central eye when hindlimb is stretched along the side of the body; heels overlapping when the flexed hindlimbs are held at right angles to the body axis; tibia length 0.47 of SVL and foot length 0.71 of SVL; relative toe length I < II < V < III < IV; tips of toes round and slightly dilated; toes with narrow lateral fringes and rudiment of webs; one indistinct subarticular tubercle at the bases of each toe; inner metatarsal tubercle long ovoid and the outer one absent.Radio-ulna length 0.26 of Dorsal skin smooth with sparse granules; flanks flabby with spiny tubercles; distinct supratympanic fold curving postero-ventrally from posterior corner of eye to a level above insertion of arm; small tubercles arranged from above the nostril, along the canthus rostralis, edge of upper eyelid and supratympanic fold, to the posterior margin of temporal region; a distinct horn-like prominent tubercle on the edge of upper eyelid; a discontinuous X-shaped ridge with several short ridges on two sides on the back; transverse skin ridges on the dorsal shank and thigh; ventral surface smooth; several tubercles on posterior hindlimbs; small pectoral gland closer to axilla; a single large femoral gland on rear of thigh.Grayish brown above in life; an dark interorbital triangle with light colored center and edge; a dark X-shaped making with light edge on the central of dorsum; dark brown transverse bands on forearms and hindlimbs; supratympanic fold light gray; dark vertical band below the eye; iris grayish brown; ventral surface grayish white; throat and chest with grayish blue latticed patches and black spots; ventral hands and feet grayish white, tips of digits creamy white, metacarpal tubercle and metatarsal tubercle grayish white; pectoral gland and femoral gland white.SVL 68.5\u201374.8 mm) are significantly larger than males (SVL 55.8\u201361.4 mm). Dorsal surfaces reddish brown and ventral surfaces with orange latticed patches and black spots in females SYS a002193, 2289.Measurement data of type series are listed in Table Megophrys (Panophrys) mirabilis sp. nov. is only known from Huaping Nature Reserve, northeastern Guangxi. The individuals were found on shrubbery branches near trail paths between elevations of 1300\u20131330 m a.s.l. from June to September. Males were not calling when found, but the collected female specimens bear mature yellowish oocytes. Tadpoles have not been found and ecological information remains unknown.Currently, Taxon classificationAnimaliaAnuraMegophryidaeLyu, Liu & Wangsp. nov.16A2525B-4362-51D6-95BB-236A8B03FFB9http://zoobank.org/E9F8A869-8923-4C0F-8750-181EE0843A07Megophrys sp29 , Yingde City, Qingyuan City, Guangdong Province, PR China.710 Figs , 5, adulParatypes. Eleven adult males from the same locality as the holotype: SYS a002077, 2081\u20132085, collected on 25\u201326 April 2013 by Run-Lin Li and Yuan-Qiu Li; SYS a004172\u20134173, collected on 27 July 2015 by Ying-Yong Wang and Yuan-Qiu Li; SYS a005448/CIB 110015 collected on 19 August 2016 and SYS a005992\u20135993 collected on 20 June 2017 by Zhi-Tong Lyu and Yong-You Zhao.shimentaina refers to its type locality, Shimentai Nature Reserve.The specific epithet Shimentai Horned Toad (in English) / Sh\u00ed M\u00e9n Ta\u00ed Ji\u0103o Ch\u00e1n (\u77f3\u95e8\u53f0\u89d2\u87fein Chinese)SVL 28.0\u201330.6  mm in adult males; (2) snout rounded in dorsal view; (3) tympanum clear, TD/ED 0.57\u20130.66; (4) presence of weak vomerine ridge and vomerine teeth; (5) margin of tongue rounded, not notched behind; (6) hindlimbs slender, heels overlapping and tibio-tarsal articulation reaching forward between tympanum to anterior corner of eye; (7) tibia 0.44\u20130.53 of SVL and foot 0.62\u20130.76 of SVL; (8) fingers with narrow lateral fringes, presence of indistinct subarticular tubercles at the bases; (9) toes with narrow lateral fringes and rudiment of webs, absence of subarticular tubercle; (10) presence of a small horn-like tubercle at the edge of upper eyelid; (11) presence of tiny, barely visible, black to dark brown spines on the whole dorsal skin, flanks, dorsal limbs, the region around cloaca, and rear of hindlimbs; (12) dorsal skin rough, a discontinuous \u201c/ \\\u201d-shaped ridge with two discontinuous dorsolateral ridges on two sides on the back; (13) several large warts on the flanks; (14) supratympanic fold distinct and white, with tiny spines; (15) light brown above, a dark brown stripe on each upper eyelid; (16) single subgular vocal sac in males; (17) weak nuptial pads with serried olive nuptial spines, on the dorsal surface of the first and second fingers in adult males.(1) Body size small, Megophrys (Panophrys) shimentaina sp. nov. with M. (Pa.) mirabilis sp. nov. and 42 recognized congeners of Panophrys are given in Table Comparative data of Megophrys (Panophrys) shimentaina sp. nov. differs from M. (Pa.) mirabilis sp. nov. by the smaller body size, SVL 28.0\u201330.6 mm in males , small horn-like tubercle at upper eyelid (vs. slightly large), presence of vomerine teeth (vs. absent), the presence of tiny spines on the whole dorsal skin, flanks, dorsal limbs, the region around cloaca, and rear of hindlimbs (vs. such spines absent), presence of vocal sac in males (vs. absent), and presence of nuptial spines in males (vs. absent).Panophrys species previously recorded from the hilly areas among Guangdong, Guangxi, and Hunan, Megophrys (Panophrys) shimentaina sp. nov. differs from M. (Pa.) acuta by the small horn-like tubercle at upper eyelid (vs. slightly large), snout rounded in dorsal view (vs. strongly remarkably pointed), presence of vomerine teeth (vs. absent), presence of tiny spines on the whole dorsal skin, flanks, dorsal limbs, the region around cloaca, and rear of hindlimbs (vs. such spines absent), and overlapping heels (vs. not meeting). M. (Pa.) shimentaina sp. nov. differs from M. (Pa.) brachykolos by the smaller body size SVL 28.0\u201330.6 mm in males , presence of vomerine teeth (vs. absent), presence of tiny spines on the whole dorsal skin, flanks, dorsal limbs, the region around cloaca, and rear of hindlimbs (vs. such spines absent), narrow lateral fringes on toes (vs. absent), and overlapping heels (vs. not meeting). M. (Pa.) shimentaina sp. nov. differs from M. (Pa.) nanlingensis by the presence of tiny spines on the whole dorsal skin, flanks, dorsal limbs, the region around cloaca, and rear of hindlimbs (vs. such spines absent), and tongue not notched behind (vs. notched). M. (Pa.) shimentaina sp. nov. differs from M. (Pa.) obesa by the smaller body size SVL 28.0\u201330.6 mm in males , presence of vomerine teeth (vs. absent), presence of tiny spines on the whole dorsal skin, flanks, dorsal limbs, the region around cloaca, and rear of hindlimbs (vs. such spines absent), narrow lateral fringes on toes (vs. absent), and overlapping heels (vs. not meeting). M. (Pa.) shimentaina sp. nov. differs from M. (Pa.) shunhuangensis by the presence of vomerine teeth (vs. absent), tibio-tarsal articulation reaching forward between tympanum to anterior corner of eye (vs. at the eye), and the presence of tiny spines on the whole dorsal skin, flanks, dorsal limbs, the region around cloaca, and rear of hindlimbs (vs. such spines absent).Compared with the five SVL 28.0\u201330.6 mm in adult males, Megophrys (Panophrys) shimentaina sp. nov. is significantly different from 15 congeners whose SVL > 35 mm in males, namely M. (Pa.) baolongensis, M. (Pa.) binlingensis, M. (Pa.) caudoprocta, M. (Pa.) hoanglienensis, M. (Pa.) huangshanensis, M. (Pa.) insularis, M. (Pa.) jingdongensis, M. (Pa.) jinggangensis, M. (Pa.) latidactyla, M. (Pa.) liboensis, M. (Pa.) omeimontis, M. (Pa.) palpebralespinosa, M. (Pa.) sangzhiensis, M. (Pa.) shuichengensis, and M. (Pa.) spinata.With a small body size, Megophrys (Panophrys) shimentaina sp. nov. can be further distinguished from the remaining 22 congeners by the following characteristics: vomerine teeth present ; tongue not notched behind ; lateral fringes on toes narrow ; rudimentary webs on toes .SVL 28.4 mm; head width slightly smaller than head length, HDW/HDL 0.95; snout rounded in dorsal view, projecting, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eyes large, ED 0.33 of HDL, pupil vertical; nostril oblique-ovoid; canthus rostralis well developed; loreal region slightly oblique; internasal distance slightly larger than interorbital distance; tympanum clear, in medium size, TD/ED 0.61; large ovoid choanae at the base of the maxilla; presence of weak vomerine ridge and vomerine teeth; margin of tongue rounded, not notched behind; presence of a single subgular vocal sac, a pair of slit-like openings at posterior of jaw.Adult male. Body size small, SVL and hand 0.26 of SVL; hand without webs, fingers with narrow lateral fringes, relative finger length I \u2248 II < IV < III; tips of fingers slightly dilated, round; one indistinct subarticular tubercle at the bases of each finger; inner metacarpal tubercle observably enlarged and the outer one smaller; nuptial pads with serried olive nuptial spines on the dorsal surface of the first and second fingers. Hindlimbs slender, tibio-tarsal articulation reaching forward to the posterior corner of eye when hindlimb is stretched along the side of the body; heels overlapping when the flexed hindlimbs are held at right angles to the body axis; tibia length 0.47 of SVL and foot length 0.67 of SVL; relative toe length I < II < V < III < IV; tips of toes round and slightly dilated; toes with distinct lateral fringes and rudiment of webs, without subarticular tubercle; inner metatarsal tubercle long ovoid and the outer one absent.Radio-ulna length 0.22 of Dorsal skin rough; numerous granules densely arranged on the top of head, loreal region, lips, temporal region, dorsal body, flanks and dorsal limbs; several tubercles on upper eyelid, including a horn-like prominent tubercle on the edge; all granules and tubercles bearing tiny, barely visible spines; clear supratympanic fold with tiny spines, curving postero-ventrally from posterior corner of eye to a level above insertion of arm; tubercles and granules forming discontinuous \u201c/ \\\u201d-shaped ridge and two discontinuous dorsolateral ridges on two sides at the central back; large tubercles and warts on the flanks; ventral surface smooth; several granules bearing black spines on the region around cloaca and rear of hindlimbs; small pectoral gland closer to axilla; a single large femoral gland on rear of thigh.Light brown above in life; a dark brown stripe on dorsal surface of each eye; narrow dark brown transverse bands on forearms and hindlimbs; supratympanic fold white; dark vertical band below the eye; iris reddish brown; all spines black or dark brown; ventral surface pale; throat flesh color; scarlet spots on the chest; a large white blotch on the belly; a pair of lateroventral longitudinal broad black stripes with several white tubercles on two sides; ventral limbs flesh color with white spots; ventral hands and ventral feet brown, tips of digits pale brown; metacarpal tubercle and metatarsal tubercle reddish; pectoral gland and femoral gland white.SYS a002082 has an \u201cX\u201d pattern on its back.Measurement data of type series are listed in Table Megophrys (Panophrys) shimentaina sp. nov. is known only from Shimentai Nature Reserve, northern Guangdong. This toad is uncommon in its distribution areas. All individuals were found from two slowly flowing mountain streams between elevations of 210\u2013500 m a.s.l. Males call on plant leaves from April to August, suggesting their breeding season corresponds to this period. Females and tadpoles have not been found.Currently, Megophrys (Panophrys) shimentaina sp. nov. were recorded from four males at 18\u201320 \u00b0C air temperature on 27 April 2016. Thirty calls with 96 notes are measured and the spectrograms are shown in Fig. N = 30) continuous click notes. Each call lasts 0.50 \u00b1 0.07 s  and each note lasts 85 \u00b1 8 ms  with an interval of 67 \u00b1 14 ms  between every two notes. The peak frequency measures at 4895 \u00b1 124 Hz .The advertisement calls of Taxon classificationAnimaliaAnuraMegophryidaeLyu, Zeng & Wangsp. nov.E8FD5215-EBFB-55AF-A138-6FA0F693AF26http://zoobank.org/F27079DE-C1AF-4B00-900F-1E1783C58762Megophrys sp2 , Shuangpai County, Yongzhou City, Hunan Province, PR China.875 Figs , 7, adulSYS a002874 and males SYS a002876/CIB 116072 and SYS a002878\u20132886, collected at the same time from the same locality as the holotype.Eleven adult specimens, female xiangnanensis is an adjective derived from Chinese Pinyin Xi\u0101ng N\u00e1n, which means southern Hunan, for the distribution area of this species.The specific epithet Southern Hunan Horned Toad (in English) / Xi\u0101ng N\u00e1n Ji\u0103o Ch\u00e1n (\u6e58\u5357\u89d2\u87fe in Chinese)SVL 38.6\u201342.0 mm  in adult males and SVL 44.4 mm in adult female; (2) snout rounded in dorsal view; (3) tympanum clear, TD/ED 0.38\u20130.49; (4) presence of weak vomerine ridge, absence of vomerine teeth; (5) margin of tongue rounded, not notched behind; (6) hindlimbs slender, heels just meeting and tibio-tarsal articulation reaching forward between eye and tympanum; (7) tibia 0.41\u20130.46 of SVL and foot 0.57\u20130.62 of SVL; (8) fingers without lateral fringes, presence of distinct subarticular tubercles at the bases; (9) toes with relatively wide lateral fringes and rudiment of webs, presence of distinct subarticular tubercles at the bases; (10) presence of small horn-like tubercle at the edge of upper eyelid; (11) dorsal skin smooth with sparse granules, a discontinuous X-shaped ridge with two discontinuous dorsolateral ridges on two side on the back; (12) sparse tubercles on the flanks; (13) supratympanic fold light colored; (14) single subgular vocal sac in males; (15) presence of nuptial pads on the dorsal surface of the first and second fingers in adult males.(1) Moderate body size, Megophrys (Panophrys) xiangnanensis sp. nov. with M. (Pa.) mirabilis sp. nov., M. (Pa.) shimentaina sp. nov., and 42 recognized congeners of Panophrys are given in Table Comparative data of Megophrys (Panophrys) xiangnanensis sp. nov. differs from M. (Pa.) mirabilis sp. nov. by the smaller body size, SVL 38.6\u201342.0 mm in males and 44.4 mm in single female , small horn-like tubercle at upper eyelid (vs. slightly large), wide lateral fringes on toes (vs. narrow), heels just meeting (vs. overlapping), presence of vocal sac in males (vs. absent), and presence of nuptial spines in males (vs. absent).Megophrys (Panophrys) xiangnanensis sp. nov. differs from M. (Pa.) shimentaina sp. nov. by the larger body size, SVL 38.6\u201342.0 mm in males , absence of vomerine teeth (vs. present), wide lateral fringes on toes (vs. narrow), and heels just meeting (vs. overlapping).Panophrys species previously recorded from the hilly areas among Guangdong, Guangxi and Hunan, Megophrys (Panophrys) xiangnanensis sp. nov. differs from M. (Pa.) acuta by the larger body size, SVL 38.6\u201342.0 mm in males and 44.4 mm in single female , small horn-like tubercle at upper eyelid (vs. slightly large), snout rounded in dorsal view (vs. strongly remarkably pointed), wide lateral fringes on toes (vs. narrow), and heels just meeting (vs. not meeting). M. (Pa.) xiangnanensis sp. nov. differs from M. (Pa.) brachykolos by the wide lateral fringes on toes (vs. absent), and heels just meeting (vs. not meeting). M. (Pa.) xiangnanensis sp. nov. differs from M. (Pa.) nanlingensis by the larger body size, SVL 38.6\u201342.0 mm in males , absence of vomerine teeth (vs. present), tongue not notched behind (vs. notched), wide lateral fringes on toes (vs. narrow) , and heels just meeting (vs. overlapping). M. (Pa.) xiangnanensis sp. nov. differs from M. (Pa.) obesa by the larger body size, SVL 38.6\u201342.0 mm in males and 44.4 mm in single female , wide lateral fringes on toes (vs. absent), and heels just meeting (vs. not meeting). M. (Pa.) xiangnanensis sp. nov. differs from M. (Pa.) shunhuangensis by the larger body size, SVL 38.6\u201342.0 mm in males and 44.4 mm in single female , wide lateral fringes on toes (vs. absent), and heels just meeting (vs. overlapping).Compared with the five SVL 38.6\u201342.0 mm in adult males, Megophrys (Panophrys) xiangnanensis sp. nov. is significantly different from 18 congeners whose SVL< 35 mm or > 45 mm in males, namely M. (Pa.) binlingensis, M. (Pa.) caudoprocta, M. (Pa.) cheni, M. (Pa.) jingdongensis, M. (Pa.) jiulianensis, M. (Pa.) kuatunensis, M. (Pa.) liboensis, M. (Pa.) lishuiensis, M. (Pa.) mufumontana, M. (Pa.) nankunensis, M. (Pa.) ombrophila, M. (Pa.) omeimontis, M. (Pa.) rubrimera, M. (Pa.) sangzhiensis, M. (Pa.) shuichengensis, M. (Pa.) spinata, M. (Pa.) wugongensis, and M. (Pa.) wuliangshanensis.With a moderate body size, Megophrys (Panophrys) xiangnanensis sp. nov. can be further distinguished from the remaining 19 congeners by the following characteristics: small horn-like tubercle at upper eyelid ; vomerine teeth absent ; tongue not notched behind ; lateral fringes on toes wide ; rudimentary webs on toes .SVL 40.9 mm; head width slightly larger than head length, HDW/HDL 1.02; snout rounded in dorsal view, projecting, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eyes large, ED 0.41 of HDL, pupil vertical; nostril oblique-ovoid; canthus rostralis well developed; loreal region slightly oblique; internasal distance slightly larger than interorbital distance; tympanum clear, TD/ED 0.44; large ovoid choanae at the base of the maxilla; presence of weak vomerine ridge, absence of vomerine teeth; margin of tongue rounded, not notched behind; presence of a single subgular vocal sac, a pair of slit-like openings at posterior of jaw.Adult male. Moderate body size, SVL and hand 0.23 of SVL; hand without webs, fingers without lateral fringes, relative finger length I < II < IV < III; tips of fingers slightly dilated, round; one distinct subarticular tubercle at the bases of each finger; inner metacarpal tubercle observably enlarged and the outer one smaller; a single nuptial pad on the dorsal surface of the first and second fingers. Hindlimbs slender, tibio-tarsal articulation reaching forward between eye and tympanum when hindlimb is stretched along the side of the body; heels just meeting when the flexed hindlimbs are held at right angles to the body axis; tibia length 0.42 of SVL and foot length 0.58 of SVL; relative toe length I < II < V < III < IV; tips of toes round and slightly dilated; toes with relatively wide lateral fringes and rudiment of webs; one distinct subarticular tubercle at the bases of each toe; inner metatarsal tubercle long ovoid and the outer one absent.Radio-ulna length 0.22 of Dorsal skin smooth with sparse granules; sparse tubercles on the flanks; a horn-like prominent tubercle on the edge; clear supratympanic fold curving postero-ventrally from posterior corner of eye to a level above insertion of arm; a discontinuous X-shaped ridge and two discontinuous dorsolateral ridges on two sides at the central back; sparse tubercles on the dorsal shank and thigh; ventral surface smooth; several tubercles on posterior hindlimbs; small pectoral gland closer to axilla; a single large femoral gland on rear of thigh.Yellowish brown above in life; a dark interorbital triangle with light colored center and edge; a dark X-shaped making with light edge on the central of dorsum; dark brown transverse bands on forearms and hindlimbs; supratympanic fold light colored; dark vertical band below the eye; iris light brown with net-like stripes; throat and anterior chest reddish gray; a longitudinal stripe on the throat; a large white blotch with scarlet spots on the belly; one pair of lateroventral longitudinal broad reddish stripes on two sides; ventral limbs flesh color; ventral hands purplish, tips of fingers pale-grey, metacarpal tubercle reddish; ventral feet purplish brown, tips of fingers pale grey, metatarsal tubercle reddish; pectoral gland and femoral gland white.SVL 44.4 mm) are slightly larger than males (SVL 38.6\u201342.0 mm).Measurement data of type series are listed in Table Megophrys (Panophrys) xiangnanensis sp. nov. is currently known only from Mt Yangming, southwestern Hunan. This toad inhabits areas near slowly flowing mountain streams surrounded by moist subtropical secondary evergreen broadleaf forests between elevations of 900\u20131400 m a.s.l. Males call from May to July, and during this time the males bear nuptial pads. Only one female individual was found, and tadpoles and other ecological information remain unknown.Megophrys (Panophrys) xiangnanensis sp. nov. were recorded from the Holotype at 16 \u00b0C air temperature on 12 June 2014. Four calls with 98 notes are measured and the spectrograms are shown in Fig. N = 4) continuous click notes. Each call lasts 9.46 \u00b1 1.77 s  and each note lasts 151 \u00b1 12 ms  with an interval of 240 \u00b1 95 ms  between every two notes. The peak frequency measures at 3033 \u00b1 123 Hz .The advertisement calls of Taxon classificationAnimaliaAnuraMegophryidaeLyu, Zeng & Wangsp. nov.0D94F88F-2455-5F77-8592-1AC34EF7D6E8http://zoobank.org/D466B824-AE2D-4EAA-94D1-A6BF39534942Megophrys sp28 , Shuangpai County, Yongzhou City, Hunan Province, PR China.887 Figs , 8, adulSYS a002877, and males SYS a2888\u20132889, 2891\u20132892, collected at the same time as the holotype; male SYS a002307 and SYS a002310/CIB 116073, collected on 8 September 2013 by Zu-Yao Liu.Seven adult specimens from the same locality as the holotype: female yangmingensis refers to its type locality, Mt Yangming.The specific epithet Mt Yangming Horned Toad (in English) / Y\u00e1ng M\u00edng Sh\u0101n Ji\u0103o Ch\u00e1n (\u9633\u660e\u5c71\u89d2\u87fein Chinese)SVL 33.2\u201337.1 mm  in adult males and SVL 45.2 mm in adult female; (2) snout rounded in dorsal view; (3) tympanum clear, TD/ED 0.42\u20130.50; (4) presence of weak vomerine ridge, absence of vomerine teeth; (5) margin of tongue rounded, not notched behind; (6) hindlimbs slender, heels overlapping and tibio-tarsal articulation reaching forward at the anterior corner of the eye; (7) tibia 0.47\u20130.51 of SVL and foot 0.64\u20130.69 of SVL in males, while tibia 0.44 of SVL and foot 0.51 of SVL in female; (8) fingers without lateral fringes, presence of distinct subarticular tubercles at the bases; (9) toes with lateral fringes and rudiment of webs, presence of subarticular tubercles at the bases; (10) presence of small horn-like tubercle at the edge of upper eyelid; (11) dorsal skin rough with sparse granules, a discontinuous X-shaped ridge with two discontinuous dorsolateral ridges on two side on the back; (12) sparse tubercles on the flanks; (13) orange-brown or light brown above, a dark interorbital triangle with light colored center and edge, a dark X-shaped making with light edge on the central of dorsum; (14) single subgular vocal sac in males; (15) presence of villiform black nuptial spines on the dorsal surface of the first and second fingers in adult males.(1) Body size small, Megophrys (Panophrys) yangmingensis sp. nov. with M. (Pa.) mirabilis sp. nov., M. (Pa.) shimentaina sp. nov., M. (Pa.) xiangnanensis sp. nov., and 42 recognized congeners of Panophrys are given in Table Comparative data of Megophrys (Panophrys) yangmingensis sp. nov. differs from M. (Pa.) mirabilis sp. nov. by the smaller body size, SVL 33.2\u201337.1 mm in males and 45.2 mm in single female , small horn-like tubercle at upper eyelid (vs. slightly large), presence of vocal sac in males (vs. absent), and presence of nuptial spines in adult males (vs. absent).Megophrys (Panophrys) yangmingensis sp. nov. differs from M. (Pa.) shimentaina sp. nov. by the larger body size, SVL 33.2\u201337.1 mm in males , absence of vomerine teeth (vs. present), and absence of tiny spines on the whole dorsal skin, flanks, dorsal limbs, the region around cloaca, and rear of hindlimbs (vs. such spines present).Megophrys (Panophrys) yangmingensis sp. nov. differs from M. (Pa.) xiangnanensis sp. nov. by the smaller body size, SVL 33.2\u201337.1 mm in males (vs. 38.6\u201342.0), heels overlapping (vs. just meeting), tibio-tarsal articulation reaching forward at the anterior corner of the eye (vs. between eye and tympanum), and narrow lateral fringes on toes (vs. wide).Panophrys species previously recorded from the hilly areas among Guangdong, Guangxi and Hunan, Megophrys (Panophrys) yangmingensis sp. nov. differs from M. (Pa.) acuta by the larger body size, SVL 33.2\u201337.1 mm in males and 45.2 mm in single female , small horn-like tubercle at upper eyelid (vs. slightly large), snout rounded in dorsal view (vs. strongly remarkably pointed), and heels overlapping (vs. not meeting). M. (Pa.) yangmingensis sp. nov. differs from M. (Pa.) brachykolos by the narrow lateral fringes on toes (vs. absent), and heels overlapping (vs. not meeting). M. (Pa.) yangmingensis sp. nov. differs from M. (Pa.) nanlingensis by the absence of vomerine teeth (vs. present), and tongue not notched behind (vs. notched). M. (Pa.) yangmingensis sp. nov. differs from M. (Pa.) obesa by the narrow lateral fringes on toes (vs. absent), and heels overlapping (vs. not meeting). M. (Pa.) yangmingensis sp. nov. differs from M. (Pa.) shunhuangensis sp. nov. by the presence of vomerine ridge (vs. absence).Compared with the five SVL 33.2\u201337.1 mm in adult males, Megophrys (Panophrys) yangmingensis sp. nov. is significantly different from nine congeners whose SVL > 40 mm in males, namely M. (Pa.) baolongensis, M. (Pa.) binlingensis, M. (Pa.) caudoprocta, M. (Pa.) jingdongensis, M. (Pa.) liboensis, M. (Pa.) omeimontis, M. (Pa.) sangzhiensis, M. (Pa.) shuichengensis, and M. (Pa.) spinata.With a small body size, Megophrys (Panophrys) yangmingensis sp. nov. can be further distinguished from the remaining 28 congeners by the following characteristics: small horn-like tubercle at upper eyelid ; vomerine teeth absent ; tongue not notched behind ; lateral fringes on toes narrow ; rudimentary webs on toes ; tympanum clear with distinct edge [vs. upper 1/4 of tympanum concealed by supratympanic fold in M. (Pa.) mufumontana]; tibio-tarsal articulation reaching forward at the anterior corner of the eye [vs. between tympanum and eye in M. (Pa.) xianjuensis].SVL 35.1 mm; head width slightly larger than head length, HDW/HDL 1.01; snout rounded in dorsal view, projecting, protruding well beyond margin of lower jaw; top of head flat; eyes large, ED 0.43 of HDL, pupil vertical; nostril oblique-ovoid; canthus rostralis well developed; loreal region slightly oblique; internasal distance slightly larger than interorbital distance; tympanum clear, TD/ED 0.43; large ovoid choanae at the base of the maxilla; presence of weak vomerine ridge, absence of vomerine teeth; margin of tongue rounded, not notched behind; presence of a single subgular vocal sac, a pair of slit-like openings at posterior of jaw.Adult male. Body size moderate, SVL and hand 0.23 of SVL; hand without webs, fingers without lateral fringes, relative finger length II < I < IV < III; tips of fingers slightly dilated, round; one distinct subarticular tubercle at the bases of each finger; inner metacarpal tubercle observably enlarged and the outer one smaller; villiform black nuptial spines on the dorsal surface of the first and second fingers. Hindlimbs slender, tibio-tarsal articulation reaching forward at the anterior corner of eye when hindlimb is stretched along the side of the body; heels overlapping when the flexed hindlimbs are held at right angles to the body axis; tibia length 0.51 of SVL and foot length 0.67 of SVL; relative toe length I < II < V < III < IV; tips of toes round and slightly dilated; toes with lateral fringes and rudiment of webs; one subarticular tubercle at the bases of each toe; inner metatarsal tubercle long ovoid and the outer one absent.Radio-ulna length 0.24 of Dorsal skin rough with sparse granules; sparse tubercles on the flanks and hindlimbs; several tubercles on upper eyelid, including a horn-like prominent tubercle on the edge; clear supratympanic fold curving postero-ventrally from posterior corner of eye to a level above insertion of arm; a discontinuous X-shaped ridge and two discontinuous dorsolateral ridges on two sides at the central back; four transverse skin ridges on the dorsal shank and thigh; ventral surface smooth; several granules on posterior hindlimbs; small pectoral gland closer to axilla; a single large femoral gland on rear of thigh.Orange-brown above in life; a triangular making with light edge between eyes; a dark X-shaped making with light edge on the central of dorsum; supratympanic fold light brown; dark vertical band below the eye; iris orange-brown; throat and anterior chest purplish brown; belly dark gray with a large white blotch on the central; ventral limbs purplish; ventral hands reddish brown with dark stripes, tips of fingers pale-grey, metacarpal tubercle reddish; ventral feet purplish, tips of fingers pale-grey, metatarsal tubercle reddish; pectoral gland and femoral gland white.SVL 45.2 mm) are distinctly larger than males (SVL 33.2\u201337.1 mm), while with relatively shorter hindlimbs. Dorsal surfaces lighter brown in SYS a002877, 2888\u20132889, 2891\u20132892.Measurement data of type series are listed in Table Megophrys (Panophrys) yangmingensis sp. nov. is only known from Mt Yangming, southwestern Hunan. This toad inhabits near flowing mountain streams over 1300 m a.s.l. Males call from early June to early September. Males found in early June bear well developed nuptial spines, while the spines are absent in males found in early September, suggesting the breeding season of this toad is before September. Only one female was found, and tadpoles and more ecological information remain unknown.Currently, Megophrys (Panophrys) yangmingensis sp. nov. were recorded from the Holotype at 16 \u00b0C air temperature on 12 June 2014. Five calls with 160 notes are measured and the spectrograms are shown in Fig. N = 5) continuous click notes. Each call lasts 7.38 \u00b1 2.08 s  and each note lasts 75 \u00b1 5 ms  with an interval of 160 \u00b1 31 ms  between every two notes. The peak frequency measures at 3424 \u00b1 82 Hz .The advertisement calls of Panophrys. Subsequently, eight of them were described as seven new species  nanlingensis after detailed morphological examination . Megophryinae as a single genus Megophrys with containing seven subgenera (corresponding to the seven molecularly resolved clades). To resolve these conflicts, Brachytarsophrys, which shows significant differences against other groups. Therefore, the recognition of genus Brachytarsophrys must be accepted, while further supported evidences for other genera are needed.The genus anophrys . However"}
+{"text": "Megophrys is described from Guizhou Province, China. Molecular phylogenetic analyses based on mitochondrial DNA and nuclear DNA sequences all strongly supported the new species as an independent clade sister to M.minor and M.jiangi. The new species could be distinguished from its congeners by a combination of the following characters: body size moderate (SVL 43.4\u201344.1 mm in males, and 44.8\u201349.8 mm in females; vomerine teeth absent; tongue not notched behind; a small horn-like tubercle at the edge of each upper eyelid; tympanum distinctly visible, rounded; two metacarpal tubercles on palm; relative finger lengths II < I < V < III; toes without webbing; heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level between tympanum and eye when leg stretched forward; in breeding males, an internal single subgular vocal sac in male, and the nuptial pads with black spines on dorsal surface of bases of the first two fingers.A new species of the genus  Megophrys Kuhl & Van Hasselt, 1822  is widely distributed in eastern and central China, throughout southeastern Asia, and extending to the islands of the Sunda Shelf and the Philippines .Three adult males and five adult females of the undescribed species were collected in Chishui National Nature Reserve, Chishui City, Guizhou Province, China  were amplified using the primers in BDNF) and recombination activating gene 1 (RAG1) were amplified using the primers and protocols in Six specimens of the undescribed species were included in the molecular analyses  and one Leptobrachiumboringii  were also downloaded  and Bayesian Inference (BI) methods, implemented in PhyML v. 3.0 (BIC) was used. As a result, the analyses selected the best partition scheme  and the GTR+ G + I model for each partition for mitochondrial DNA dataset, and as well, selected the best partition scheme  and the GTR+ G + I as the best model for all codon position of RAG1 and BDNF genes. For the ML tree, branch supports were drawn from 10000 non-parametric bootstrap replicates. In BI analyses, two runs each with four Markov chains were run for 40 million generations with sampling every 1000 generations. The first 25% of generations were removed as the \u201cburn-in\u201d stage followed by calculation of Bayesian posterior probabilities and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity. Finally, genetic distance between species under uncorrected p-distance model was estimated on 16S gene sequences using MEGA v. 6.06 .Phylogenetic relationships were reconstructed based on the mitochondrial DNA data and nuclear DNA data, respectively. Phylogenetic analyses were conducted using maximum likelihood ;FL foot length (distance from tarsus to the tip of fourth toe);HDL head length (distance from the tip of the snout to the articulation of jaw);HDW maximum head width (greatest width between the left and right articulations of jaw);HLL hindlimb length ;IND internasal distance ;IOD interorbital distance (minimum distance between the inner edges of the upper eyelids);LAL length of lower arm and hand ;LW lower arm width (maximum width of the lower arm);SL snout length (distance from the tip of the snout to the anterior corner of the eye);SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent);TFL length of foot and tarsus ;THL thigh length (distance from vent to knee);TL tibia length (distance from knee to tarsus);TYD maximal tympanum diameter;TW maximal tibia width;UEW upper eyelid width (greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis).Megophrys congeners. Comparative data were obtained from related species as described in literature  and M.nankunensis Wang, Zeng & Wang, 2019; Suppl. material Genetic distances on16S gene with uncorrected Taxon classificationAnimaliaAnuraMegophryidae6C144FD5-3754-53EB-A648-CF06D30DC197http://zoobank.org/20B6A80B-E937-4443-88A2-E357B77DB6CAHolotype. CIBCS20190518031 , collected by Shi-Ze Li on 18 May 2019.031 Figs , 5, adulParatype. Two adult males and five adult females from the same place as holotype, collected by Shi-Ze Li and Jing Liu. Two females CIBCS20190518022 and CIBCS20190518023 collected by Jing LIU on 18 May 2019, two adult males CIBCS20190518019 and CIBCS20190518021 and three adult females CIBCS20190518025, CIBCS20190518027 and CIBCS20190518030 collected by Shi-Ze Li on 18 May 2019.Megophryschishuiensis sp. nov. is assigned to the genus Megophrys based on molecular phylogenetic analyses and the following generic diagnostic characters: snout shield-like; projecting beyond the lower jaw; canthus rostralis distinct; chest glands small and round, closer to the axilla than to midventral line; femoral glands on rear part of thigh; vertical pupils  body size moderate  vomerine teeth absent; (3) tongue not notched behind; (4) a small horn-like tubercle at the edge of each upper eyelid; (5) tympanum distinctly visible, rounded; (6) two metacarpal tubercles on palm; (7) relative finger lengths II < I < V < III; (8) toes without webbing; (9) heels overlapping when thighs are positioned at right angles to the body; (10) tibiotarsal articulation reaching the level between tympanum and eye when leg stretched forward. In breeding male, (11) an internal single subgular vocal sac; (12) nuptial pads with black spines on dorsal surface of bases of the first two fingers.Figs , 5. SVL Forelimbs slender, the length of lower arm and hand 42.4% of SVL; fingers slender, relative finger lengths: II < I < V < III; tips of digits globular, without lateral fringes; subarticular tubercle distinct at the base of each finger; two metacarpal tubercles, prominent, the outer one long and thin, the inner one oval-shaped.Hindlimbs slender, 1.48 times SVL; heels overlapping when thighs are positioned at right angles to the body, tibiotarsal articulation reaching tympanum to eye when leg stretched forward; tibia length longer than thigh length; relative toe lengths I < II < V < III < IV; tips of toes round, slightly dilated; subarticular tubercles absent; toes without webbing; no lateral fringe; inner metatarsal tubercle oval-shaped; outer metatarsal tubercle absent.Dorsal skin rough, with numerous granules; several large warts scattered on flanks; a small horn-like tubercle at the edge of each upper eyelid; tubercles on the dorsum forming a weak X-shaped ridge, the V-shaped ridges disconnect; two discontinuous dorsolateral parallel ridges on either side of the X-shaped ridges; an inverted triangular brown speckle between two upper eyelids; several tubercles on the flanks and dorsal surface of thighs and tibias and forming four transverse tubercle rows; supratympanic fold distinct.Ventral surface smooth; chest with small and round glands, closer to the axilla than to midventral line; femoral glands on rear of thighs, numerous white granules on outer thighs; posterior end of the body distinctly protruding and forming an arc-shaped swelling above the anal region.Fig. . An inveFig. . Color oIn CIBCS20190518027, the back is brown with some brick-red granules Fig. ; in CIBCN = 10) notes. Call duration was 2.10\u20133.18 second . Call interval was 0.92\u20131.32 seconds . Each note had a duration of 0.07\u2013 0.12 seconds  and the intervals between notes 0.038\u20130.085 seconds . Amplitude modulation within note was apparent, beginning with moderately high energy pulses, increasing slightly to a maximum by approximately mid note, and then decreasing towards the end of each note. The average dominant frequency was 5859 \u00b1 118.02.61 .The call description is based on recordings of the holotype CIBCS20190518031 Fig.  from theAdult females with SVL 44.8\u201349.8 mm, larger than adult males with 43.4\u201344.1 mm. Adult males have a single subgular vocal sac. In breeding males, brownish red nuptial pads are present on dorsal surface of the bases of the first and second fingers with black spines obvious under microscope.Megophryschishuiensis sp. nov. differs from M.aceras Boulenger, 1903, M.auralensis Ohler, Swan & Daltry, 2002, M.carinense Boulenger, 1889, M.caudoprocta Shen, 1994, M.chuannanensis , M.damrei Mahony, 2011, M.edwardinae Inger, 1989, M.feae Boulenger, 1887, M.flavipunctata Mahony, Kamei, Teeling & Biju, 2018, M.gigantica Liu, Hu & Yang, 1960, M.glandulosa Fei, Ye & Huang, 1990, M.himalayana Mahony, Kamei, Teeling & Biju, 2018, M.intermedia Smith, 1921, M.jingdongensis Fei & Ye, 1983, M.kalimantanensis Munir, Hamidy, Matsui, Iskandar, Sidik & Shimada, 2019, M.lekaguli Stuart, Chuaynkern, Chan-ard & Inger, 2006, M.liboensis , M.major Boulenger, 1908, M.mangshanensis Fei & Ye, 1990, M.maosonensis Bourret, 1937, M.medogensis Fei, Ye & Huang, 1983, M.omeimontis Liu, 1950, M.oreocrypta Mahony, Kamei, Teeling & Biju, 2018, M.orientalis , M.periosa Mahony, Kamei, Teeling & Biju, 2018, M.popei , M.sangzhiensis Jiang, Ye & Fei, 2008, M.shapingensis Liu, 1950, M.shuichengensis Tian & Sun, 1995, and M.takensis Mahony, 2011 (maximum SVL < 49.8 mm in the new species vs. minimum SVL > 53 mm in the latter), and differs from M.acuta Wang, Li & Jin, 2014, M.angka , M.caobangensis Nguyen, Pham, Nguyen, Luong & Ziegler, 2020, M.damrei Mahony, 2011, M.dongguanensis Wang & Wang, 2019, M.cheni, M.jiangi, M.jinggangensis , M.jiulianensis Wang, Zeng, Lyu & Wang, 2019, M.kuatunensis Pope, 1929, M.lini , M.lishuiensis , M.mufumontana , M.minor, M.nanlingensis , M.obesa Wang, Li & Zhao, 2014, M.pachyproctus Huang, 1981, M.palpebralespinosa Bourret, 1937, M.serchhipii Mathew & Sen, 2007, M.shunhuangensis Wang, Deng, Liu, Wu & Liu, 2019, M.vegrandis Mahony, Teeling & Biju, 2013, M.wuliangshanensis Ye & Fei, 1995, M.wushanensis Ye & Fei, 1995, M.zunhebotoensis Mathew & Sen, 2007, M.xianjuensis Wang, Wu, Peng, Shi, Lu & Wu, 2020, and M.zhangi Ye & Fei, 1992 (vs. maximum SVL < 42 mm in the latter).By having medium body size, Megophryschishuiensis sp. nov. differs from M.aceras, M.ancrae Mahony, Teeling & Biju, 2013, M.carinense, M.baluensis , M.caudoprocta, M.chuannanensis, M.damrei, M.daweimontis Rao & Yang, 1997, M.dongguanensis, M.fansipanensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong & Rowley, 2018, M.flavipunctata, M.glandulosa, M.hoanglienensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong & Rowley, 2018, M.himalayana, M.insularis , M.intermedia, M.jingdongensis, M.jinggangensis, M.jiulianensis. M.kalimantanensis, M.kobayashii Malkmus & Matsui, 1997, M.lancip Munir, Hamidy, Farajallah & Smith, 2018, M.lekaguli, M.liboensis, M.ligayae Taylor, 1920, M.longipes Boulenger, 1886, M.major, M.mangshanensis, M.maosonensis, M.medogensis, M.megacephala Mahony, Sengupta, Kamei & Biju, 2011, M.montana Kuhl & Van Hasselt, 1822, M.nasuta , M.nankunensis, M.nanlingensis, M.omeimontis, M.oropedion Mahony, Teeling & Biju, 2013, M.oreocrypta, M.palpebralespinosa, M.parallela Inger & Iskandar, 2005, M.parva , M.periosa, M.popei, M.robusta Boulenger, 1908, M.rubrimera Tapley, Cutajar, Mahony, Chung, Dau, Nguyen, Luong & Rowley, 2017, M.sangzhiensis, M.stejnegeri Taylor, 1920, M.takensis, M.zhangi, and M.zunhebotoensis (vs. present in the latter).By the absence of vomerine teeth, Megophryschishuiensis sp. nov. differs from M.binchuanensis Ye & Fei, 1995, M.binlingensis, M.damrei, M.gigantica, M.minor, M.monticola , M.nasuta, M.nankiangensis Liu & Hu, 1966, M.oropedion, M.pachyproctus, M.spinata, M.stejnegeri, M.takensis, M.wuliangshanensis, M.wushanensis, M.zhangi, and M.zunhebotoensis (vs. lacking tubercle in the latter), and differs from M.carinense, M.feae, M.gerti , M.hansi , M.intermedia, M.kalimantanensis, M.koui Mahony, Foley, Biju & Teeling, 2017, M.latidactyla, M.liboensis, M.microstoma , M.palpebralespinosa, M.popei, M.shuichengensis, and M.synoria  (vs. having a prominent and elongated tubercle in the latter).By having a small horn-like tubercle at the edge of each upper eyelid, Megophryschishuiensis sp. nov. differs from M.ancrae, M.baolongensis Ye, Fei & Xie, 2007, M.binlingensis, M.boettgeri , M.carinense, M.cheni, M.chuannanensis, M.damrei, M.dringi Inger, Stuebing & Tan, 1995, M.fansipanensis, M.feae, M.feii Yang, Wang & Wang, 2018, M.flavipunctata, M.gerti, M.glandulosa, M.hoanglienensis, M.huangshanensis Fei & Ye, 2005, M.insularis, M.jiulianensis. M.jingdongensis, M.kalimantanensis, M.kuatunensis, M.liboensis, M.mangshanensis, M.maosonensis, M.medogensis, M.minor, M.nankiangensis, M.nanlingensis, M.omeimontis, M.oropedion, M.pachyproctus, M.parallela, M.popei, M.robusta, M.sangzhiensis, M.shapingensis, M.shuichengensis, M.spinata, M.vegrandis, M.wawuensis Fei, Jiang & Zheng, 2001, M.zhangi, and M.zunhebotoensis (vs. tongue notched behind in the latter).By having a tongue not notched behind, Megophryschishuiensis sp. nov. differs from M.acuta, M.auralensis, M.baolongensis, M.binchuanensis, M.boettgeri, M.carinense, M.cheni, M.chuannanensis, M.elfina Poyarkov, Duong, Orlov, Gogoleva, Vassilieva, Nguyen, Nguyen, Nguyen, Che & Mahony, 2017, M.feae, M.feii, M.flavipunctata, M.gigantica, M.glandulosa, M.hansi, M.intermedia, M.jingdongensis, M.jinggangensis, M.kuatunensis, M.latidactyla, M.lini, M.major, M.maosonensis, M.nankiangensis, M.omeimontis, M.palpebralespinosa, M.popei, M.rubrimera, M.sangzhiensis, M.serchhipii, M.shapingensis, M.shuichengensis, M.spinata, M.vegrandis, M.xianjuensis, M.zhangi, and M.zunhebotoensis (vs. present in these species).By lacking lateral fringes on the toes, Megophryschishuiensis sp. nov. differs from M.brachykolos Inger & Romer, 1961, M.carinense, M.flavipunctata, M.jingdongensis, M.jinggangensis, M.lini, M.major, M.palpebralespinosa, M.popei, M.shuichengensis, M.spinata (vs. at least one-fourth webbed).By having toes without webs at bases, Megophryschishuiensis sp. nov. differs from M.acuta, M.brachykolos, M.dongguanensis, M.huangshanensis, M.kuatunensis, M.nankunensis, M.obesa, M.ombrophila Messenger & Dahn, 2019, and M.wugongensis Wang, Lyu & Wang, 2019 (vs. not meeting).By heels overlapping when thighs are positioned at right angles to the body, Megophryschishuiensis sp. nov. differs from M.baolongensis, M.nankiangensis, M.pachyproctus, M.shuichengensis and M.tuberogranulata Shen, Mo & Li, 2010 (vs. just reaching posterior corner of the eye in the latter); differs from M.daweimontis, M.glandulosa, M.lini, M.major, M.medongensis, M.obesa, and M.sangzhiensis (vs. reaching the anterior corner of the eye or beyond eye or nostril and tip of snout in the latter); differs from M.leishanensis Li, Xu, Liu, Jiang, Wei & Wang, 2018 (vs. reaching middle part of eye in this group of species); and differs from M.mufumontana .With tibiotarsal articulation reaching to the level between tympanum and eye when leg is stretched forward, Megophryschishuiensis sp. nov. differs from M.caudoprocta, M.shapingensis, and M.shuichengensis .By having an internal single subgular vocal sac in male, Megophryschishuiensis sp. nov. differs from M.acuta, M.feii, M.shapingensis, and M.shuichengensis (vs. lacking in these species).By having nuptial pads and nuptial spines on dorsal surface of the base of the first two fingers in breeding males, M.carinense, M.jiangi, M.leishanensis, M.liboensis, M.shuichengensis, and M.spinata have sympatric distribution with Megophryschishuiensis sp. nov. , vomerine teeth absent (vs. present), horn-like tubercle at the edge of each upper eyelid small (vs. prominent), tongue not notched behind (vs. notched behind), lacking lateral fringe in toes (vs. present), and toes without webs at bases (vs. one-fourth webbed). The new species vs. M.jiangi: body size bigger , and relative finger lengths II < I < V < III vs. I < II < V < III. The new species vs. M.leishanensis: body size bigger , and tibiotarsal articulation reaching forward to the region between tympanum and eye when hindlimb is stretched along the side of the body vs. reaching middle part of eye. The new species vs. M.liboensis: body size smaller in adult females , vomerine teeth absent vs. vomerine teeth present, and horn-like tubercle at the edge of each upper eyelid is small vs. prominent. The new species vs. M.shuichengensis: body size smaller , horn-like tubercle at the edge of each upper eyelid is small vs. prominent, tongue not notched behind vs. tongue notched behind, lacking lateral fringe in toes vs. present, toes without webs at bases vs. one-fourth webbed, having an internal single subgular vocal sac in male vs. absent, and having nuptial pads and nuptial spines on the dorsal base of the first two fingers in breeding male vs. lacking. The new species vs. M.spinata: body size is smaller , horn-like tubercle at the edge of each upper eyelid is small vs. lacking tubercle, tongue not notched behind vs. notched behind, lacking lateral fringe in toes vs. present, and toes without webs at bases vs. one-fourth webbed.The congeners Megophryschishuiensis sp. nov. is phylogenetically closest to M.minor, and this new species could be identified from the latter distinctly by having larger body size , having a small horn-like tubercle at the edge of each upper eyelid (vs. absent in the latter), tongue not notched behind (vs. notched in the latter), tibiotarsal articulation reaching the level between tympanum to eye when leg stretched forward (vs. reaching the level between eye and tip of snout in the latter), and having two metatarsal tubercles in each hand (vs. absent in the latter).Megophryschishuiensis sp. nov. is known from the type locality, Chishui National Nature Reserve , Chishui City, Guizhou Province, China at elevations between 270\u2013604 m. The individuals of the new species were frequently found in bamboo forest nearby the streams , Zhangixalusomeimontis , and Ranaomeimontis Ye & Fei, 1993.chishuiensis refers to the distribution of this species, Chishui City, Guizhou Province, China. We propose the common name \u201cChishui horned toad\u201d and its Chinese name as Chi Shui Jiao Chan (\u8d64\u6c34\u89d2\u87fe).The specific name Megophryschishuiensis sp. nov., resembles M.minor and M.jiangi, and detailed comparison with different data sets are important for recognizing them. Our molecular phylogenetic data on mitochondrial DNA and nuclear DNA, and morphological comparisons both separated the new species from the two closely related species. Megophrysminor were reported to be distributed widely through the provinces of Sichuan, Guizhou, Chongqing, Yunnan, Guangxi, Jiangxi and north of Vietnam (Megophryschishuiensis sp. nov. and M.jiangi). In recent years, a lot of new species of the genus Megophrys have been gradually described, of which, a large part of number of species were found in China (Megophrys, 51 species were discovered in China. Even so, dozens of cryptic species need to be described (Chen et al. 2016; The new species,  Vietnam , but detin China . To now,Megophryschishuiensis sp. nov. with a narrow distribution also fits the \u201cmicro-endemism\u201d model like many other congeners ("}
+{"text": "In Parkinson's disease, the excess of beta oscillations in cortical-basal ganglia (BG) circuits has been correlated with normal movement suppression. In this paper, a physiologically based resonance model, generalizing an earlier model of the STN-GPe circuit, is employed to analyze critical dynamics of the occurrence of beta oscillations, which correspond to Hopf bifurcation. With the experimentally measured parameters, conditions for the occurrence of Hopf bifurcation with time delay are deduced by means of linear stability analysis, center manifold theorem, and normal form analysis. It is found that beta oscillations can be induced by increasing synaptic transmission delay. Furthermore, it is revealed that the oscillations originate from interaction among different synaptic connections. Our analytical results are consistent with the previous experimental and simulating findings, thus may provide a more systematic insight into the mechanisms underlying the transient beta bursts. Parkinson's disease ranks as the second most common neurodegenerative disorder after Alzheimer's disease \u20133. AccorA commonly acknowledged opinion is that these symptoms are associated with the degeneration of dopaminergic neurons in the substantia nigra par compacta, which releases the neurotransmitter dopamine to the basal ganglia . ParticuSeveral computational models have been proposed to elucidate the generation of pathologically exaggerated beta oscillations in Parkinson's disease \u201321. WithNote that the resonance model is mainly explored by data fitting and parameter identification , thus thS(t), G(t), E(t), and I(t) are the firing rates of STN, GPe, excitatory, and inhibitory populations, respectively. Str denotes the constant inhibitory input from striatum to GPe, and C denotes the constant component of extrinsic and intrinsic excitatory input to cortical excitatory neurons. The parameters Tij and wij represent transmission delay and connection weight, respectively. Here, the subscript i indicates the population from which the signal originates, and the subscript j indicates where the signal is received. \u03c4x denotes the membrane time constants for population x, describing how rapidly the population reacts to its inputs. Notice that the \u201cresonance\u201d is mainly reflected in the hypothesis that the oscillations in basal ganglia resonate to the excitatory cortical input.We begin with a review of the resonance model proposed by Alex Pavlides et al. , which iFX are the sigmoid activation functions expressing the relationship between firing rate and synaptic input, shown as followsMX is the maximum firing rate of population X, and BX is the firing rate in the absence of the synaptic input. The curves of these activation functions and their derivatives are shown in Figures The terms The values of all parameters in this paper, except for the transmission delay and connection weights, are summarized in \u03c4X were taken to have an average value of \u03c4 = 10ms; (ii) all transmission delays were taken to be equal, denoted by the single variable T. Then, we could get the following system:It is well known that one of the classical mechanism for occurring oscillations is the Hopf bifurcation, in which the attractive limit cycle and asymptotically stable equilibrium point could transform as parameters change. And these two states correspond to the Parkinsonian and healthy state in the STN-GPe model, respectively. Thus, we will investigate the conditions for Hopf bifurcation of the resonance model here. Since the model is difficult to analyze mathematically, we make two simplifications: (i) the membrane time constants u0 = T, and the model (3) around the equilibrium point can be formally rewritten intou(t) = (S(t) \u2212 S\u2217, G(t) \u2212 G\u2217, E(t) \u2212 E\u2217, I(t) \u2212 I\u2217)T. Here, f = T is deduced from the higher-order terms of the vector field and B1 = \u2212(1/\u03c4)I4\u00d74denoting the equilibrium point of the system as du/dt = B1u(t) + B2u(t \u2212 T), the characteristic equation is given byFor the linearized system i\u03c9(\u03c9 > 0), the stability should be changed, and a Hopf bifurcation may occurs. So, we consider \u03941(i\u03c9) = 0 and \u03942(i\u03c9) = 0 next. \u03bb = i\u03c9(\u03c9 > 0) is a root of \u03941(\u03bb), i.e.,Suppose that As well known, if Eq.  has a paSeparating real parts and imaginary parts of Eq. , one canThen, one can further obtain2(\u03c9T) + cos2(\u03c9T) = 1, we haveAccording to Eqs.  and 12)12) and sz = \u03c92, then Eq. . Then, Eq. . Eq.  has at lhen, Eq.  has fourg to Eq. , we can i\u03c9k is a pair of purely imaginary roots of Eq. (Tkj. \u03bb = i\u03c9(\u03c9 > 0) is a root of \u03942(\u03bb), i.e.,(ii) Suppose that Then \u00b1s of Eq.  with TkjSo, we know that i\u03c90.Finally, let the critical time delay as Next, we need to verify the transversality condition. Differentiating the two sides of Eq.  on time Thus,PRQR + PIQI \u2260 0 holds, Re{(d\u03bb/dT)\u03bb=i\u03c90\u22121} \u2260 0, the transversality condition for Hopf bifurcation is satisfied.Therefore, if (H2) On the other hand, we need to prove that the remaining roots of Eq.  have str\u03c4i \u2265 0 and pji) are constants. As  varies, the sum of orders of the zeros of p in the open right half plane can change only if a zero appears on or crosses the imaginary axis., 29. ConT = 0, Eq. -(H3) hold, system (3) undergoes a Hopf bifurcation at the equilibrium T0. At first, let v(t) = u(Tt), T = T0 + \u03bc, \u03bc \u2208 R, then the system (4) can be transformed into the following formL\u03bc : C\u27f6R4, F : R \u00d7 C\u27f6R4are given byIn \u03b7, \u03b8 \u2208 , such thatBy the Riesz representation theorem, there exists a function \u03b7 = (T0 + \u03bc)[B1\u03b4(\u03b8) + B2\u03b4(\u03b8 + 1)], where \u03b4(\u03b8) is the Dirac delta function, i.e.,In fact, we can choose \u03c6 \u2208 C1, , we defineFor Then, the system (27) can be transformed into the following operator equation:A\u2217 of A is given byThe adjoint operator i\u03c90T0 are eigenvalues of A(0) and A\u2217(0), let q(\u03b8) = TeiT0\u03c90\u03b8(\u22121 < \u03b8 \u2264 0) be the eigenvectors of A(0) corresponding to eigenvalue i\u03c90T0, andq\u2217(s) = (1/\u03c1)TeiT0\u03c90s(0 \u2264 s < 1) be the eigenvectors of A\u2217(0) corresponding to the eigenvalue \u2212i\u03c90T0. With a simple computation, we can obtainAccording to the discussion in q\u2217, q\u232a = 1, And from the definition of the bilinear inner product\u03bb = \u00b1i\u03c90, and all other eigenvalues have strictly negative real parts. So the whole infinite-dimensional state space C could be decomposed into two complementary subspaces, namely, C = EC + ES [EC is the two-dimensional subspace spanned by the eigenvectors corresponding to \u00b1i\u03c90, termed the center eigenspace. And ES corresponds to the subspace complementary to EC, in which the real part of all eigenvalues is negative. Then, based on the center manifold theorem, there exist a two-dimensional center manifold C0, and the dynamical flow of the system (24) on it can be transformed intoz is the local coordinate for C0 in the direction of q for the solution of Eq. plane readsThus, our system in the (o(|\u03be|3) represents all terms of fourth and higher order in |\u03be|, andgij(i + j = 2) and g21 can be explicitly determined. Let us rewrite o(|z|3) including all terms of fourth and higher order in |z|; then, the calculation of gij(i + j = 2) is straightforward.Then, we apply the normal form theory to deduce the Poincare normal form for the Hopf bifurcation, i.e.,F = f2(vt(\u22121)) and inserting By keeping t in Eq. , we getg21 by two parts. The calculation of g20. Insertion of On the other hand, consideringinto Eq.  yields(EC to its complementary subspace ES. With the second equation of Eq. (W20(\u22121) and W11(\u22121) can be computed based on the following equations, respectively.The calculation of n of Eq.  in mind,follows:Hz,z\u00af=H20s of Eq. , we haveThen, insertinginto Eq.  to get(gij(i + j = 2) and g21 all available, one can obtain c1(0) as Eq. (\u03bc(\u03b5) which determine the exists of the periodic solution, the period T(\u03b5) and the nontrivial Floquet exponent near 0 of the periodic solution are given byWith ) as Eq. , and the\u03bc2 determines the direction of the Hopf bifurcation: if \u03bc2 > 0 (\u03bc2 < 0), the Hopf bifurcation is supercritical The sign of T2 determines the period of the bifurcating periodic solutions: if T2 > 0 (T2 < 0), the period increases (decreases)The sign of \u03b22 determines the stability of the bifurcating periodic solutions: if \u03b22 < 0 (\u03b22 > 0), the bifurcation periodic solutions are stable (unstable)The sign of Therefore, we have the following results:wSG = 2.56, wGS = 3.22, wCC = 2.75, wCS = 6.60, wGG = 0.90, C = 277.94 and Str = 40.51. In the calculations, we distinguish between the supercritical and subcritical Hopf bifurcation by ramping up and ramping down the parameter across the critical point with the Euler integration, respectively [Synaptopathy is the earliest step in the Parkinson's disease cascade . To clarectively .T0 = 3.6807ms, with \u03bc2 = 5.5252 \u00d7 10\u22124, \u03b22 = \u22121.4874 \u00d7 10\u22125, and T2 = 8.6223 \u00d7 10\u22126. Here, the signs of \u03bc2, \u03b22, and T2 imply that the Hopf bifurcation is supercritical, the periodic solution is stable, and the period increases with increasing time delay, respectively. To confirm the theoretical results, let us resort to T0 = 3.6807ms, starting from a stable equilibrium point  is located in domain I, the system converges to a stable equilibrium point, but excessive oscillations at beta frequencies occur when the parameter is within domain II. Hence, the system always stays at a healthy state for small wCS or wSG. This is equally to say that the beta oscillations cannot be generated in the single STN-GPe circuit but can originate from interaction among different neuronal population circuits, which is in agreement with Ref [The above numerical results show that the critical points for Hopf bifurcation in the resonance model can be influenced not only by STN-GPe connection but also the excitatory input from the cortex to STN. To illustrate how the beta oscillation onset depends on both the STN-GPe and cortex-STN connection strength, we depict the codimension-two bifurcation diagram in with Ref . TherefowCS and the transmission delay T is shown in wCS = 8.0 fixed, the variation of the nonzero oscillation frequency with transmission delay in The codimension-two bifurcation diagram with the connection weight We have investigated critical conditions for pathological beta oscillation onset in the resonance model based on the center manifold theorem and normal form analysis. It is confirmed that the model undergoes a supercritical Hopf bifurcation as the synaptic transmission delay increases, which governs the transitions from the healthy state to the Parkinsonian state. It is found that a strong excitatory connection from STN to GPe is favorable for the generation of beta oscillations, while excessive excitatory input from cortex to STN would suppress beta oscillations. Particularly, the codimension-two bifurcation diagram suggests that the beta oscillation onset depends on the interaction of the STN-GPe circuit and Cortex-STN synaptic connection. Our investigation has demonstrated that a suitable transmission delay is responsible for the emergence of the beta oscillation. The investigation could be inspiring for clinical physician in treating Parkinsonian patients.In the near future, this study can be extended to generalized models with more biological conditions such as the feedback connection from the STN-GPe circuit to the cortex. Note that the model of this study considers only four populations, namely, the excitatory population and the inhibitory population of cortex, the subthalamic nucleus and globus pallidus external segment, thus, for more insight in this regard, one may consider the more complicated model  which ca"}
+{"text": "Varicocele is a common cause of male infertility with multifactorial etiology. Inflammation is acharacteristic pathological event that occurs in the testis tissue following the varicocele. The aim of this study was toinvestigate expression of nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome componentsand cytokines in semen of varicocele and control subjects.In this case-control study, seminal plasma was collected from 32 varicocele patients (withgrades 2 and 3) and 20 fertile men as control group. Semen analysis was performed in all subjects. Concentrationsof interleukin-1b (IL-1b), IL-18 and caspase-1 in seminal plasma were measured by enzyme-linked immunosorbentassay (ELISA). Apoptosis-associated speck-like protein containing a caspase activation and recruitment domain, inaddition to NALP3 were identified in seminal plasma by Western blot. Statistical significance between the meanvalues was determined by student\u2019s t test.According to our data, the level of IL-1b was significantly (P=0.03) increased in the seminal plasma ofvaricocele patients, compared to the control subjects. We analyzed amount of IL-18 in the both groups. The level ofthis interleukin was markedly (P=0.002) decreased in varicocele patients. No change was observed in the level ofcaspase-1 in both groups. Western blot analysis revealed that apoptosis associated speck-like protein and NLRP3 (P=0.005) were significantly elevated in the semen of varicocele patients.This study provides the first evidence of activation of NLRP3 components in semen of men with varicocele. Varicocele is one of the most common causes of maleinfertility. Approximately, 15% of healthy men and 40%of infertile men suffer from varicocele . VaricocIn varicocele disease, heat stress induces undesirableadverse effects on testis tissue, such as spermatogenesisimpairment, increase in production of reactive oxygenspecies (ROS) and apoptosis . On the Inflammation is an immune response to pathologicalevents, such as bacterial/viral infection and tissue damageto protect other cells from injury . InflammNLRP3 mRNA in rat testis tissue wasinduced seven days after spinal cord injury (SCI)  .In addiry (SCI) . Recentlry (SCI) . Due to ry (SCI) , 11, we This study was performed from December 2017to September 2018. Sample size was calculated by thefollowing formula:where type one (\u03b1) and type two errors (\u03b2) were 0.05 and 0.20, respectively according to previous studies, 18. BasSemen samples were collected from varicocele andcontrol subjects by masturbation after at least 48 hoursof sexual abstinence. After liquefaction for 30 minutes,semen parameters including volume, pH, concentration,morphology and motility were analyzed according to theWorld Health Organization criteria . AnalysiSemen samples were centrifuged at 1000 xg for 15minutes at room temperature. The supernatant was thencollected and stored at -70\u00b0C for further analysis .Concentrations of the mature IL-1\u03b2, IL-18 and caspase-1 were measured by an enzymelinked immunosorbent assay (ELISA) kit following themanufacturer\u2019s protocol. Seminal plasma samples werethawed at room temperature and placed in plates precoated with a specific monoclonal antibody for each ofIL-1b, IL-18 or caspase-1. Each sample was duplicatelyassayed.Seminal plasma samples were thawed at roomtemperature. One microliter of seminal plasma from eachsubject was mixed with loading buffer , and heated at 95\u00b0Cfor 10 minutes. Protein concentrations were determinedusing the BCA\u2122 Protein Assay Kit according to the manufacturer\u2019s protocol. The sameamount of protein samples was loaded, separated on8-12% (v/v) discontinuous sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) andtransferred onto a polyvinylidenefluoride (PVDF)membrane . After blocking with 5%skimmed milk in Tris-buffered saline containing 0.05%Tween 20 (TBS-T) for 1 hour at room temperature, PVDFmembranes were incubated with anti-ASC antisera or anti-NLRP3antisera  overnight at 4oC.After washing with TBS-T, membranes were incubatedwith a peroxidase-conjugated goat anti-rabbit  secondary antibody for 2 hours atroom temperature. Visualization was performed usingthe enhanced chemiluminescence method  according to the manufacturer\u2019sprotocol. For densitometric quantification, intensity of thespecific bands was normalized to \u03b2-actin  in the same blot using Image J software .The results are expressed as means \u00b1 standard errors(SE). The Shaprio-wilktest was used to determine normaldistribution. Independent sample t test was applied tocheck the matched factor between the case and controlgroups. Statistical significance between the mean valueswas determined by paired t test and P\u22640.05 was consideredstatistically significant.In this study, we analyzed relationship of spermparameters with NLRP3, ASC, IL-18 and caspase-1 inboth control and varicocele groups. We could not find anycorrelation in our study.The semen volume (5.1 \u00b1 1.8 ml in control vs. 4.06 \u00b11.5 in varicocele) and pH (7.8 \u00b1 0.2 in control vs. 7.7 \u00b10.1 in varicocele) were not significantly different betweenthe control and varicocele subjects. The median spermconcentration in the varicocele group (44 million/ml) wassignificantly lower than the control group .The median sperm total motility was equal inthe both groups (61% in control vs. 55% in varicocele)while the sperm progressive motility in varicocele patientsshowed a significant decrease (P=0.0003) compared to thecontrol subjects. In addition, varicocele patients had moreabnormal sperm  morphologycompared to the control group (99% in varicocele vs. 80%in control).The levels of IL-1\u03b2, IL-18 and caspase-1 wereinvestigated in seminal plasma by ELISA. Our datashowed that IL-1\u03b2 was significantly increased (P=0.03)in seminal plasma of varicocele patients in comparisonwith control subjects . In these patients,concentration of caspase-1 showed no obvious change, while sTo investigate whether inflammasome components areexpressed and changed in seminal plasma of varicocelepatients, we quantified respectively ASC and NLRP3protein levels by Western blot. NLRP3 (P=0.005) andASC (P=0.0002) protein levels were significantlyelevated in varicocele patients versus control subjects.In this study, quality of semen in all varicocele patientswas lower than the control group. Varicocele patients hadabnormal semen quality  which miIn this study our results showed that ASC and NLRP3levels in semen of varicocele subjects were significantlyelevated compared to the control subjects. Additionally,concentration of IL-1\u03b2 was higher in varicocele versuscontrol subjects, whereas IL-18 was decreased in seminalplasma of varicocele patients and caspase-1 was notchanged. In addition, we could not find any significantcorrelation between sperm parameters and NLRP3inflammasome components.Sahin et al.  acclaimeIn this study, we expected that IL-18 level wouldbe increased in varicocele subjects , while iWestern blot analysis showed high level of NLRP3 andASC protein expressions in seminal plasma of varicocelepatients. In our previuos work, we showed high levels ofASC, NLRP3 and caspase 1 expression in the testis tissueof varicocele-induced rats, three months after surgery .Novelty is the strength of this study, as this is the firstevidence for the existence and presence of the NLRP3inflammasome in seminal plasma of varicocele patients.Thus, it highlights the importance of anti-inflammasometherapies to improve the fertility rate in varicocele patients.However, a limitation is about the control subjects. It wasbetter to choose fertile varicocele subjects as control group.The other weak point is that the immunohistochemistrywas not done in this study to localize this complex. Findings obtained from this study suggest that NLRP3activation occurs in varicocele and it might be responsiblefor pathological procedure occurring in varicocele patients.Details of the NLRP3 inflammasome activation processhas not been clarified yet. At present, it is not clear whetherNLRP3 is causally related to the onset of varicocele or theresult of pathological damages appearing in the courseof this gonadal disease. Further study is underway todetermine time course of activation of inflammasome invaricocele disease."}
+{"text": "The reliability and safety of power transmission depends first and foremost on the state of the power grid, and mainly on the state of the high-voltage power line towers. The steel structures of existing power line supports (towers) have been in use for many years. Their in-service time, the variability in structural, thermal and environmental loads, the state of foundations (displacement and degradation), the corrosion of supporting structures and lack of technical documentation are essential factors that have an impact on the operating safety of the towers. The tower state assessment used to date, consisting of finding the deviation in the supporting structure apex, is insufficient because it omits the other necessary condition, the stress criterion, which is not to exceed allowable stress values. Moreover, in difficult terrain conditions the measurement of the tower deviation is very troublesome, and for this reason it is often not performed. This paper presents a stress-and-strain analysis of the legs of 110 kV power line truss towers with a height of 32 m. They have been in use for over 70 years and are located in especially difficult geotechnical conditions\u2014one of them is in a gravel mine on an island surrounded by water and the other stands on a steep, wet slope. Purpose-designed fiber Bragg grating (FBG) sensors were proposed for strain measurements. Real values of stresses arising in the tower legs were observed and determined over a period of one year. Validation was also carried out based on geodetic measurements of the tower apex deviation, and a residual magnetic field (RMF) analysis was performed to assess the occurrence of cracks and stress concentration zones. Power transmission lines extend over hundreds of kilometers. The collapse of one or more towers may have serious consequences, resulting in a local shutdown or even a blackout. Therefore, the systematic testing, maintenance and repair of tower elements are necessary. Unfortunately, power line operators often fail to do so. This especially applies to older structures which have been in use for over 30 years. The repair or replacement of a tower structure element is difficult because it is usually done manually or using a small winch. The tower\u2019s location on a steep mountainside or an island surrounded by water often creates an additional difficulty.Periodic testing is performed by measuring deformation or the tower apex deviation. An interesting analysis of a high-voltage power line tower structure deformation is presented in . The anaNumerical as well as numerical-and-experimental simulations of power line towers were also performed, which were described, for example, in the works of Yin  and Lam An analysis of damage to a power transmission tower due to strong wind was conducted in ,18. BothDepending on the standard, they are included in the range of 250\u2013270 MPa for the tower legs and 100\u2013140 MPa for the diagonal elements. The results of the analyses indicate that more emphasis should be put on the design of diagonal elements. The analyzed tower was designed using steel angle sections. A combination of an interesting analysis of a full-scale test and a numerical simulation of a power transmission tower with a height of 46.05 m and a 7.14 m \u00d7 7.14 m base under the impact of wind is presented in ,22,23.The full-scale test is the most reliable way to reflect the power transmission tower\u2019s mechanical properties. Obviously, the experimental cost is very high and the test is only possible for new tower types ,25,26.Ma et al.  proposedA system monitoring the deformation of the structure of power transmission towers was built. The system was based on fiber Bragg grating  strain sensors. The essence of the solution was to introduce FBG sensors in certain points of the tower structure as a kind of \u201cnervous system\u201d. In the case of supporting structures of power transmission lines, the appropriate places were those with the highest bending moment and the biggest compressive stresses. The places were pre-determined using numerical simulations and they were located at the tower base. The entire optical fiber system intended for the strain-and-stress state measurement was duly calibrated. Special grips had been designed earlier to make it possible to fix the strain sensor to the selected tower leg repeatedly. The FBG strain sensor is presented in Based on preliminary testing, the measuring base L = 1000 mm was adopted. Using special grips, the sensor axis was placed in the center of gravity of the tower leg angle section.A and B calibration parameters. A = 7.8648540 \u00d7 10\u22127 [\u03bc\u03b5\u22121]; B = 5.99813502 \u00d7 10\u22126 [\u00b0C].The measuring part of each strain sensor was a Bragg grating with a specific wavelength embedded in an optical fiber. One sensor was used for each tower leg, i.e., there were four sensors per tower. Due to the optical interrogator traceability, a difference of at least 5 nm between the wavelengths of each Bragg grating had to be kept. A change in strains in the tested structural element, i.e., in the tower leg, caused a change in the wavelength in the Bragg grating. This relation is described by the following formula (1):(a)testing the tower structural elements together with installed FBG SC-01 sensors with a measuring base of 1000 mm. The testing was carried out on the tower leg fragment in the form of 80 mm \u00d7 80 mm \u00d7 8 mm and 75 mm \u00d7 75 mm \u00d7 6 mm angle sections with special grips fixing the optical fiber sensors and enabling the appropriate initial tension;(b)for the assumed constant 5 kN increment in force set by the strength-testing machine, strains corresponding thereto and established experimentally using the FBG SC-01 sensor were determined.Another important factor was to select an appropriate initial tension of the sensor. The sensor\u2019s initial tension was set precisely using right-and-left nuts with a fine thread. Apart from the dedicated optical fiber sensors, the system included a 2 kHz FBG-800 optical interrogator , a recorder, special software, a multiplier and telecommunication fibers. An option was also possible with a wireless transfer of measurement results from the interrogator. The constructed optical fiber system intended for the measurement of strains arising in the tower legs was first tested in laboratory conditions. The tests consisted of setting the known force and strain values to a fragment of a tower leg cut-out together with the FBG sensor fixed on it. The fixing manner ensured that the cut-out response was identical with that of the actual tower leg. Considering the required spacing between the traverses of the strength-testing machine with Accuracy Class 0.5, the tests were performed in the Research and Supervisory Center of Underground Mining. The diagram of the tension and compression test rig is presented in The testing results proved unequivocally that the optical fiber sensor and the method of fixing the sensor enabled a correct measurement of strains arising in the tested angle section of the tower leg.The angle section with the attached grips and the installed FBG sensor was subjected to a tensile force and a compressive force included in the same range as in the tower leg real conditions. The results of the testing are illustrated by the load-strain characteristic plotted for the tested leg cf. .For every 5 kN increment in force, there was an increment in strain totaling 23 \u00b5strain. It can be observed that constant increments in load result in constant increments in strain. The testing results prove unequivocally that the optical fiber sensor and the method of fixing the sensor to the tower leg angle section enabled a correct measurement of strains in the case of both tensile and compressive forces.Using the results of the laboratory testing, performed on the strength-testing machine , of the angle sections used as the power line tower supporting elements and of the designed grips with the FBG sensors fixed to them, the angle sections were verified and re-designed to obtain the final form, as presented in The change in design made the assembly of the grips easier and ensured an appropriate and constant initial tension of the sensors at a level of about 600 \u00b5strain.The strain testing results were composed of two parts. The first presented the results of measurements of strains arising in the tower legs under a simulated short-term lateral load of 500 N. The load was applied in the direction of the tower two axes  and along its diagonals (III). A diagram illustrating the loading is presented in The load applied to the tower in direction II resulted in compressive strains of \u221214 \u00b5strain in legs 1 and 2. A tensile strain of 10 \u00b5strain arose in the legs on the tower\u2019s opposite side. The strain measurement results are presented in This meant the occurrence of tensile stresses of 2.1 MPa and compressive stresses of about \u22122.82 MPa. If a load was applied in direction III, the values of strain for leg XP1 total \u221219 \u00b5strain, and for the leg located diagonally, 14 \u00b5strain. The highest strain values were recorded if the tower was loaded along the axis in relation to which the tower apex was shifted. The recorded strain values totaled 21 \u00b5strain cf. , which, The second part of the testing concerned the observation of strains arising in selected legs of the tower over a period of one year. In the case of the legs of tower A, located on an island in a gravel mine area, strains rose in the winter period to 230\u2013270 \u00b5strain and then returned, in the case of leg 1, to the initial level of 150 \u00b5strain. In the case of tower B, the course of the changes was similar, but the strain values did not return to the initial state and kept at the level of 230 \u00b5strain. The tower leg was shortened, which meant that the stresses arising in the two legs were compressive. The strain values for the other two legs were similar but with the opposite sign, which meant that there was tension. By determining real strain values in the tower legs, it was possible to define the performance of the tower foundation. The tower rotation axis (direction II) at the tower base is marked in The changes in stresses were correlated with the changes in temperature cf. . AnalyziThe tower located in the mountains did not display such regularity cf. .This is especially visible in the case of leg 2, for which the relations were the opposite, i.e., a rise in temperature in the initial period involved a drop in stresses by 15 MPa and a change in the nature of the stress, whereas a temperature drop changed the sign of stresses and increased them to the value of 5 MPa. It can be seen that as leg 4 was subjected to tension, leg 4 was compressed. It can be supposed that this is due to the landform features and# the fact that the terrain was unstable and wet. Most probably, the tower foundation rotated in relation to the axis along one of the tower base diagonals. Moreover, each of the tower legs had a separate foundation. The tower\u2019s hypothetical rotation at the tower base is presented in Validation by means of geodetic measurements was carried out on the tower apex deviation from the plumb line. The method of direct projection onto a leveling staff placed on the tower foundation was applied. A Leica TCR 407 tacheometer  and a measuring staff were used. The measurements revealed a tower axis deviation from the plumb line of 3 and 13.5 cm, respectively, in the direction parallel (component X) and perpendicular (component Y) to the course of the 110 kV high-voltage power transmission line .The results of the measurements of the tower deviation confirmed the results of the measurements of strains in the tower legs. They indicated that the tower structure inclined towards legs 2 and 3 and caused higher compressive stresses.The tower legs were also tested using the residual magnetic field (RMF) method. The method is described in ,29 and mThe conducted research proved an essential influence of difficult geotechnical and environmental conditions of the power line tower foundation on the strain state in the tower legs. The testing of supporting structures of high-voltage power transmission lines by means of optical FBG systems provided a lot of essential information about the real values of strains and stresses occurring in the legs of the analyzed towers of overhead power transmission lines. This is of particular importance in the case of lines which have been in use for many years. The obtained information may also contribute to an improvement in the operating safety of power line towers. The presented fiber-based system intended for the measurement of strains enabled dynamic measurements  as well as long-term measurements. The results of the strain-state measurements of the tower legs were confirmed by geodetic measurements of the tower apex deviation from the plumb line. Using various research methods, e.g., combining the FBG system to measure strains with the assessment of the tower\u2019s strain-and-stress state using the RMF method, made it possible to evaluate the performance of a tower structure in very difficult geotechnical conditions."}
+{"text": "Urothelial carcinoma (UC) is a unique entity with different histological variants: squamous, glandular, small cell, micropapillary, sarcomatoid, and plasmacytoid. Each of those subtypes behaves differently. As such, and in many scenarios, an accurate histological diagnosis is of paramount importance\u00a0to dictate the therapeutic approach. We hereby present a unique case of urothelial carcinoma that differentiated into two distinct histological subtypes, squamous and glandular, in three different organs within the genitourinary tract. We also describe\u00a0the pathological and clinical differences entailed between the two histological variants in bladder and upper urinary tract urothelial carcinoma. Urothelial carcinomas (UC) are known to exhibit different types of histological variations .\u00a0Each hiThis is a case of a 62-year-old gentleman with a medical history of hypertension and dyslipidemia who presented several years ago for multiple episodes of gross hematuria. Initial workup included an ultrasound, followed by an enhanced computed tomography scan with delayed phases (CT urography) revealing a 2 cm polypoid lesion arising from the left lateral wall of the bladder. Both kidneys and upper urinary tracts appeared free from filling defects with the absence of hydronephrosis. Following that,\u00a0transurethral resection of the bladder wall tumor (TURBT) was done with a subsequent pathology of high-grade papillary urothelial carcinoma invading the lamina propria, with no involvement of the muscularis propria, consistent with a tumor, node, metastasis (TNM) staging of pT1 high grade. One month later, a second-look TURBT was negative for any residual carcinoma. As such, the patient was started on intravesical Bacillus Calmette-Gu\u00e9rin (BCG) therapy. A six-week induction course was followed by\u00a0three-year maintenance.The patient was regularly followed up for three consecutive years with a yearly CT urography and diagnostic cystoscopies initially at three-month\u00a0intervals and then at six-month\u00a0intervals. There was no evidence of tumor recurrences, up until a follow-up CT urography scan three years after diagnosis demonstrated a small filling defect within the distal portion of the left ureter Figure .Retrograde pyelography with ureteroscopy was done; a polypoid lesion within the distal ureter was noted, measuring around 1.5 cm. A lesion biopsy revealed the presence of a low-grade noninvasive urothelial carcinoma. At the time, random bladder wall biopsies were negative for recurrence.Taking into consideration his overall health status with a baseline serum creatinine level of 1.3 mg/dL, a multidisciplinary meeting decided to proceed with a left distal segmental ureterectomy with ureteral re-implantation into a Boari flap\u00a0at the anterior aspect of the bladder wall. Pathological studies indicated the presence of a high-grade, noninvasive urothelial carcinoma of the ureter.Throughout the next three years, the patient underwent four TURBTs, consistently revealing low-grade, non-muscle, invasive urothelial carcinoma, whilst bilateral upper urinary tract evaluations by ureteroscopy and retrograde pyelography were consistently negative.A year later, he presented with lethargy, nausea, and decreased PO intake. Acute onset of kidney injury with a serum creatinine level of 2.4 mg/dL, up from a previous level of 1.4 mg/dL, was noted. After adequate hydration and a drop of serum creatinine back to baseline, a follow-up CT urography scan was done and showed urothelial thickening along the left distal ureter, almost obliterating the ureteral lumen at the site of the previous re-implantation. Severe hydronephrosis and hydroureter were noted along with effaced kidney parenchyma . Postoperatively, his serum creatinine settled at 1.6 mg/dL.Another multidisciplinary meeting decided to treat with an adjusted dosage of a platinum-based regimen of cisplatin and gemcitabine. His course was complicated by an abdominal aortic thrombus with 50% luminal narrowing at the level of the renal arteries Figure .As such, chemotherapy was halted and he was started on therapeutic anticoagulation. The panel\u2019s decision was to proceed for aggressive surgical management involving a left radical nephroureterectomy, cystoprostatectomy, and an ileal conduit for urinary diversion.Final bladder pathology at the anterior wall of the bladder revealed an infiltrating, high-grade urothelial carcinoma with glandular and squamous differentiation, whereas the distal portion of the left ureter revealed an infiltrating high-grade urothelial carcinoma with glandular differentiation. The left kidney pathology revealed an infiltrating high-grade urothelial carcinoma of the renal pelvis with squamous differentiation Figure .To note, the remaining part of the bladder wall showed superficial foci of high-grade urothelial carcinoma infiltrating the dense fibroconnective tissue with carcinoma-in-situ.The patient\u2019s postoperative course was complicated by acute kidney injury with serum creatinine reaching 9 mg/dL that was treated conservatively with adequate intravenous hydration. He was discharged on postop day 15 when his creatinine dropped back to his preoperative baseline of 1.6 mg/dL.Urothelial carcinoma comprises around 90% of bladder tumors .\u00a0They exUrothelial carcinoma with squamous differentiation comprises approximately 60% of UC variants, whereas UC with glandular differentiation comprises approximately 10% of all UC .\u00a0UrothelUrothelial carcinoma with glandular differentiation is characterized by the presence of enteric gland-like or intratumoral tubular spaces .\u00a0GlandulThe uniqueness of this case lies in the presence of synchronous urothelial carcinomas in the genitourinary tract with distinct histological differentiation: glandular and squamous. The presence of different histological variants within the same patient is associated with a worse prognosis -2.\u00a0MoreoPatient outcomes in pure urothelial carcinoma of the bladder are not significantly different from those of patients with glandular and/or squamous differentiation .\u00a0In addiEarly radical cystectomy remains the definitive treatment of choice for patients with a T2 tumor stage or higher disease of pure or variant UC .\u00a0HoweverIn conclusion, urothelial carcinomas are tumors capable of divergent differentiation. The identification of the various histologic variants is essential in determining therapeutic approaches."}
+{"text": "The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2\u201310 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2\u2032 position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2\u2032-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation. Electron paramagnetic resonance (EPR) spectroscopy can offer unique insight into the structure and dynamics of biomolecules such as DNA by observation of the behaviour of free radicals incorporated into the target of interest . The pulThe interpretation of interspin distances in biomolecular EPR typically relies on the use of a complementary technique to correlate spin label position with the EPR-derived most probable distance value Figure . X-ray cSolid-state structures can also be influenced by intermolecular crystal packing effects. Solution-state nuclear magnetic resonance (NMR) spectroscopy is not susceptible to this limitation, and can detect conformational ensembles as well as dynamic structural changes . NMR hasComputational investigations are the most common technique used to aid interpretation of EPR measurements. A detailed picture of a conformational ensemble can be obtained through molecular dynamics (MD) simulations, where consideration of the dynamic behaviour of the spin label, the biomolecular scaffold, and the linker connecting the two, can enable the prediction of one or more conformer classes ,24\u201333. IIn previous work, we described the synthesis of 2\u2032-alkynylnucleotides and their incorporation into DNA duplexes , a stratHere we address this challenge through a synergistic combination of EPR spectroscopy, X-ray crystallography, NMR spectroscopy and MD simulations, which accurately identify spin label location on a DNA duplex. As well as affording the first X-ray crystallographic structures of B-form spin-labelled DNA duplexes, the combination of these four techniques demonstrates the minimally perturbing nature of deoxynucleic acid 2\u2032-spin-labelling. Our results show that EPR spectroscopy with 2\u2032-triazole spin-labelled nucleotides can be used as a tool for accurate and precise distance measurements in DNA, providing a sound basis for the exploration of nucleic acid conformation and dynamics.For all methods, further details and raw data are provided in the Supplementary Material.et\u00a0al.  Q-band (34 GHz) spectrometer at 50 K. Samples were flash frozen from room temperature (293 K), or from 253 K. A 4-pulse DEER experiment was used to obtain distance distributions (The 4-pulse DEER experiments were performed on 200 \u03bcM (based on ssDNA) spin-labelled duplexes ibutions ,56. Furtibutions  was usedibutions .Duplexes were prepared from the purest HPLC fraction of each oligonucleotide to a concentration of 1 mM duplex in a 10 mM solution of KCl. These stock solutions were used to set up Natrix HT crystallization screens (Hampton Research) in 96-well Greiner plates, using 100 nl DNA solution and 100 nl crystallization reagent solution dispensed by a Microsys instrument (Cartesian Technologies). Plates were sealed and kept at 21 \u00b0C in a Formulatrix Storage and Imaging system for 20 days, until suitable crystals had grown.6) and 6QJR (duplex 7). Data were indexed and scaled with XDS (The Natrix HT (Hampton Research) screen was used to identify suitable crystallization conditions. Crystals were flash-cooled in liquid nitrogen, and all diffraction data were collected at the Diamond Light Source synchrotron science facility, Harwell, UK, at beamline I04  for thymidine. Lyophilized oligonucleotides were dissolved in 20 mM sodium phosphate, 80 mM KCl, pH 7 in 99.9% D2O + 23 \u03bcM DSS , and duplexes 9 and 10 were formed by mixing equimolar amounts of 1 with 2 or 3, followed by slow annealing from 60 \u00b0C. All NMR spectra were recorded at 14.1 T on an Agilent DD2 spectrometer using a 3 mm inverse triple resonance HCN cold probe. After recording spectra of the nitroxide-containing duplexes, 5 \u03bcl aliquots of 1 M sodium dithionite dissolved in the same buffer were added to reduce the nitroxide to the hydroxylamine diamagnetic state, and NMR spectra were recorded again on the reduced (hydroxylamine) form of the duplexes (NOH9 and NOH10).Three 14-mer oligodeoxynucleotides containing the Mbp1 binding site  were syn1, Figure 2, containing a 2\u2032-alkynyluridine (U) in place of thymidine at the 9-position, was synthesized according to our previous studies (3) or a tetramethylpiperidinoxyl radical  afforded duplexes 5 (containing two 5U modifications) and 6 (two 6U modifications). These nitroxides were selected on the basis of our previous work on different duplexes, where well-defined, but distinct conformations were observed for each nitroxide , and suitable for analysis by EPR spectroscopy. A variant of the Dickerson\u2013Drew dodecamer was selected which, unmodified, has been crystallized in the B-form . Duplex 6 displayed a near symmetric distribution with a most probable interspin distance of 2.99 nm, suggesting a distribution of very similar conformations of the spin label; the distribution for duplex 5 was less symmetric, implying a broader conformational ensemble, with a most probable interspin separation of 3.12 nm. The distributions show that the spin labels occupy well-defined locations on the duplexes.From the analysed DEER data, both spin labels were found to exhibit narrow distributions  showed only minor perturbation of the global structure, despite significant differences in crystal packing, as assessed by their all-atom RMSDs . Th. Th6 andking see . By cont8 Figure  shows th6 and 7 exhibit overall B-form geometry, some local structural parameters varied at the modified uridines. Most notably, the puckering and glycosyl torsion angles of these residues are A-form-like: the pseudorotation phase angles P of the 6U sugars are 49\u00b0 and 36\u00b0 (C4\u2032-exo and C3\u2032-endo), and 45\u00b0 for the 5U sugar (C4\u2032-exo), whilst the glycosyl torsion angles are \u2013141\u00b0 and \u2013146\u00b0 for the 6Us, and \u2013146\u00b0 for 5U  which converted to the B-form within 3 ns, the other from simulating a B-form duplex over 1000 ns (sugars C2\u2032-endo)  .In the former conformation, the label associates with the minor groove, and exhibits a distance distribution for all conformations (between 200.5\u20131000.5 ns) that correlates very well with the DEER-derived distance distribution , or associates with the major groove . The experimental maximum is now seen as a significant shoulder in the simulation  and C3\u2032-endo (13\u00b0), conformations that were also observed crystallographically . As with the X-ray structures, the triazole ring primarily occupies the least sterically hindered environment, with the triazole N3 atom positioned under the ribose ring; the C\u2013C bond linked to the spin label again adopts a gauche conformation . These angles are similar to that observed for the 5U nucleotide in the crystal structure of the singly modified duplex 7 , resonances were assigned for all nucleotides , double spin-labelled duplexes troscopy . In the ides see . The cheides see . Further strands .U nucleotides, but also the sugar puckering. In native duplex 11, all nucleotides are predominantly C2\u2032-endo with a small admixture of C3\u2032-endo (3J1\u20322\u2032 \u223c10 Hz and 3J2\u20323\u2032 \u223c6 Hz) , and 6.5 and 6.0 Hz for 6U3 (in NOH10).The NOESY peak intensities and coupling constants of the sugar protons  . This isendo or pure C3\u2032-endo, but can be rationalized by a similar proportion of C2\u2032-endo and O4\u2032-endo states, reflecting the tendency of these sugars towards A-form like conformations. The 5/6U glycosyl torsion angles were estimated to be \u2013140 to \u2013150\u00b0, comparing well with those determined by crystallography and MD simulations. The chemical shifts of the reduced duplexes are very similar, including at the sites of modification, as are the cross-peak intensities in DQF-COSY and short mixing time (50 ms) NOESY spectra, showing minimal effect of the labels on the overall duplex conformations. The signals corresponding to the methyl groups of the reduced spin labels were sharp, and showed few nOes to the DNA, which is consistent with their occupying the minor groove.These values differ significantly from those expected for C2\u2032-U3-containing strand (C1\u2013G6), or for the final five on the complementary strand , MD simulations and NMR spectroscopy, with the first two of these methods revealing conformations that correlate well with the interspin distance distributions measured by EPR spectroscopy .Interspin distance measurements have the potential to afford detailed information on duplex structure, provided that accurate models can be constructed that enable confident interpretation of EPR results. Analysis of the positioning of 6-Me and 5-Me nitroxides on single and double spin-labelled duplexes showed a remarkable degree of consistency between the results of X-ray crystallography   ,49.5/6U modification. Our results show that the more rigid linker / spin label 4 (6-Me) imparts similar perturbation of the DNA structure to 3 (5-Me); in the absence of other considerations such as reduction stability, 4 would thus be the label of choice for 2\u2032-alkynyl spin-labelling, given its narrower and near-symmetric EPR distance distributions. Taken together, the three techniques used in this study each establish minor groove positioning of the 2\u2032-triazolyl nitroxide spin labels, thus allowing confident interpretation of associated EPR data and enabling the reliable use of this spin-labelling strategy to probe nucleic acid structure.NMR spectroscopic investigations of two single spin-labelled duplexes in the paramagnetic (nitroxide) and diamagnetic (hydroxylamine) forms support adoption of the B-form in solution, with conformations very similar to an unmodified duplex. The spin label likely occupies the minor groove, and once again an A-form like conformation was observed at the modified sugar. Only minor conformational changes were seen at the nucleotides neighbouring the These observations lead to a model that explains spin label conformation. The introduction of a 2\u2032-substituent onto the deoxyribose ring leads to a more A-form like pucker. The triazole ring and carbocyclic spin label then occupy a conformationally restricted environment on the sugar that minimizes torsional and steric strain; finally, further conformational restrictions imparted by the constrained environment of the minor groove result in a well-defined location of the spin label. Indeed, it is possible that a significant proportion of the EPR measured distance distribution derives from a range of duplex conformations, as much as from label and linker flexibility itself.Using experimental and computational methods to support EPR spectroscopic observations, we demonstrate that spin-labelling of DNA via 2\u2032-triazole-linked nitroxides is minimally perturbing and affords DEER data with narrow (sub-nanometre) distance distributions due to well-defined minor groove nitroxide conformations. This multi-faceted approach offers unprecedented insight into spin label localization, enabling more confident interpretation of DEER-derived distance distributions of 2\u2032-triazolyl-mounted spin labels. Overall, this work provides the foundation for further applications of 2\u2032-alkynyl spin-labelling in the study of nucleic acid structure, dynamics and interactions with other biomolecules.10.17630/c4d64b89-d19b-4f9e-8409-762b430d72f5. Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under accession numbers 6QJS and 6QJR. NMR data have been deposited with the Biological Magnetic Resonance Data Bank under accession number 27958.EPR data is deposited at DOI:\u00a0gkaa086_Supplemental_FileClick here for additional data file."}
+{"text": "Arabidopsis enhancer-tagged carboxylesterase 20 (AtCXE20) line was identified in a drought tolerance screen. Ectopic expression of AtCXE20 in Arabidopsis and maize resulted in phenotypes characteristic of strigolactone (SL)-deficient mutants, including increased branching and tillering, decreased plant height, delayed senescence, hyposensitivity to ethylene, and reduced flavonols. Maize silk growth was increased by AtCXE20 overexpression, and this phenotype was partially complemented by exogenous SL treatments. In drought conditions, the transgenic maize plants silked earlier than controls and had decreased anthesis-silking intervals. The purified recombinant AtCXE20 protein bound SL in vitro, as indicated by SL inhibiting AtCXE20 esterase activity and altering AtCXE20 intrinsic fluorescence. Homology modeling of the AtCXE20 three-dimensional (3D) protein structure revealed a large hydrophobic binding pocket capable of accommodating, but not hydrolyzing SLs. The AtCXE20 protein concentration in transgenic maize tissues was determined by mass spectrometry to be in the micromolar range, well-above known endogenous SL concentrations. These results best support a mechanism where ectopic expression of AtCXE20 with a strong promoter effectively lowers the concentration of free SL by sequestration. This study revealed an agriculturally important role for SL in maize silk growth and provided a new approach for altering SL levels in plants.Severe drought stress can delay maize silk emergence relative to the pollen shedding period, resulting in poor fertilization and reduced grain yield. Methods to minimize the delay in silking could thus improve yield stability. An  Maize production worldwide ranks first among the cereal crops, but drought stress can significantly limit maize yield . DroughtThe \u03b1/\u03b2 hydrolase fold superfamily of proteins includes carboxylesterases, cholinesterases, carbohydrate esterases, lipases, and peptidases . MembersArabidopsis has 20 carboxylesterase genes that have been phylogenetically organized into seven clades  are \u03b2-carotene derived plant hormones  that infof crops . The SL of crops . After bof crops .Arabidopsis carboxylesterase in maize provides silk phenotypes that are relevant for drought tolerance, most likely via a SL sequestration mechanism.This study demonstrates that strong ectopic expression of an Arabidopsis with T-DNA vectors containing multiple copies of the cauliflower mosaic virus (CaMV) 35S enhancer is an effective way to increase expression of genes near the insertion site (Arabidopsis library tagged with four copies of the CaMV 35S gene enhancer (AtCXE20 (At5g62180), and a gene encoding an RNA helicase  also had the greatest increase in tiller numbers.The maize ubiquitin promoter and sorghum gamma kaffirin terminator were used to drive strong constitutive expression of in maize . Seven sbundance . Controlbundance . Approxic events . Six of c events . In thesAtCXE20 were grown outdoors in tree pots supported by wooden racks in 2014 and 2016 in Johnston, Iowa. Drought stresses were then imposed by withholding irrigation and letting pots dry, as described previously , and the esterase activity was inhibited by addition of SL  carboxylesterase 1 , we built a model of 4-nitrophenyl methylphosphonate covalently bound to S166, a transition state mimic of the substrate used here for AtCXE20 activity assays, p-nitrophenyl acetate. The model showed that all the polar atoms around the catalytic center properly form hydrogen bonds with -NHs of \u201coxyanion hole\u201d residues and with the catalytic triad residue H302. The model revealed that the acetyl methyl group of this transition state mimic forms a direct Van der Waals contact with the F200 benzene ring, suggesting that any potential substrates with acyl groups larger than an acetyl group, including p-nitrophenyl butyrate and strigolactones, would be unlikely to be hydrolyzed by AtCXE20. Larger acyl groups would move the ester bond position of an ester substrate, or the ether linkage of SLs, further away from the AtCXE20 catalytic triad. Although such substrates may bind to the hydrophobic gorge, their larger acyl groups would not allow sufficient proximity and orientation to the catalytic triad to allow efficient hydrolysis.As observed previously with AeCXE1, the AtCXE20 substrate binding gorge is deep, hydrophobic, and wide open and its walls are made of flexible loops including the \u03b14-\u03b15 loop, the \u03b28-\u03b18 loop, and the N-terminal loop . The gorin vitro in the presence or absence of GR24  had improved drought tolerance as demonstrated by increased survival of drought treatments and decreased rate of water loss in detached flag leaves  suggested that AtCXE20 would have sufficient abundance and binding affinity to sequester appreciable amounts of at least some SLs in transgenic tissues. The complementation of the enhanced silk growth phenotype by exogenous SL treatments was also consistent with a SL sequestration mechanism. Finally, the observation that the increased branching in Myzus persicae (peach-potato aphid) increased the abundance of a carboxylesterase that binds carbamate and organophosphate insecticides . The protein contained a 20-amino acid N-terminal peptide fusion (mgsshhhhhhssglvprgsh) that included a 6-histidine tag to facilitate purification. The E. coli cultures were grown at 37\u00b0C in 2X YT media to an OD600nm of 0.6. Transgene expression was then induced with 0.5 mM IPTG and the culture was grown an additional 6 h at 20\u00b0C. The fusion protein was purified from E. coli extracts using cobalt affinity chromatography. The pellet from a 300 ml culture was lysed at room temperature in 10 ml of 50 mM Tris-HCl pH 8, 100 mM NaCl, 0.1% TritonX-100, 0.1 mg/ml lysozyme, and 100 \u03bcl of Protease Inhibitor Cocktail Set III . After 20 min, 5 \u03bcl Benzonase Nuclease (Novagen) was added for an additional 10 min to reduce viscosity. The lysate was centrifuged at 4\u00b0C for 15 min at 15,000 g and the supernatant was then incubated with 4 ml of HisPur Cobalt Resin (Thermo Scientific) for 20 min before pouring into a column and allowing unbound protein to flow through. The column was washed with 20 ml of 50 mM Tris-HCl pH 8, 200 mM NaCl, and 10 mM imidazole, followed by elution with 50 mM Tris-HCl pH 8, 200 mM NaCl, and 60 mM imidazole. Aliquots of the purified protein containing 10% glycerol were quickly frozen in liquid nitrogen and then stored at \u221280\u00b0C. Aliquots were thawed as needed and dialyzed against 50 mM Tris-HCl pH 8, followed by addition of 5 mM TCEP and storage at 4\u00b0C. Addition of the TCEP reductant was important, because substantial loss of activity was observed during storage at 4\u00b0C in the absence of reductant. Dialyzed protein was quantitated by absorbance at 280 nm, using a value of 1 OD (280 nm) = 0.92 mg/ml, obtained from Vector NTI. Esterase assays with p-nitrophenyl acetate as substrate were done in 50 mM Tris-HCl, pH 8, with 1 \u03bcg of protein in an assay volume of 200 \u03bcl, using 96-well flat bottom microtiter plates. The increase in absorbance at 405 nm was monitored, and rates of control reactions without enzyme were subtracted to correct for background due to autohydrolysis of substrate. Kinetic parameters, SEs, and Ki values were determined using the enzyme kinetics module of SigmaPlot 12.5.Recombinant AtCXE20 protein was obtained by expression with the pET28a vector (Novagen) in The AeCXE1 structure (PDB:2o7v) was selected as modeling template. Based on the target-template alignment, 20 different three-dimensional (3D) models were generated by MODELER 9.15 in Discovery Studio  and the model with the lowest discrete optimized protein energy (DOPE) score was used for ligand docking and analysis. The quality of the model was verified with MODELER, which yielded a normalized DOPE score of <\u22121.66 and showed that all side chains except S166 resided in the allowed area in the Ramachandran plot. Molecular Dynamic simulation of the AtCXE20 homologous model was carried out with GROMACS 5.1.4  on LinuxArabidopsis thaliana (ecotype Columbia) enhancer-tagged library were grown in flats until 15\u201320 days after planting, and then watering was stopped until bolting stage approximately 2 weeks later. Green leaf area was monitored during the drought treatment by using LemnaTec HTSBonitUV software as described previously (Plants from an eviously . WaterinAgrobacterium tumefaciens-mediated transformation of maize inbred line PHR03 was carried out with the AtCXE20 transformation cassette described in AtCXE20 expression. The selectable marker was maize optimized phosphinothricin acetyltransferase (MOPAT) which provided resistance to the herbicide glufosinate. Hybrid seed for the 2014 field studies was produced by crossing the transformed PHR03 inbred events with inbred PH12SG. Subsequently, a 2nd maize inbred line PH184C was transformed with a similar cassette as AtCXE20 transgene and the MOPAT selectable marker, except for PHR03 event E3 which had two copies of the AtCXE20 transgene.A randomized complete block design was used with two-row plots and four field blocks. Row length was 4.4 m, and row width was 76.2 cm. Each block included one plot for each transgenic event and two plots for null. The maize hybrid was PHR03 \u00d7 PH12SG. The number of tillers/plant was determined at growth stage V12, using all plants in the plot except for the end plants of each row.Leaf samples for transgene expression analysis were obtained at growth stage R2 and were immediately frozen in liquid nitrogen. Total RNA was extracted with RNeasy reagents (Qiagen), and cDNA was synthesized with a mixture of oligo(dT) and random hexamer primers using Applied Biosystems HCAP Reverse Transcription kit (Life Technologies). Quantitative RT-PCR (qRT-PCR) was performed with a TaqMan hydrolysis probe using the TaqMan Universal Master Mix (Life Technologies). Relative quantification values were determined using the difference in Ct between the target gene and the internal control, maize eIF4-\u03b3.The quantity of AtCXE20 protein in maize leaves and roots was determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Leaf samples from plots for three transgenic events  and null were obtained at growth stage V9 by pooling one leaf punch per plant for 10 plants per plot, and quick freezing in liquid nitrogen. Primary root samples from 1-week old seedlings grown in the dark in moist paper rolls were obtained from transgenic events E2 and E3, and null. The samples were ground in liquid nitrogen, and the frozen powder was lyophilized. The protein from 10 mg of lyophilized powder per sample was extracted with 600 \u03bcl PBST buffer, normalized to a concentration of 0.8 mg/ml protein, reduced with dithiothreitol, alkylated with iodoacetamide, and digested for 18 h overnight at 37\u00b0C with trypsin (1:25 trypsin/protein ratio). Formic acid was added to stop the digestion and to assist in ionization followed by the addition of isotopically labeled internal standard. Digested extract (10 \u03bcl) was injected onto a 2.1 mm x 50 mm ultra-performance liquid chromatography (UPLC) column from Waters (BEH C18 1.7 \u03bcm) set at 60\u00b0C. Mobile phases were 0.1% formic acid in water (MPA) and 0.1% formic acid in acetonitrile (MPB). Separation was achieved with a 1.5 min linear gradient of 8\u201330% MPB. The LC-MS/MS system included a SCIEX 5500 Q-TRAP with a Turbo ion-spray source. Recombinant AtCXE20 protein was spiked into null matrix to produce a standard curve with a linear dynamic range of 50X. The AtCXE20 tryptic peptide DLNAIVVSPSYR was used to quantitate AtCXE20 protein abundance. Targeted analyses of MRM 667.3/807.4 for DLNAIVVSPSYR and MRM 670.3/813.4 for DLNAIV*VSPSYR internal standard were completed using Analyst software (SCIEX) for data acquisition and quantification. The parent ions for the analyte and internal standard are noted as follows: DLNAIVVSPSYR, 667.3 and DLNAIV*VSPSYR, 670.3. The heavy-labeled Valine (noted as V*) was labeled with carbon 13 (C13) and nitrogen 15 (N15), which increases the mass of valine by 6 Daltons (Da). Because the internal standard is a +2 charge state, the addition of the heavy-labeled valine  increases the parent ion mass by 3 Da . The product ions for the analyte and internal standard are noted as follows: DLNAIVVSPSYR, 807.4 and DLNAIV*VSPSYR, 813.4. The y7 product ion was measured and corresponds to the following analyte and internal standard fragment sequences, VVSPSYR and V*VSPSYR. Because the product ion is a +1 charge state, the addition of the heavy-labeled valine  increases the product ion mass by 6 Da .2O5, 15% K2O, 5% Ca, 2% Mg, 0.0187% B, 0.0187% Cu, 0.075% Fe, 0.0375% Mn, 0.0075% Mo, and 0.0375% Zn. Weather information and drought treatment timing are presented in Hybrid maize plants were grown outdoors in 2014 and 2016 in Johnston, Iowa in 10-L pots supported by wooden racks, with one plant per pot. The hybrids used were PHR03 \u00d7 PH12SG for 2014 and PH184C \u00d7 PH1V69 for 2016. The pots  were filled with an equal weight of 4,500 g potting soil (Fafard 3B mix). The pots were rectangular with dimensions at the top of 19.1 cm x 45.7 cm (7.5''x18''). Each wooden support rack was 250 cm long, 60 cm wide, and 55 cm high and held two rows of 12 pots each. Pots were irrigated with Peters Excel fertilizer (Everris NA Inc.) adjusted to provide 100 ppm N. The fertilizer contained 15% N, 5% PijnmsY) of rack R)j, event (E)n, genotype (G)m, and plant s, were modeled as a function of an overall mean \u03bc, factors for rack, rack by replication, event, rack by event, rack by replication by event, genotype, genotype by event, rack by genotype, and a residual within each rack For the field pot study, the experimental design was a multi-rack split-plot design. Events were randomized in main plots, and event positive and negative pairs were randomized in sub plots to ensure that throughout the experiment, each transgenic plant was adjacent to an appropriate control plant. Each rack contained multiple replications . A linear mixed model was applied to model traits for each environmental treatment separately. Data for trait .where event and genotype were treated as fixed effect, and all the other effects except the residual were treated as independent normally distributed random variables with means of zero. insY) of replication (R)i, event (E)n, and plant s, were modeled as a function of an overall mean \u03bc, factors for replication, event, and a residual \u025bins. The model can be specified as:For the field plot study, the experimental design was a randomized complete block design with events randomized within blocks (replications). A linear mixed model was used. Data for trait .where event was treated as fixed effect, and all the other effects were treated as independent normally distributed random variables with means of zero. t-tests, assuming equal variances (Microsoft Excel).For the lab studies, the branching, ACC sensitivity, esterase activity, fluorescence, and silk growth, experiments were statistically analyzed by two sample 2/s was used to provide a photoperiod of 16 h light/8 h dark. Daytime and nighttime temperatures were kept at 26 and 20\u00b0C, respectively, and relative humidity was kept at 60%. At 1 day after silking, ears were removed from greenhouse-grown plants and placed in a bucket of water for transport to the lab. In a sterile hood, husks were removed and 3-cm lengths of silks were excised from the area shown in Transgenic and segregating null plants of maize hybrid PHR03 \u00d7 PH12SG event E3 were grown in a greenhouse in 20-L pots. Supplemental lighting at an intensity of 800 \u03bcmol photons/mThe rac-GR24 (product number CX23880) was purchased from Chiralix (The Netherlands). The 5-deoxystrigol  and karrikin 2 (product number 0256823) were purchased from OlchemIm Ltd (the Czech Republic). Other hormones and p-nitrophenyl acetate were purchased from Sigma-Aldrich (United States).The original contributions presented in the study are included in the article/Research designed by CL, RW, KR, JT, QX, LL, and JH. Research performed by CL, JT, QX, PV, ZH, MO, and KR. Data analyzed by LL, QX, CL, and KR. Project supervised by RW and JH. Manuscript written by KR with input from all authors. All authors contributed to the article and approved the submitted version.All authors are or were employees of Corteva Agriscience or its parent companies when they carried out this research. Corteva Agriscience is a for-profit agricultural technology company. Patent applications related to this work have been filed."}
+{"text": "Uncertainty regarding \u201ccage-free\u201d housing guidelines have left egg producers unsure about how to transition to cage-free housing. A primary driving force behind cage-free housing is the perceived animal welfare concerns for caged birds. Therefore, it is of great importance to perform a systematic investigation on specific cage-free facility types with an emphasis on bird comfort assessment. Thus, the goal of this study was to investigate alternative ventilation schemes of a cage-free house to provide practical designs for a comfortable interior environment at the hen level. By modeling four different ventilation schemes in a one-eighth section of a typical floor-raised layer house\u2014indoor temperature, air speed, and static pressure were compared and analyzed quantitatively. Distribution contours and quantitative analysis of airflow, temperature, and pressure suggested that indoor conditions could be maintained at a suitable range uniformly, especially at the hen level. In addition, the ventilation rates of the hen house within four ventilation schemes fell at the higher end of the desired ventilation range, indicating that the barn could be expected to maintain good air quality during cold weather. This study demonstrated that computational fluid dynamics modeling was a powerful tool that facilitated researchers to address animal welfare issues in animal housing designs.3/s (4100 ft3/min) in the desired range for cold weather (0 \u00b0C). Simulation results and subsequent analyses demonstrated that these alternative models had the capacity to create satisfactory comfortable temperature and air velocity at the hen level. A full-scale CFD model with individual hen models presented robustness in evaluating bird welfare conditions.This work investigated alternative ventilation schemes to help define a proper ventilation system design in cage-free hen houses with the goal of assuring bird welfare through comfortable conditions. Computational fluid dynamics (CFD) modeling was employed to simulate indoor and outdoor airflows to quantify the effectiveness of ventilation systems in maintaining suitable and uniform living conditions at the hen level. Four three-dimensional CFD models were developed based on a full-scale floor-raised layer house, corresponding to ventilation schemes of the standard top-wall inlet, sidewall exhaust, and three alternatives: mid-wall inlet, ceiling exhaust; mid-wall inlet, ridge exhaust; and mid-wall inlet, attic exhaust with potential for pre-treatment of exhaust air. In a sophisticated and powerful achievement of the analysis, 2365 birds were individually modeled with simplified bird-shapes to represent a realistic number, body heat, and airflow obstruction of hens housed. The simulated ventilation rate for the layer house models was 1.9\u20132.0 m Poultry facilities are going through significant transitions to cage-free production modes to address bird welfare concerns with caged housing. Aviary systems, convertible cages, and floor housing systems are three representative cage-free housing systems that are commonly used . ApparenEnvironmental control ventilation systems are vital in cage-free poultry production . A bird\u2019Computational fluid dynamics (CFD) has been used as a powerful tool to model fluid flow in diverse applications. For decades, CFD modeling has been employed to address indoor environmental problems and optimize the design of ventilation systems in a variety of agricultural facilities . Most enThe need to address indoor environment problems in poultry housing has encouraged researchers to improve the design of ventilation systems by CFD modeling. Research conducted by Mistriotis and Jong investigated a broiler house with natural ventilation by developing a two-dimensional CFD model, and the simulation results revealed that the uniformity of indoor temperature and air velocity distribution was improved by installing a solar chimney . Blanes-This study applied CFD simulations in characterizing three alternative ventilation schemes applied to a floor-raised layer house, in comparison with the standard ventilation scheme  by evaluFour CFD models and corresponding simulations were conducted using the commercial software package FLUENT v19.1 . The stace model ,17 with ce model ,19,20,212/bird (1.20 ft2/bird).The modeled layer house was located at Lititz, Pennsylvania, with a typical floor-raised housing configuration ,22. The The current ventilation system of the layer house included 84 rectangular ventilation inlets [each 1.17 m (46 in.) by 0.20 m (8 in.)] located at the top of each sidewall near the eaves along both sides of the building. Four exhaust fans (0.91 m (36 in.) in diameter) installed along one long sidewall of the barn were operated during brooding, and for cold and mild weather. In addition, a tunnel ventilation system was equipped (not modeled in this study) for warmer and hot weather. Fresh outdoor air was drawn into this negative pressure house through the inlets under the barn eaves with sidewall exhaust fans in the standard ventilation scheme used in North America that we referred to as \u201ctop-wall inlet sidewall exhaust\u201d [TISE] [Initially, a two-dimensional computational domain was employed to simulate the indoor air conditions within TISE ventilation scheme, though the real ventilation performance of the layer house could not be accurately reflected . TherebyA three-dimensional geometry was developed with realistic dimensions provided by collaborators to represent one-eighth of the entire layer house, resulting in a reasonable model size with ventilation features that included an exhaust fan and the proportional quantity of inlets. Only a central section of the layer house was modeled to minimize end-wall effects. The house was modeled using dimensions obtained from construction blueprints. Details of the barn dimensions were described in our previous publication .The computational domain of each model included the barn itself and ambient air to properly simulate airflows inside and outside the building . All modFour ventilation schemes were modeled and investigated . Other t3 [A significant aspect of the study was to determine conditions at the hen level for welfare comfort conditions, in addition to overall environment patterns in the building air space. For this reason, hen models were included in the simulation. In total 2365 individual hen models were included in each model to represent approximately 1/8 of the total hens housed in the 1/8 house section. Assuming all hens were evenly distributed , an esti3 [\u201cWalls\u201d: the ground, ceiling, roof, slatted floor, nesting area, litter area, inlet baffles, sidewalls, animal surfaces, and the top surface of the computational domain . Note th3 .z-axis and both near and far ends of the house were defined as \u201csymmetry\u201d boundary conditions, whereas those surfaces represented internal faces that accounted for 1/8 of the actual scenario.The front and back surfaces of the computational domain along the Two faces of each inlet that were perpendicular to the wind direction and two faces (functioning parts) of the exhaust fan were assigned boundary conditions of \u201cinterior\u201d to represent an interior portion of the computational domain through which air could flow .\u201cVelocity inlet\u201d was assigned to the left end of the entire domain with a specified wind magnitude of 2.0 m/s (393.7 ft/min) along the positive x-axis .\u201cPressure outlet\u201d was assigned to the right end of the entire domain representing where the flow exits to atmospheric pressure (0 Pa).A \u201c3D fan zone\u201d was assigned to the body of exhaust fan where the entire fan volume was considered a fluid cell zone, which simulated the effect of an axial fan by applying a distributed momentum source . ConstanFor this study, the temperature of the atmosphere was specified as 0 \u00b0C (32 \u00b0F). Six types of boundary conditions or cell zones were adopted in the CFD simulation inside and outside the layer house ,22:\u201cWall\u03b5 is a relative error indicator, f1 is the variable value calculated using a fine mesh, and f2 is the variable value at the same point calculated using a coarse mesh. The mesh refinement ratio r is calculated in Equation (3), where fineN and coarseN are the total number of cells of the fine and coarse mesh, respectively.ANSYS meshing was employed to perform discretization of the computational domain. Prior to launching CFD simulation, index of mesh skewness was checked to assess meshing quality. In addition, standard mesh convergence studies were performed using the Grid Convergence Index (GCI) method . GCI valr = 1.2 was the mesh refinement ratio from the middle mesh to the coarse mesh, and r = 1.4 was the ratio from the fine mesh to the coarse mesh. The GCI at three selected points P1, P2, P3 were analyzed and compared with the variables of air speed, temperature, and pressure, respectively. The GCI value decreased when r increased from 1.21 to 1.44  [For cold weather, the desirable range of ventilation rate for the modeled layer house ought to be 0.39 to 1.95 mft3/min) . Each inft3/min)  to maintft3/min) ,29.Predicted environmental data of CFD simulations were quantitatively compared across five hen-occupied zones at every single reference plane. Note that only one data output was exported at a given location since it did not vary with time after convergence for a steady-state analysis. Therefore, exported data points in each zone were treated as repeated measurements captured throughout that hen-occupied zone. The simulation data were fit to a mixed-effects model as shown in Equation (4):0H: there was no difference in the means of factor Model.0H: there was no difference in the means of factor Zone.0H: there was no interaction between factors Model and Zone.To compare the simulation data between models, the primary interest was testing whether different models had different results at the same plane. Thus, the interpretation for Equation (4) is: p-value significance level \u03b1 = 0.05 was used for determining whether The hypotheses were tested using analysis of variance (ANOVA) using R Studio v1.2 . A p-valrocedure .Four ventilation schemes were analyzed and compared with contours of airflow patterns, temperature distribution, and static pressure difference. In addition, environmental data of five different zones at hen level were statistically analyzed to compare and assess the bird welfare suitability of indoor air conditions provided by each design.Three-dimensional rendering of air velocity magnitude of the entire layer house and velocity vectors at each reference plane were created to visualize indoor airflow patterns . In geneThe patterns of indoor airflow varied with the type of ventilation schemes at Plane I. The standard TISE ventilation system possessed the strongest incoming air jets compared to three alternative designs, because the TISE model had inlets at the top of the sidewalls along a flat ceiling . WithoutAir movement patterns varied dramatically at Plane N that contained no ventilation features . VigorouThe air movement patterns at Plane F exhibited the influence of the exhaust fan on performance of each model . The TISAlthough no artificial heating was supplied in the layer house in a cold weather, indoor temperature was maintained warm due to birds\u2019 body heat. The distribution of temperature at each plane was analyzed by temperature contours .The incoming fresh air (dark blue) with an initial temperature of 0 \u00b0C (32 \u00b0F) at both inlets ran into a quick color transition after mixing with indoor air, approaching the nest-boxes area . At PlanThe reason for the different patterns between the TISE model and the other three models was the position of inlets. The inlet at a lower, mid-wall position in the house of MICE, MIRE, and MIAE provided cold air that was quickly mixed with the warm air that was heated by the birds. Hence, the warmed air did not have a chance to rise toward upper areas of the interior due to mixing into the cooler inlet air. Otherwise, the incomplete mixing of cold incoming air and warm house air in TISE left warmer air stratified near the ceiling location of low airflow. This explained why the obvious warm region was not observed in the other models.The indoor temperature distribution at Plane N  containeThe temperature contours of TISE at Plane F showed large areas of relatively low temperature  with the majority of the house appearing green on the color-temperature scale . The thrStatic pressure difference between interior and exterior of the study layer house varied slightly over four ventilation schemes with a total range from \u221230 to 0 Pa (the atmospheric pressure was set at 0 Pa) . The uniIndoor static pressure in the TISE model was \u221224.3 Pa at Plane I on average. The redness at both inlets indicated the attachment to outdoor atmosphere. The pressure of MICE and MIRE fell in the range of \u221220 to \u221224 Pa, while the indoor static pressure of MIAE had the smallest magnitude, ranging between 18 to 22 Pa. Overall average static pressure fell in a normal range . IdenticIn addition, the indoor static pressure at Plane F showed similar patterns compared to the other two planes excluding the white region close to the exhaust fan where the static pressure lower than \u221230 Pa .Environmental conditions in terms of air speed, temperature, and static pressure at the hen level were analyzed quantitatively to evaluate the performance of each ventilation scheme in ensuring hen comfort. Simulation outputs were analyzed at each hen-occupied zone separately. Because all these data varied by indoor locations, referring to the type of planes, comparisons across four models ought to be conducted at the same plane. Thereby, statistical analyses were performed to test the significance of key factors\u2019 effects on the simulation data, including the type of model, the zone, and their interactive effects.p-value, accordingly.At Plane I, in total, 35,340 data points were exported for thorough analysis from four models\u2019 simulation results. The ANOVA analysis suggested that for all environment parameters, the effects of model type, hen-occupied zone, and the interactions among them, were statistically significant  accordinSame ANOVA procedure was conducted to analyze the data at Plane N and Plane F exported from simulation results of each model . The datAs follow-up analyses, Tukey\u2019s tests were applied in pairwise comparisons for parameters of interest. The results of Tukey\u2019s test were reflected in At Plane I, MIAE had the fastest average air speed in hen-occupied area compared to the other models , and theAverage temperature of TISE was the warmest at Plane I, about 22.90\u00b0C at the hen level, which was 2.26 \u00b0C higher than the lowest of MICE. Interestingly, the average temperature of MIAE was the second highest, though it had the fastest air speed on average. Average temperature at each zone was exceptionally close between four models as several crossbars indicating the lack of statistical significance at Plane I . For insIndoor static pressure averages at hen level were of extraordinary uniformity within each zone for individual model as small standard deviations suggested . MoreoveSimulation data from Plane N revealed different patterns at hen-occupied zones . InteresThe average temperatures of each model were fairly uniform at Plane N. The highest mean temperature was in the MICE model as it had the slowest air speed on average . AdditioThe static pressure at Plane N was identical to the performance at Plane I for each model. The lowest pressure magnitude on average was 21.05 Pa from the data of MIAE, which was the same with that of Plane I. Additionally, the highest magnitude was 24.53 Pa of TISE model.Simulation results at Plane F suggested all four models have fairly uniform air speeds with relatively smaller magnitudes . All fouAverage temperature at Plane F increased slightly due to slower air speeds overall. The temperature of TISE was the lowest on average about 20.10 \u00b0C, while MIRE had the highest temperature of 23.65 \u00b0C, which was consistent with its low average air speeds . The difIndoor static pressure at hen level at Plane F was identical to the other two planes  and was 3/s (4174 ft3/min), 1.93 m3/s (4089 ft3/min), 1.96 m3/s (4153 ft3/min), and 1.91 m3/s (4047 ft3/min) for the study layer house respectively, which were on the high end of the recommended range (0.39 to 1.95 m3/s). Furthermore, the alternative designs provided indoor air movements similar to the standard model, even better at the hen level (Plane I). Air speeds on average were maintained at 0.35 m/s (69 ft/min) at hen level in the majority of the house for TISE, MIRE, and MIAE. Airflow pattern visualization showed two large circular air eddies that included the incoming air jets in TISE whereas the alternative models had these large eddies along with more numerous, smaller circulation patterns.Three alternative ventilation schemes presented comparable performances with the standard TISE, in terms of sufficient indoor airflow and air mixing. By observing air velocity vector contours, MICE, MIRE, and MIAE were able to provide strong incoming air jets that can reach the central region of the house almost as well as the TISE model. Simulation results of the four ventilation schemes revealed a total ventilation rate of 1.97 mTemperature contours of individual models indicated that the uniformity of indoor temperature distribution was satisfactory overall. The entire indoor temperature was kept above 15 \u00b0C on average, assuming the house was ideally insulated. Nonetheless, all four models presented the capacity to ensure birds comfortable temperatures. The average temperature at the hen level varied within a range of approximately 21 to 24 \u00b0C for the three alternative models, which matched the comfort zone for birds nearly perfectly. Furthermore, the average temperature at hen-occupied areas of the three alternative models was slightly higher than that of the TISE model at reference planes N and F. In particular, within Zone-3 (where nest-boxes were located) should be of interest, as the TISE model offered higher air speed and lower temperature than the alternative designs. Microenvironment in and around the nest-boxes may be used to encourage nest-box use by hens rather than laying eggs in other parts of the house.The static pressure of the house was quite uniform for each model and within a normal range. Slight differences among models up to 4 Pa were found. The TISE model had the largest magnitude of static pressure around 25 Pa, while the MIAE model had the smallest magnitude of static pressure. In addition, static pressure at different locations for the same model was quite consistent.Future studies may be conducted with regard to design details of cage-free hen housing accordingly. As revealed in this work, airflow patterns and the formation of indoor air circulations were highly dependent on the trajectory of incoming air. The relative location of inlets and exhaust fans, the dimensions of baffles, and so on, all play an indispensable role in the formation of indoor environment. In addition, further CFD analysis can be used to examine the uniformity of temperature and air speeds as well as the environmental parameters at the hen level to reason and address practical problems, such as floor eggs.One standard and three alternative ventilation schemes were modeled at full-scale to simulate indoor environmental conditions in a commercial floor-raised cage-free hen house with the goal of evaluating the performance of ventilation scheme in maintaining comfortable conditions for good bird welfare.CFD simulation results suggested three alternative ventilation schemes have competitive performance compared to the standard scheme, and demonstrated impacts of the ventilation schemes on characteristics and uniformity of indoor environmental conditions. Data of environmental parameters at the hen level were documented to ensure hen comfort. The performance of alternative ventilation systems was assessed accordingly. Planes I and N had the most variability of air speeds due to the impact of fresh air inlet in or adjacent. The middle zones of each house  in Planes I and N had the highest air velocities and largest variation in air velocities, yet they were below the threshold for being considered chilling drafts on the birds. Higher air speed in this central area was a result of cooler, fresh incoming air circulating into that portion of the layer house. However, fortunately, there was not a trend toward cooler temperatures within those bird-occupied zones at the middle of the house, which indicated the central nest-boxes area would be favored for laying eggs. Temperatures within the animal zones were variable across all zones (high standard deviations), but on average showed reasonably uniform temperatures within each model and across models. Static pressure had the smallest variation among planes and zones within a model. The static pressure data suggested consistency at each zone of an individual model, implying a clear trend of the average magnitude of pressure from high to low: TISE, MICE, MIRE, and MIAE, ranging from only 21 to 25 Pa.This study recognizes CFD modeling is a robust methodology to analyze ventilation performance and assess bird welfare conditions. Full-scale modeling with individual simplified animal models enforces the usefulness of CFD simulation and restores the model\u2019s realism. The four models herein can be fine-tuned to assess other existing ventilation schemes or evaluate proposed ventilation options for various types of poultry houses. In summary, CFD modeling facilitates investigators to tackle animal welfare problems and explore sophisticated solutions related to animal housing."}
+{"text": "Snord116, maintains increased expression of hypothalamic Nhlh2, a basic helix\u2013loop\u2013helix transcription factor. We have previously also shown that obese mice with a deletion of Nhlh2 respond to a conjugated linoleic acid (CLA) diet with weight and fat loss. In this study, we investigated whether mice with a paternal deletion of Snord116 (m+/p\u2212Snord116) would respond similarly. We found that while m+/p\u2212Snord116 mice and mice with a deletion of both Snord116 alleles were not significantly obese on a high-fat diet, they did lose body weight and fat on a high-fat/CLA diet, suggesting that the genotype did not interfere with CLA actions. There were no changes in food intake or metabolic rate, and only moderate differences in exercise performance. RNA-seq and microbiome analyses identified hypothalamic mRNAs, and differentially populated gut bacteria, that support future mechanistic analyses. CLA may be useful as a food additive to reduce obesity in humans with PWS.Prader\u2013Willi Syndrome (PWS) is a human genetic condition that affects up to 1 in 10,000 live births. Affected infants present with hypotonia and developmental delay. Hyperphagia and increasing body weight follow unless drastic calorie restriction is initiated. Recently, our laboratory showed that one of the genes in the deleted locus causative for PWS,  SNORD116 cluster , and the IPW gene which also encodes a non-coding RNA of little known function -Snord116tm1.1Uta/J Stock No: 008149|1-loxp (KO), Snord116del) were obtained from Jackson Laboratories and maintained on a C57Bl/6 background. Genotyping and breeding were performed as reported  + [15 min glucose \u00d7 15) + [30 min glucose \u00d7 22.5]) + [60 min glucose \u00d7 30]) + [90 min glucose \u00d7 30]) + [120 min glucose \u00d7 15]).Rotarod balance measurements, metabolic measures, mouse functional muscle testing, euthanasia with blood collection, tissue collection, and histology were all performed only as post-measurements at the end of the study during Weeks 13\u201314. Mice continued on their study diet until euthanasia.For rotarod analysis, animals were tested using a Columbus Instruments  Economex Rota-Rod apparatus. All mice in the study were given four trials each day during the day (between 11 a.m. and 1 p.m.) for four consecutive days from 0 rpm to a maximum speed of 20 rpm, with an acceleration slope of 2.65%. Animals were tested for a maximum of 5 min of running per test, or until the animals fell off the device. The Economex Rota-Rod apparatus is equipped with a pressure-sensitive landing area so that the time spent on the rotating rod is automatically recorded when the animal falls. The procedure has been previously described by our laboratory [2 consumption and VCO2 production in individual mice were measured using metabolic chambers. Air going into the TSE system was at 20.9% oxygen, 0.05% CO2, and the airflow rate was 0.4 L/min . Data were collected every 15 min. Body composition was measured, as described above, prior to assessment of the animals using the TSE system. A photobeam-based activity monitoring system detected and recorded ambulatory movements. The results were used to calculate the respiratory exchange ratio (RER) and total energy expenditure/gram lean mass. Energy expenditure (kJ/h) was calculated using the formula VO2 \u00d7 (3.815 + (1.232 \u00d7 RER)) \u00d7 4.1868 [N = 7 WT control, N = 5 WT CLA, N = 6 PWS control, N = 6 PWS CLA, N = 4 PWS-KO control, N = 4 PWS-KO CLA. These reduced n values may have contributed to the non-significant finding for the indirect calorimetry assessment.Indirect calorimetry and locomotor activity measurements were performed using a Labmaster Mouse Calorimetry and Locomotor system  in the Metabolism Core Virginia Tech. VO\u00d7 4.1868  and normThe procedures for in vivo muscle testing were recently published . Briefly\u00ae using a rotor stator homogenizer. TRIzol samples were frozen at \u221220 \u00b0C in microfuge tubes until purified (2 weeks to 5 months). Thawed TRIzol samples were purified using the TRIzol + Purelink RNA minikit , following manufacturer\u2019s instructions for the TRIzol\u00ae Plus Total Transcriptome Isolation protocol. Purified RNA was then DNAse-treated using TURBO DNA-free\u2122 Kit (ThermoFisher #AM1907) according to manufacturer\u2019s instructions, diluted to 60 ng/\u03bcL in nuclease-free water, and stored at \u221280 \u00b0C.Fresh hypothalamus tissue was lysed in TRIzolRNA sequencing and library preparation was performed by Virginia Tech\u2019s Genomics Sequencing Center facility at the Fralin Life Sciences Institute. Total RNA with an RNA Intensity Number \u2265 8.0 was converted into a strand-specific library using Illumina\u2019s TruSeq Stranded mRNA HT Sample Prep Kit , for subsequent cluster generation and sequencing on Illumina\u2019s NextSeq. The library was enriched by 14 cycles of PCR, validated using Agilent TapeStation, and quantitated by qPCR. Individually indexed cDNA libraries were pooled and sequenced on NextSeq 75 SR. The Illumina NextSeq Control Software v2.1.0.32 with Real Time Analysis RTA v2.4.11.0 was used to provide the management and execution of the NextSeq 500 and to generate binary base call (BCL) files. The BCL files were converted to FASTQ files, adapters trimmed, and demultiplexed using bcl2fastq Conversion Software v2.20. FASTQ files were aligned to mouse genome GRCm38.p6 using the Geneious RNA assembler 2 January 2020 from Geneious Prime with map quality 30 (99.9% confidence) and spanning intron annotations. Raw read counts were quantified to gene and/or transcript annotations with loss of strand-specificity. Differential expression analysis across experimental conditions was performed using the DESeq2 plugin in Geneious Prime ,24,25. D\u00ae Green RNA-to-CT\u2122 1-Step Kit (ThermoFisher #4389986) was used according to manufacturer\u2019s instructions. Reactions of 10 \u03bcL were performed using 150 nM final primer concentration. Primers were assessed for efficiency using a dilution series and fell within 90\u2013110% efficiency. A 90 ng measure of RNA was used per 10 \u03bcL reaction. Two to three technical replicates were performed. Control reactions for each sample (minus reverse-transcriptase and minus template controls) were used for quality control. On the ViiA 7 Real-Time PCR System (ThermoFisher), 384-well plates were run according to RT-QPCR mix instructions, and thermocycling conditions were not modified from suggested protocol . Quality-control measures including melt-curve analysis, technical replicate analysis, etc., were analyzed by thermocycler software and by operator; any major errors were excluded from analysis when deemed appropriate by the quality-control software, and/or new samples and plates were run. Candidate reference genes for ddCT analysis were analyzed and mouse beta-actin was chosen as the reference gene control for all experiments.For RT-QPCR, a Power SYBRCecal samples were flash-frozen in liquid nitrogen and stored at \u221280 \u00b0C. DNA was isolated from cecal content using a DNeasy PowerSoil Pro Kit (Qiagen #47014) and the TissueLyser II (Qiagen #85300). Fifty nanograms of genomic DNA was utilized for amplification of the V4 variable region of the 16S rRNA gene using 515F/806R primers. Forward and reverse primers were barcoded to accommodate multiplexing up to 384 samples per run as described by Kozich and colleagues . Paired-Demultiplexing, adapter trimming, and generating the fastq files were performed automatically using the Miseq Reporter on the instrument computer. Bioinformatics analysis was then conducted using the QIIME 2 platform . Denoisi2 asphyxiation and placed into 4% paraformaldehyde, overnight, with rocking at 4 \u00b0C. The tissues were then rinsed in 70% ethanol and stored in 70% ethanol at 4 \u00b0C until processing. The Virginia Tech Veterinary Teaching Hospital at the Virginia-Maryland College of Veterinary Medicine processed the tissues for histology and stained with hematoxylin\u2013eosin stain for microscopy. Representative samples were visualized using a 40\u00d7 ocular on a Nikon Eclipse 50i microscope and captured using an Olympus Q-color3 camera.Tissues were isolated immediately following euthanasia by COp-values were made using JMP Pro15 software , with Tukey\u2019s post hoc analysis for multiple comparisons when overall p-values in the comparison were significant. For the responses of weight, fat, lean mass, temperature, and food intake shown in figures, a fitted least squares regression with effects of genotype, treatment, and genotype \u00d7 treatment was analyzed for Week 12 data only. Responses of these dependent variables were also analyzed across the 12-week time period, adding in effects of week, week \u00d7 treatment, and week \u00d7 genotype. These results are shown in All values were expressed as mean \u00b1 SEM unless indicated otherwise. Comparison of means between groups and calculation of ddCT method of relative quantification was used. Statistical significance tests were performed on respective ddCT values, from which Relative Quantification values were derived. A two-way ANOVA with Bonferroni correction was used for relative expression of RNA normalized to WT or control conditions.All RT-QPCR data were analyzed using Microsoft Excel 16 for Microsoft 365, IBM SPSS Statistics 26 for Windows, and GraphPad Prism 9.0.0. The numbers of samples in statistical tests are described in respective figures. The 2In vivo plantarflexor torque\u2013frequency and fatigue curves were analyzed using GraphPad Prism 9.2.0. A two-way ANOVA was performed with multiple comparisons across groups. For torque\u2013frequency figures, a nonlinear model was fit using the sigmoidal curve with variable slope. p-values corrected for multiple testing using the false discovery rate (FDR) approach [Statistical analyses for microbiome data were performed in R v4.0.5. Differences in beta diversity were evaluated using permutational multivariate analysis of variance (PERMANOVA) with 999 permutations and including the weighted Unifrac distance matrix. The Kruskal\u2013Wallis test was used to evaluate alpha metrics and taxonomic differences and the Dunn\u2019s test was used to evaluate pairwise multiple comparisons, with approach . F = 234.19, p < 0.001), treatment , and week  were all highly significant. Additionally, the cross effect of genotype \u00d7 treatment was significant , with post hoc analysis indicating significant differences between WT on control diet and all other genotypes and treatments (p < 0.001). Treatment by week response was significant (p = 0.044), as was genotype by treatment (p = 0.016). Analysis of the end of study (Week 12) data for body weight indicates that there was a significant effect of genotype  and treatment . Post hoc analysis of genotype shows significance .The 20% fat diet only results in a slight body weight and fat increase over a normal chow diet, and the highest weight gain occurs in the WT animals on control diet A,B. Overeatments A. In theificance B. Althouificance , CLA dieificance . Post hoificance A indicatF = 109.77, p < 0.001), treatment  , and genotype by week  were all highly significant, while the interaction of genotype by week was not (p = 0.8962), indicating that genotypes all responded to the treatment similarly. In analyzing body fat at the end of the study, WT mice on the control diet had ~4 g more body fat than WT mice on the CLA diet. Comparing fat levels to differences in body weight indicate that increased weight in control-diet groups was nearly all due to increased body fat  , week , without a treatment effect , but with both an effect of week , and an interaction of genotype \u00d7 treatment . No other interactions were significant. Post hoc analysis of the genotype \u00d7 treatment interaction through the entire study indicated that CLA-treated PWS-KO and PWS mice had lower overall lean mass when compared to CLA-treated WT mice , with Tukey post hoc analysis showing WT lean mass (22.31 g) significantly higher than both PWS (19.36 g) and PWS-KO (19.16 g) lean mass , and nor was treatment , but the effect of week was significant . However, there were no cross-interaction effects. Post hoc analysis using Student\u2019s t to examine the overall effect of week, going from pre- to post-week data, showed that there was a significant reduction in overall body temperature from Week 1 to Week 12 . However, by Week 12, body temperature showed no significant differences for genotype by treatment   D, while  = 0.14) .F = 41.9, p <0.0001), treatment , and week . Post hoc analysis of genotype indicated that WT mice ate approximately 2\u20133 g more food overall than PWS and PWS-KO mice , treatments , or the interaction of genotype \u00d7 treatment .Over the entire study, CLA diet showed a significant effect of treatment on food intake for genotype , with post hoc tests revealing a significant increase in fasting glucose for PWS-KO mice over WT, but not PWS mice , with the post hoc analysis showing significant differences between PWS and PWS-KO genotypes, but not WT mice . Post hoc analysis revealed that fasting glucose for animals fed CLA diet  was significantly higher compared to control-diet-fed animals  or on KJ per fat-free mass energy expenditure levels  in humans given CLA, although mice treated with CLA show consistently higher RMRs and respiratory quotient, a measure of fat oxidation . TSE meae levels  . Post hoc analysis demonstrated greater wheel revolutions for WT compared to PWS-KO mice in the pre-study period . At theF = 16.42, p < 0.0001) or lean body mass . When normalized to lean body mass or total body mass, torque was significantly reduced in WT mice on the control diet, compared to all other groups except PWS-KO . Interestingly, CLA treatment increased torque output in WT mice, regardless of normalization method (p < 0.0001). PWS mice on the control diet showed a significant increase in torque over PWS mice on the CLA diet when data were normalized to lean mass (p < 0.0336), but not when total body mass was used. (p = 0.9993). Resistance to fatigue was greatest in the PWS-KO group . WT  and PWS  mice on CLA diet both showed greater fatigue than most other genotypes and treatments  analysis (p = 0.07). However, the gamma-aminobutyric acid (GABA) A receptor, subunit gamma 2 (Gabrg2) mRNA maintained its significant increase in PWS mice compared to WT mice, although the increase of 1.16-fold by QPCR is modest (p = 0.02). In comparing the effect of diets on both genotypes, charged multivesicular body protein 1B (Chmp1b) mRNA was 1.27-fold increased with CLA diet for RNA-seq, and this increase was maintained in the verification QPCR results (p = 0.01). Reticulophagy regulator family member 2 (Retreg2) showed a significant 0.89-fold reduction in the RNA-seq analysis of CLA diet treatment on PWS mice, but this modest reduction was not significant in the validation QPCR results (p = 0.22).NHLH2 and SNORD116 are both highly expressed in the adult hypothalamus ,41. As hfindings . Howeveranalysis  revealedanalysis . When onanalysis . Only twanalysis . Four diUsing a mix of isomers similar to those used in our study, Li and colleagues demonstrated a significant reduction in pro-inflammatory bacteria in CLA-fed high-fat-diet mice . Other sp \u2265 0.58) but differed between the control and CLA treatments , whereas genotype had no effect. At phylum level, the relative abundance of Cyanobacteria was lower in mice fed the CLA diet compared to those fed the control diet (p = 0.04), whereas the relative abundance of the other phyla was similar (p > 0.09) between diets. Amongst genotypes, PWS-KO mice had significantly lower relative abundance of Bacteroidetes compared to WT mice (p = 0.04), but similar compared to PWS (p = 0.26). Reduced abundance of Bacteroidetes phylum is generally associated with obesity [Proteobacteria, PWS mice had a lower relative abundance (p = 0.05) compared to WT mice, but similar compared to PWS-KO (p = 0.73) . Turicibacter is a genus in the Firmicutes phylum, with this phylum generally making up the largest portion of the gut microbiome [Turicibacter sp. In all genotypes , although the Firmicutes phylum overall was not significantly affected .\u03b1-diversity describes the structure of a microbial community in relation to the number of taxonomic groups and their respective abundance. \u03b1-diversity metrics were similar by genotype ( obesity . For Pro = 0.73) B. Only t = 0.73) C, with ahe colon , and in crobiome . CLA treSnord116 . The Snord116 locus encodes multiple small nucleolar RNAs and is one of the three genes within the minimal causative genomic region for PWS [Snord116, as well as a deletion of both alleles of Snord116, were included in this study. Our findings demonstrate that deletion of Snord116 either hemi- or homozygously does not interfere with the weight- and fat-reducing properties of CLA. Despite the lack of overt obesity of the PWS and PWS-KO mice, even on a 20% fat diet, both genotypes showed significant body weight loss due to body fat and not lean (muscle) mass loss. Of note, Tonalin\u2122, which is a 50:50 mixture of CLA isoform, has mixed results overall in weight reduction for humans, perhaps due to the wide variability in dosage per gram body mass given in each study [Obesity and the complications of increased body weight and fat mass represent one of the main adult phenotypes impacting health and well-being for patients with PWS. The main goal of the study was to determine if CLA treatment would result in body weight and fat loss in mice with a single or double deletion of  for PWS . Mice wich study . In humach study . Thus, fPatients with PWS experience increased body weight due to both hyperphagia and reduced physical activity ,52,53. TPatients with PWS display overall muscle hypotonia and functional weakness. Several studies report reduced cross-sectional area and unusual morphology of PWS-affected muscles ,59. Few Several studies have demonstrated that CLA can cause browning of white adipose tissue . As broIn further attempting to characterize physiological differences in CLA-treated PWS and PWS-KO mice, fasting glucose and glucose tolerance tests, as well as liver histology, were measured. Not unexpectedly, CLA treatment significantly increased fasting glucose, and overall AUC in GTT tests, regardless of genotype. Likewise, CLA treatment increases the incidence of liver steatosis regardless of genotype. Several reports have demonstrated that it is the t10, c12 CLA isomer that is responsible for liver steatosis development, and that the mechanism appears to be due to increased circulating free fatty acids and glucose that are picked up by the liver, and not adequately oxidized . ProteomMael, was \u201crescued\u201d by CLA in PWS mice. Mael plays a central role in spermatogenesis [Chmp1b. The protein product of Chmp1b is an endosomal-associated protein involved in the endosomal sorting complexes required for transport (ESCRT) that may be involved in the multi-vesicular body (MVB), a specialized endosome [Chmp1b was increased with CLA diet, these data suggest there may be more membrane protein turnover or fatty-acid oxidation with CLA. However, more studies are needed to confirm this finding. The RBM3/CIRBP RNA binding protein GO pathway, which was significant for WT versus PWS mice, includes Rbm3 and Cirbp, which are two RNA binding proteins induced by hypothermia, and modulated during circadian rhythms [Nhlh2 mRNA stability is modulated by Snord116, possibly through a motif that is very near to the end of the transcript [Nhlh2 mRNA oscillation with cold-temperature exposure [Nhlh2 in PWS and hypothalamic circadian and sleep patterns. Finally, for WT versus PWS mice  two genes were further analyzed by qPCR, but only one, the Gagbr2 gene encoding GABA receptor Type A, subunit gamma2, was significantly upregulated in PWS mice with secondary analysis. In the PVN of the hypothalamus, Gabrg2 protein has been implicated in diurnal rhythmicity in metabolism and diet-induced obesity through upstream regulation by the circadian gene Bmal1 [Nhlh2, Pomc, Npy, and others. We hypothesize that a cell-type specific approach under specific signaling conditions may better capture sensitivity to Snord116-mediated regulation. Overall, the mRNA-seq and follow-up RT-QPCR show very modest changes, if any. The weak effect size for all differentially expressed genes in the current RNA-seq study also leads to difficulty in validation of differentially expressed genes, as RT-QPCR is limited by the sample size in the current study and their natural variability. Similarly, previous RNA expression studies of hypothalamus tissue from PWS mouse models have shown modest effect sizes [We undertook an RNA-seq approach to attempt to identify Snord116-deletion-specific effects and CLA-specific effects on mRNA expression in the hypothalamus, possibly linking these to relevant pathways. This is the first RNA-seq study examining the hypothalamus in mice with a CLA-supplemented diet, to the authors\u2019 knowledge ,16,40,43ogenesis , but theendosome ,69,70. Cendosome . As Chmp rhythms . This is rhythms . The twoanscript , as wellexposure . Future ne Bmal1 . Loss ofne Bmal1 . Additione Bmal1 . Interesne Bmal1 . Upregulne Bmal1 . Of notect sizes ,80,81.m+/p\u2212Snord116) genotype, or the interaction of these could have effects on gut microbiota. Previous analyses of individuals with PWS have found that those individuals with obesity share a similar microbiome compared to individuals with non-syndromic obesity [Bifidobacterium sp. [Bifidobacterium sp. than others in the study, including individuals with irritable bowel syndrome [Tenericutes were significantly higher in those individuals with PWS, which was not replicated in our mouse model. Turicibacter sp. decreased in CLA diet, and while not much is published on this genus, it was also found to be low in Type I diabetic children [Bacteriodetes and Firmicutes showed the trend towards decrease and increase (non-significant), respectively, in PWS and PWS-KO compared to WT mice. This is consistent with overall Bacteriodetes/Firmicutes profile in obese vs. normal-weight individuals. Firmicutes are known for their energy harvesting capabilities and tend to show increase in obese individuals [As this was a dietary intervention, microbiome analysis was performed to determine if CLA diet, the PWS ( obesity ,83, and syndrome . Howeverchildren . Relativividuals . The micSnord116 was confirmed. Consistent with the literature on CLA diet, there was no detectable change in food intake throughout the 12-week study. While fasting glucose was, as expected, increased with CLA diet in all genotypes, there was an unexpected difference in hepatic steatosis, with about half of the PWS and PWS-KO genotype mice not showing fatty liver, compared to all of the WT mice on CLA diet. This difference appears to be genotype-specific, although more work is needed to determine the mechanism. Interestingly, neither lean mass nor muscle function were compromised or improved by CLA treatment. Metabolism was also unchanged, leading us to be unable to conclude the mechanism for CLA-induced body fat loss. Studies that were designed to tease out CLA-induced or -suppressed pathways through either mRNA regulation in the hypothalamus or microbiome analysis in the gut have provided intriguing leads for future studies.Overall, the original hypothesis that CLA diet would reduce body weight and body fat in mice carrying a paternally inherited deletion of VTIP 21-068: Reversal of Prader\u2013Willi Syndrome Symptoms with Tonalin Conjugated Linoleic Acid, U.S. Provisional Application No. 63/184,001, to D.J.G."}
+{"text": "Structural cohesion is maintained by an array of C\u2014H\u22efO, N\u2014H\u22efCl and O\u2014H\u22efCl hydrogen bonds.In the racemic title compound, the [Co(en) 2H8N2)3]Cl3.{[Na(H2O)6]Cl}0.5, are reported. The trivalent cobalt atom, which resides on a crystallographic threefold axis, is chelated by a single ethyl\u00adene di\u00adamine (en) ligand and yields the tris-chelate [Co(en)3]3+ cation with distorted octa\u00adhedral geometry after the application of crystal symmetry. The sodium cation (site symmetry 2O)6]+ cation after the application of symmetry. One of the chloride ions lies on a general position and the other has \u22efO, N\u2014H\u22efCl and O\u2014H\u22efCl hydrogen bonds exists between the ethyl\u00adene di\u00adamine ligands, the water mol\u00adecules of hydration, and the anions present, thereby furnishing solid-state stability.The synthesis and crystal structure of the title racemic compound, [Co(C In all cases, the 3+ and [Na(H2O)6]+ cationic complexes that result each adopt distorted octa\u00adhedral geometries.The title compound crystallizes in the centrosymmetric trigonal space group c1 Fig.\u00a01. The asy3sp-hybridization of the C atoms and an expected tetra\u00adhedral coordination environment around those C atoms, bond angles around each should be near the expected 109.5\u00b0. The values obtained from the crystal structure  indicate a degree of distortion.Within the chelating en ligand, given the \u22efO, N\u2014H\u22efCl and O\u2014H\u22efCl hydrogen bonds between the [Co(en)3]3+ and [Na(H2O)6]+cations and the chloride anions. Unlike the structure of enanti\u00adopure \u039b-[tris\u00ad(ethyl\u00adenedi\u00adamine)\u00adcobalt(III) trichloride]\u00b70.5NaCl\u00b73H2O 3 cations demonstrate similar energies  plane. As a result of crystal symmetry, the full Cl3, other chemically-similar salts have been crystallized that demonstrate hydrogen-bonding arrays involving the ethyl\u00adenedi\u00adamine ligands, inter\u00adstitial water mol\u00adecules, and the counter-ions. These included the \u039b-enanti\u00adomer of the monohydrate Cl and I salts, which crystallize in the tetra\u00adgonal space group 3212P4, in 1969 and 2001, respectively 3]3+. The structures of the nitrate salts, obtained both as a racemic crystal in the 1Pca2 space group 5(NO)]\u00b72H2O 3]2[HPO4]3\u00b79H2O was determined to exist in the ortho\u00adrhom\u00adbic space group Pnma 3]\u00b7Cl3\u00b70.5NaCl\u00b73H2O 1 was prepared following the method of Girolami  was dissolved in water (20\u2005mL) with stirring. Upon addition of ethyl\u00adenedi\u00adamine di\u00adhydro\u00adchloride , the solution became pink and cloudy. Sodium hydroxide pellets  were next added slowly while the solution stirred. Each pellet initially turned blue and then completely dissolved within a few minutes. The pH was then tested using litmus paper and determined to be 8. Hydro\u00adchloric acid (6 M) was added dropwise until the pH was approximately 7\u20137.5, which changed the color of the solution to rusty orange. A hydrogen peroxide solution (20\u2005ml of 3% solution) was added dropwise over a couple of minutes and the solution became dark orange. The solution was slowly brought to a boil while stirring. The stir bar was removed and the beaker placed into an ice bath for 30 minutes to cool. The crystals were collected through filtration and washed with 95% ethanol (50\u2005ml) and subsequently diethyl ether (20\u2005ml) to yield a bright-orange powder . Large single crystals (ca 3 \u00d7 3 \u00d73\u2005mm) of 1 were grown by slow evaporation from water and cut to size using a razor blade.The title complex 1 are summarized in Table\u00a02E, which was constrained to ride on the water O atom (O1), all other H atoms were located in the difference-Fourier map and freely refined with 0.91 < C\u2014H < 0.99\u2005\u00c5, 0.81 < N\u2014H < 0.84\u2005\u00c5, and O\u2014H = 0.89\u2005\u00c5.Crystal data, data collection and structure refinement details for 10.1107/S2056989021009336/hb7980sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021009336/hb7980Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021009336/hb7980Isup3.cmlSupporting information file. DOI: 2042981CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:"}
+{"text": "Serum hepatitis B virus (HBV) pregenomic RNA (pgRNA) is correlated with covalently closed circular DNA. We aimed to investigate the utility of serum HBV pgRNA in chronic hepatitis B patients receiving nucleos(t)ide analogue treatment and those achieving HBsAg loss. One hundred and eighty-five patients were enrolled for studying long-term HBV pgRNA kinetics during treatment. Twenty patients achieving HBsAg loss after treatment were enrolled for examining HBV pgRNA kinetics around HBsAg loss. HBV pgRNA significantly decreased in the high baseline HBV pgRNA (\u22656 log copies/mL) group but significantly increased in the low baseline HBV pgRNA (<4 log copies/mL) group after 3-month entecavir treatment. Among the 20 patients achieving HBsAg loss, 13 (65%) patients had serum HBV pgRNA higher than the limit of detection  when they achieved HBsAg loss. Finally, all 20 patients had HBV pgRNA going below the LOD within 3 years after achieving HBsAg loss. In conclusion, baseline serum HBV pgRNA alone is insufficient for predicting the trajectory of HBV pgRNA. Most patients still had HBV pgRNA higher than the LOD when they achieved HBsAg loss. Further studies on HBV pgRNA kinetics around HBsAg loss would provide an enhanced basis for further applications of HBV pgRNA. Chronic hepatitis B virus (HBV) infection is an important health problem worldwide. The WHO estimated that 257 million people were living with chronic HBV infection in 2015 . CurrentSerum HBsAg, which is derived from cccDNA and integrated HBV DNA sequences, could be found in complete virions, subviral particles, empty virions, and HBV RNA-containing virion-like particles . QuantifHBV pregenomic RNA (pgRNA), which is transcribed from cccDNA, is the template for both reverse transcription of relaxed circular DNA and translation of viral polymerase and core proteins . CirculaPatients were retrospectively enrolled from our clinics, previous clinical trials, and databases in National Cheng Kung University Hospital. All the CHB patients in Part I and Part II received at least 2 years of consecutive entecavir treatment, which was the only antiviral therapy during the study period. The indication for antiviral therapy mostly followed the Asian Pacific Association for the Study of the Liver HBV treatment guideline . The excThe patients in Part I and II were followed up at 3- to 6-month (12- to 24-week) intervals through liver biochemistry and serum HBV DNA tests, as well as abdominal sonography. In HBeAg-positive patients, HBeAg was assessed every 3 to 6 months until negative results were obtained. Liver cirrhosis was diagnosed by liver biopsy, abdominal sonography, computed tomography (CT), magnetic resonance imaging (MRI), or portal hypertension . Hepatocellular carcinoma was diagnosed by histological examination or dynamic imaging studies (CT or MRI). The upper limit of normal (ULN) of alanine aminotransferase (ALT) was 50 U/mL in male patients and 35 U/mL in female patients at National Cheng Kung University Hospital.For studying the kinetics of serum HBV pgRNA, HBV DNA, and HBsAg during entecavir therapy, serum HBV pgRNA and HBV DNA quantification was performed at baseline and the 3rd month (12th week), 6th month (24th week), 12th month (48th week), and 60th month (240th week) of treatment, and serum HBsAg quantification was performed at baseline and the 1st year (48th week) and 5th year (240th week) of treatment. Virological response was defined as undetectable serum HBV DNA (<60 IU/mL) after entecavir therapy.For analyzing the factors associated with virological relapse, these patients were monitored for up to 3 years after cessation of entecavir therapy. Serum HBV pgRNA, HBV DNA, and HBsAg levels were determined at the end of treatment. Virological relapse was defined as serum HBV DNA level >2000 IU/mL after cessation of entecavir therapy.Serum HBsAg levels were measured using the Architect HBsAg assay , with a limit of detection (LOD) of 0.05 IU/mL. For statistical analysis, serum samples with HBsAg levels below LOD were recorded as 0.04 IU/mL (\u22121.40 log IU/mL).Serum HBV DNA levels were determined using the Roche Cobas Amplicor (LOD: 60 IU/mL); Roche Cobas TaqMan 48 analyzer (LOD: 29 IU/mL); Roche Cobas AmpliPrep/Cobas TaqMan HBV Test, version 1.0 (LOD: 12 IU/mL); and Roche Cobas AmpliPrep/Cobas TaqMan HBV Test, version 2.0 (LOD: 20 IU/mL). For statistical analysis, serum samples with HBV DNA values below LOD and serum samples in which HBV DNA was not detected were recorded as LOD \u2013 1 IU/mL and 1 IU/mL, respectively.HBV RNA was extracted from 150 \u03bcL of serum using the Total RNA Extraction Miniprep System Kit according to the manufacturer\u2019s instructions  and was treated with DNase I . Isolated HBV RNA was reverse transcribed using RevertAid reverse transcriptase  with an HBV pgRNA-specific reverse transcription (RT) primer . Before \u00ae Green Master Mix , 0.5 \u03bcL of the forward primer (10 \u03bcM), 0.5 \u03bcL of the reverse primer (10 \u03bcM), 1 \u03bcL of the cDNA template, and 8 \u03bcL of double distilled water (ddH2O). The reaction mixture was denatured at 95 \u00b0C for 5 min, followed by 40 cycles at 95 \u00b0C for 20 s and 60 \u00b0C for 40 s. The LOD of serum HBV pgRNA was 1466 copies/mL, as calculated by probit analysis  by using a SYBR Green method. The primers used to detect 3.5 kb HBV pgRNA are provided in analysis . For staThe HBV genotype was determined through melting curve analysis using LightCycler hybridization probes, as described previously .t-test or the Mann\u2013Whitney test, as appropriate. Continuous variables were compared between three groups using one-way ANOVA or the Kruskal\u2013Wallis test, as appropriate. The distributions of categorical variables were compared using Pearson\u2019s chi-squared test or Fisher\u2019s exact test when the expected value was less than 5 in 2 \u00d7 2 tables. The cumulative incidence of virological response, virological relapse, and HBsAg loss was derived using the Kaplan-Meier analysis and was tested using the log-rank test. Multivariate analysis was performed using Cox proportional hazards regression to identify the factors associated with virological responses, virological relapse, and HBsAg loss. Linear mixed models with a random intercept were used for analyzing the longitudinal changes in serum HBV pgRNA, HBsAg, and HBV DNA levels. In this model, groups and time points were considered categorical variables and represented by dummy variables. Statistical analysis was performed using Stata 16.1 . The results were considered statistically significant at p < 0.05.Continuous variables were compared between two groups using Student\u2019s In Part I, 185 CHB patients were enrolled for the analysis of long-term HBV pgRNA kinetics during entecavir therapy. The mean age was 51.0 \u00b1 12.0 years, 70.3% were men, and 29.2% were HBeAg-positive. At baseline, the mean ALT, HBsAg, HBV DNA, and HBV pgRNA levels were 3.84 \u00b1 5.84 \u00d7 ULN, 3.15 \u00b1 0.78 log IU/mL, 5.83 \u00b1 1.72 log IU/mL, and 5.07 \u00b1 1.98 log copies/mL, respectively. For patients with available data on HBV genotypes, 88 had genotype B and 76 had genotype C. The median treatment duration was 4.85  years. As expected, HBeAg-positive patients were younger and had higher baseline HBsAg, higher baseline HBV DNA, higher proportion of genotype C, lower proportion of liver cirrhosis, and later virological responses than HBeAg-negative patients .For analyzing serum HBV pgRNA kinetics during entecavir treatment, patients were categorized into three groups according to baseline HBV pgRNA levels: high, medium, and low baseline HBV pgRNA groups . In the high baseline HBV pgRNA group, serum HBV pgRNA levels decreased rapidly in the first 3 months and remained constant thereafter. In the medium baseline HBV pgRNA group, HBV pgRNA levels decreased gradually in the first 12 months and remained constant thereafter. In the low baseline HBV pgRNA group, HBV pgRNA levels increased in the first 3 months and remained constant thereafter. Compared with the medium and low HBV pgRNA groups, the high baseline HBV pgRNA group had higher HBV pgRNA levels at baseline, 12 months, and 60 months. The high baseline HBV pgRNA group also had higher HBV pgRNA levels at 3 months and 6 months than the low HBV pgRNA group. The medium baseline HBV pgRNA group had higher HBV pgRNA levels at baseline and 3 months compared with the low HBV pgRNA group. No significant differences were detected in HBV pgRNA levels at 6 months, 12 months, and 60 months between the medium and low HBV pgRNA groups .Moreover, no significant differences were found in age, sex, HBeAg status, HBV genotype, baseline ALT, and liver cirrhosis status between the high, medium, and low HBV pgRNA groups .p = 0.0018, p = 0.034; HBeAg negativity vs. positivity: HR: 2.03, 95% CI: 1.27\u20133.25, p = 0.003; HBV pgRNA < 6.4 vs. \u2265 6.4 log copies/mL: HR: 1.57, 95% CI: 1.06\u20132.31, p = 0.023, The virological response to entecavir treatment was assessed in 171 CHB patients, after excluding 8 HBeAg-positive and 6 HBeAg-negative patients with limited serum samples within the first year, as the limited serum samples prevented the virological response from being precisely determined. Kaplan-Meier analysis showed that baseline HBV pgRNA < 6.4 log copies/mL was associated with earlier virological responses compared with baseline HBV pgRNA \u2265 6.4 log copies/mL . By contrast, HBV pgRNA was not correlated with HBV DNA or HBsAg in HBeAg-negative patients. HBV DNA was positively correlated with HBsAg in both HBeAg-positive and HBeAg-negative patients . After 1 year of entecavir treatment, HBV pgRNA was not correlated with HBsAg in either HBeAg-positive or HBeAg-negative patients  years. At the end of treatment, the mean age, ALT, HBsAg, and HBV pgRNA levels were 46.8 \u00b1 11.9 years, 0.74 \u00b1 0.85 \u00d7 ULN, 2.76 \u00b1 1.30 log IU/mL, and 3.90 \u00b1 0.97 log copies/mL, respectively. The median HBV DNA level was 0.00  log IU/mL at the end of treatment. For the patients with available data on HBV genotypes, 20 had genotype B and 24 had genotype C. Compared with HBeAg-negative patients, HBeAg-positive patients were younger and had higher baseline HBsAg, baseline HBV DNA, and HBsAg levels at the end of treatment .p = 0.040 and p = 0.038, p = 0.039, Kaplan-Meier analysis showed that \u201cHBV pgRNA \u2265 1466 copies/mL\u201d and \u201cHBsAg \u2265 2 log IU/mL or HBV pgRNA \u2265 1466 copies/mL\u201d at the end of treatment were associated with earlier virological relapse after cessation of entecavir therapy ide analogue treatment, 21 CHB patients who achieved HBsAg loss spontaneously, 20 healthy subjects with negative HBsAg but positive anti-HBc, and 17 healthy controls with negative HBsAg and negative anti-HBc were enrolled for determining the expression of serum HBV pgRNA after HBsAg loss. Serum HBV pgRNA was not detected in CHB patients who achieved HBsAg loss spontaneously, healthy subjects with negative HBsAg but positive anti-HBc, or healthy controls with negative HBsAg and negative anti-HBc. Notably, 10 CHB patients who received nucleos(t)ide analogue had serum HBV pgRNA higher than the LOD (1466 copies/mL) at or after HBsAg loss, with a median HBV pgRNA of 5.80  log copies/mL .In Part IV, 20 CHB patients who achieved HBsAg loss after nucleos(t)ide analogue treatment with serial serum samples before and after HBsAg loss were enrolled. Five (25%) and two (10%) patients had HBV pgRNA going below the LOD (1466 copies/mL) before and at HBsAg loss, respectively. The other 13 (65%) patients had serum HBV pgRNA higher than the LOD when they achieved HBsAg loss, with a median HBV pgRNA of 5.90  log copies/mL. Finally, all of these 20 (100%) patients had HBV pgRNA going below the LOD within 3 years after achieving HBsAg loss . Anti-HBp < 0.0001, p = 0.031; HBsAg decline \u22651.5 log IU/mL vs. HBsAg decline <1.5 log IU/mL: HR: 53.59, 95% CI: 3.83\u2013749.5, p = 0.003, In Part V, a total of 161 CHB patients were enrolled for analyzing the factors associated with HBsAg loss after nucleos(t)ide analogue treatment . Kaplan-The study was the largest one to investigate long-term HBV pgRNA kinetics in CHB patients receiving nucleos(t)ide analogue treatment and the first one to study HBV pgRNA kinetics in those patients achieving HBsAg loss.Serum HBV pgRNA levels decreased rapidly in the first 3 months in the high baseline HBV pgRNA group. The levels decreased gradually in the first 12 months in the medium baseline HBV pgRNA group. By contrast, the levels increased in the first 3 months in the low baseline HBV pgRNA group. The high baseline HBV pgRNA group had higher HBV pgRNA levels at baseline, 12 months, and 60 months than the medium and low HBV pgRNA groups. No significant differences were observed in HBV pgRNA levels at 6 months, 12 months, and 60 months between the medium and low HBV pgRNA groups. Our study demonstrated that baseline serum HBV pgRNA alone is insufficient for predicting the trajectory of HBV pgRNA. A more accurate description of HBV pgRNA trajectory would be provided by serial HBV pgRNA levels at baseline, 6 months, and 12 months after treatment.HBV pgRNA is the template for reverse transcription and is normally degraded during the process of reverse transcription. Nucleos(t)ide analogues inhibit reverse transcription. While reverse transcription is inhibited, HBV pgRNA accumulates and is released in the circulation as HBV pgRNA-containing viral particles. Wang et al. showed the levels of HBV pgRNA virion increased after entecavir treatment in cell culture models and HBV transgenic mice . GoncalvEven more remarkably, among the 20 patients achieving HBsAg loss, 13 (65%) patients had serum HBV pgRNA higher than the LOD (1466 copies/mL) when they achieved HBsAg loss, with a median HBV pgRNA of 5.90  log copies/mL. Finally, all of these 20 patients had HBV pgRNA going below the LOD within 3 years after achieving HBsAg loss. Mak et al. showed that 3 of 19 (15.8%) treatment-naive patients with HBsAg loss had detectable serum HBV RNA and HBcrAg . FurtherPrevious studies have shown that serum HBV pgRNA was positively correlated with HBV DNA before antiviral treatment in both HBeAg-positive and HBeAg-negative patients ,24,36. OSeveral studies have shown that serum HBV pgRNA was associated with treatment responses in patients with CHB receiving pegylated interferon or nucleos(t)ide analogue therapy ,41,42,43Our study revealed that age below 50 years and HBsAg decline greater than 1.5 log IU/mL within the first year were independently associated with HBsAg loss after nucleos(t)ide analogue therapy. These findings are compatible with previous studies. Lee et al. indicated that younger age, lower baseline HBsAg, rapid HBsAg decline at week 24, and achievement of sustained virological response were predictors of HBsAg loss after peginterferon therapy . Jeng etBai et al. showed that extracellular HBV RNAs comprised full-length pgRNA and 3\u2032 RNA fragments degraded by the RNase H domain of polymerase from incomplete RT. These RNAs were localized in naked capsids and virions in cell culture supernatants, and they circulated as unenveloped capsids in the form of capsid\u2013antibody complexes and virions in the blood of hepatitis B patients . AnotherOur study has a few limitations. First, this was a retrospective study using stored serum samples. Second, only Asian patients infected with HBV genotype B or C were included. Third, serum HBV pgRNA levels were determined using an in-house method, which utilized DNase I for removal of viral DNA, with an LOD of 1466 copies/mL. Future prospective studies using commercial kits with a higher specificity and a lower LOD in large and diverse populations would provide more comprehensive descriptions of HBV pgRNA kinetics during antiviral treatment, especially for patients with low serum HBV pgRNA levels ,53. FourIn summary, our study demonstrated the kinetics and clinical utility of serum HBV pgRNA in CHB patients receiving long-term entecavir therapy and those achieving HBsAg loss. Baseline serum HBV pgRNA alone is insufficient for predicting the trajectory of HBV pgRNA. Serum HBV pgRNA was associated with virological responses during treatment and virological relapse after treatment withdrawal. Moreover, most patients had serum HBV pgRNA higher than the LOD (1466 copies/mL) when they achieved HBsAg loss. Further studies clarifying the production mechanisms of viral particles containing HBV pgRNA would provide an enhanced basis for further applying serum HBV pgRNA as a biomarker in antiviral treatment."}
+{"text": "The present study aimed to compare the effects of paracetamol and ibuprofen on pain, bleeding, nausea, and vomiting following adenotonsillectomy in children.This was a prospective, double-blinded, randomized clinical trial. Block randomization was used to assign 50 patients to two groups of paracetamol and ibuprofen. In the paracetamol group, subjects received 15 mg/kg oral paracetamol 30 minutes before the induction of anesthesia, followed by the same dosage every 6 hours postoperatively. Meanwhile, the ibuprofen-treated group took 10 mg/kg oral ibuprofen 30 minutes before and every 6 hours after the operation. The subjects in both groups received the medication for three postoperative days. The postoperative pain score was assessed 6 hours after the surgery and during the second and the third postoperative days. Nausea and vomiting episodes were recorded in the first postoperative day and first postoperative week. Based on the results, intraoperative and postoperative bleeding in both groups was not significantly different. The mean score of pain in the first postoperative day (6 hours after the surgery) and the second and the third postoperative days did not show any statistical difference. The ibuprofen group experienced fewer vomiting episodes, compared to the paracetamol group during the first postoperative day (P=0.011). Vomiting episodes in the first postoperative week did not illustrate any significant difference.As evidenced by the results of the current study, Ibuprofen had the same effect on the alleviation of postoperative pain, caused fewer vomiting episodes, and did not cause excessive bleeding as an NSAID. Therefore, oral administration of ibuprofen is suggested for pain relief and management of other complications following adenotonsillectomy in children. Tonsillectomy is one of the most common surgical procedures in children ,2, and rThese complications delay the postoperative oral fluid intake, which leads to dehydration. The management of these complications helps to maintain oral fluid intake and avoid a prolonged hospital stay, leading to a speedy return to normal activities. Opioids, as powerful analgesic drugs, are not favorable due to opioid-induced respiratory depression, sedation, as well as the high incidence of nausea and vomiting. Non-steroidal anti-inflammatory drugs\u00a0(NSAIDs) and paracetamol are good alternatives to opioids. Based on the literature, NSAIDs (other than Ketorolac) are not associated with increased postoperative bleeding. It seems that these drugs can be safely used for the management of pain after tonsillectomy -9. AccorA prospective, double-blinded, randomized trial (IRCT2013071814047N1) was initiated to investigate the comparative effects of paracetamol and ibuprofen on pain relief following adenotonsillectomy. Between March 2015 and February 2017, consecutive children who were candidates of elective adenotonsillectomy in Children\u2019s Medical Centre were assessed for eligibility. The inclusion criteria were as follows: the age range of 4-10 years old, American Society of Anesthesiologists classes I or II, no allergy to NSAIDs, no history of coagulopathy, no renal and hepatic disorder, no severe asthma, no family history of coagulopathy, and no evidence of mental retardation. According to a similar article and sample size formula, a total of 52 patients were included in the study. Block randomization using a block size of 4 was used to assign patients to two groups of paracetamol and ibuprofen. A nurse performed the randomization by opening the envelope which contained the randomization number (1 or 2). There were two syrup bottles with identical shapes which were labeled as 1 or 2 . In the paracetamol group, subjects received 15 mg/kg oral paracetamol 30 minutes before the induction of anesthesia, followed by the same dosage every 6 hours postoperatively. Meanwhile, the ibuprofen-treated group took 10 mg/kg oral ibuprofen 30 minutes before and every 6 hours after the operation.The subjects in both groups received the medication for three postoperative days. The patients and parents were blinded to the type of medications that had been prescribed and the surgeon. The Wong-Baker visual analog pain scale (VAS) graded from 0-10 was used for the assessment of pain. In the preoperative visit, all patients and their parents were trained on the use of the VAS. The patients who reported a missing dose of medications were excluded from the study. All procedures regarding human subjects were conducted in accordance with the declaration of Helsinki. The Ethics Committee of Tehran University of Medical Sciences approved the study protocol. Written informed consent was acquired from all parents prior to enrolment.General anesthesia was induced using sevoflurane 8%. Endotracheal intubation was facilitated by atracurium 5 mg/kg, thiopental 5 mg/kg, and fentanyl 1 \u00b5g/kg. Anesthesia was maintained using 1.5%-2% sevoflurane and 50% N2O. Dexamethasone (as an antiemetic drug) was administered in a single intraoperative dose (0.5 mg/kg) for all patients. Adenoidectomy is performed using a curve adenoid curette, and tonsillectomy is carried out using an extracapsular technique with a cold knife and bipolar cautery. All the operations were performed by the same surgeon. Intraoperative bleeding was assessed by the number of used gauzes and the amount of blood in the suction pump bottle. The occurrence of postoperative bleeding was recorded in the first postoperative day and first postoperative week.The postoperative pain score was assessed 6 hours after the surgery (inpatient setting), as well as during the second and the third postoperative days (via a questionnaire recorded by patients). Nausea and vomiting episodes were recorded in the first postoperative day and first postoperative week (via a questionnaire). Moreover, the time that the patient tolerated the usual diet and the time that they returned to usual activity was recorded (via a questionnaire). The data were analyzed in SPSS software (version 19.0) using t-test, chi-square test, and Mann-Whitney U-test for group, frequency, and pain score comparison, respectively. Continuous variables were expressed as Mean\u00b1SD. A p-value of less than 0.05 was considered statistically significant.A total of 52 patients met the criteria to participate in this study, out of whom two patients (n=1 from each group) were lost to follow-up. The analysis was performed on the remaining 50 subjects  .The indications for adenotonsillectomy included: recurrent tonsillitis (52%), sleep apnea (36%), and obstructive adenotonsillar hypertrophy (12%).Based on the results, 24 (48%) patients were female and 26 (52%) cases were male, 13 (52%) patients in the paracetamol group and 13 (52%) subjects in the ibuprofen group were male . The mean age scores of all participants, the paracetamol group, and the ibuprofen group were reported as 6.94\u00b11.83 6.8\u00b11.75, and 7.08\u00b11.93 years, respectively (P=0.595). The mean body weight scores of all the patients, the paracetamol group, and the ibuprofen group were obtained at 24.38\u00b14.12, 24.04\u00b14.08, and 24.72\u00b14.22 kg, respectively (P=0.566). No significant differences were observed between the two groups in age, gender, and mean body weight. Mean scores of intraoperative bleeding of all patients, paracetamol group, and ibuprofen group were 3.30\u00b10.81, 3.20\u00b10.73, and 3.42\u00b10.89 ml/kg, respectively (P=0.36). Five patients experienced postoperative bleeding in the first postoperative day (two patients in the paracetamol group and three patients in the ibuprofen group (P=1.00). Bleeding was controlled in the ward, and no surgical intervention was required. No bleeding was recorded in the first postoperative week. The mean score of pain in the first postoperative day (6 hours after the surgery), as well as the second and the third postoperative days, did not show any statistical difference . There was no patient who required additional analgesia due to insufficient pain control in both groups.The ibuprofen group experienced fewer vomiting episodes (0.24\u00b10.52), compared to the paracetamol group (0.72\u00b10.73) during the first postoperative day. This finding was statistically significant (P= 0.011). Vomiting episodes in the first postoperative week did not demonstrate any significant difference. The mean postoperative day for the initiation of oral fluid, as well as semi-solid and solid intake, in both groups was not significantly different . This randomized clinical trial study aimed to compare the effect of acetaminophen and ibuprofen on the management of pain and other postoperative complications following adenotonsillectomy. Along with other studies, adenotonsillectomy, in the present research, is mainly performed due to recurrent throat infection, sleep apnea, and obstructive adenotonsillar hypertrophy ,12. SincAcetaminophen was orally administered in the acetaminophen group since this route is more effective. A repeated dosage of acetaminophen and ibuprofen (every 6 hours) was administered for maintaining better pain management. As described by Humanen et al., a single dosage of analgesic is not sufficient for postoperative pain relief . The res\u00a0 The mechanism of post-tonsillectomy nausea and vomiting can be ascribed to blood stasis in the pharynx, as well as oropharyngeal sensitivity due to surgical manipulation and tracheal intubation. According to the literature -11, the  As illustrated by the results of the present study, Ibuprofen had the same effect on the alleviation of postoperative pain, caused fewer vomiting episodes, and did not cause excessive bleeding as an NSAID. Therefore, oral administration of ibuprofen is suggested for pain relief and management of other complications following adenotonsillectomy in children."}
+{"text": "The resulting PLYS-Fe3O4@TiO2 nanosheets possess well defined yolk\u2013shell structures with a large BET surface area (\u223c187.26 m2 g\u22121) and a strong magnetic susceptibility (\u223c17.4 emu g\u22121). The reaction rate constant was 24.2 \u00d7 10\u22122 min\u22121 as a result of oxidative decomposition of BPA using UV/PLYS-Fe3O4@TiO2/H2O2 system. This is 1.1 and 8.34 times faster than the BPA decomposition reaction rate constant in UV/TiO2/H2O2 and UV/Fe3O4/H2O2 systems, respectively. The synthesized catalyst also exhibited excellent recycle capability and excellent acid decomposition performance.Uniform pea-like yolk\u2013shell (PLYS) structured magnetic TiO 2(PLYS-Fe3O4@TiO2) nanosheets have been prepared via a combined kinetics-controlled mechanical force-driven and hydrothermal etching assisted crystallization method and characterized.Uniform pea-like yolk\u2013shell (PLYS) structured magnetic TiO However, the narrow working pH range, difficulties to recover the dissolved metal ions and necessity for further treatment of ferric hydroxide sludge greatly hinder its wide application for practical water treatment.7,8 To this end, heterogeneous Fenton process is developed as a valid approach to overcome these kinds of drawbacks.Emerging organic contaminants (EOCs) are a burgeoning and extremely diverse class of contaminants that are not routinely monitored but have the great potential to enter the environment and may cause known or suspected adverse ecological and human health effects. EOCs of major concern include endocrine disrupting chemicals, pharmaceuticals and personal care products, surfactants, and various industrial additives as well as hormones. BPA, classified as an endocrine disruptor, is a contaminant of importance because it is extensively used in the production of polycarbonates, epoxy resins, and other plastics.et al.9 prepared an effective photo-Fenton catalyst of Fe/TiO2 by deposition\u2013precipitation method for degradation of thiacloprid. However, the catalyst separation issue seems to be a big challenge. Although Lejin et al.10 synthesized Fe3O4 magnetic particles via the co-precipitation of Fe2+ and Fe3+ method and observed the effect of different parameters, the degradation efficiency of 2,4-dichlorophenol is not high. Xiaoliang et al.11 demonstrated the heterogeneous Fenton-like process for the treatment of methylene blue (MB), whereas the core\u2013shell structured Fe3O4@C nanoparticles only worked well in acidic environment. Sheng-Tao et al.12 showed the core\u2013shell structured Fe3O4@SiO2 nanoparticles as efficient Fenton-like catalyst in neutral environment for the degradation of MB but the mechanism still requires farther investigations. In addition, yolk\u2013shell material with a distinctive core@void@shell configuration, has stimulated considerable interest because of the void space between the core and the shell which can provide as a reactor. Dan et al.13 reported that yolk\u2013shell structured Fe3O4@TiO2 nanoparticles as a high-performance catalyst for the combination of photo-Fenton degradation of tetracycline. However, due to Fe3O4 was fully covered by the TiO2 shell, it can cause lots of disadvantages.Recently, Nemanja 3O4@TiO2 nanosheets as a heterogeneous photocatalytic photo-Fenton catalyst. Instead of TiO2 shell, TiO2 nanosheets are coated which cannot only increase the surface area, but also shorter the penetration pathway of both H2O2 and light. Most importantly, even after 3 times cycle test, the catalyst still shows high activity for the degradation of BPA under a neutral condition.In this study, we design and prepare an advanced PLYS-Fe2.2.13\u00b76H2O), sodium citrate tribasic dehydrate, sodium acetate (NaAc), bisphenol-A, concentrated ammonia solution (28 wt%), t-butanol, titanium(iv) isopropoxide (TIPO), TiO2  and tetraethyl orthosilicate (TEOS) were analytical grade and purchased from Sigma-Aldrich (USA). Sodium hydroxide (NaOH), hydrochloric acid , perchloric acid (HClO4), potassium bi-phthalate , ammonium molybdate ((NH4)6Mo7O24\u00b74H2O, 99.0%), ethylene glycol and ethanol were purchased from Samchun Pure Chemicals. All chemicals were analytical grade and used as received without further purification. Corp. deionized water was used for all experiments.Iron chloride hexahydrate , sodium citrate tribasic dehydrate (1.3 g), and sodium acetate  were dissolved in ethylene glycol (80 mL) with agitation. The mixture was stirred vigorously for 1 h at room temperature and then transferred into a Teflon-lined stainless-steel autoclave (100 mL). The autoclave was heated at 200 \u00b0C for 10 h, and then allowed to cool to room temperature. The black products were washed with deionized water and ethanol for 3 times, respectively.The superparamagnetic Fe2.2.23O4@SiO2 nanospheres were prepared according to a St\u00f6ber sol\u2013gel method.12,15 For a typical synthesis, an ethanol dispersion of the Fe3O4 magnetite particles obtained above  was added to a three-neck round-bottom flask with ethanol (70 mL), deionized water (30 mL) and concentrated ammonia solution . The mixed solution was sonicated for 20 min. Then, 1.0 mL of TEOS was added dropwise in 5 min, and the reaction was allowed to proceed for 1 h under continuous mechanical stirring at room temperature. The resultant products (denoted as Fe3O4@SiO2) were separated and collected with a magnet, followed by washing with deionized water and ethanol for 3 times, respectively.The core\u2013shell Fe3O4@SiO2 nanospheres were further coated with a TiO2 shell through a kinetic-controlled St\u00f6ber method. Briefly, the core\u2013shell Fe3O4@SiO2 nanospheres (0.1 g) were dispersed in ethanol (100 mL), and mixed with concentrated ammonia solution  under ultrasound for 15 min. Subsequently, 0.75 mL of TIPO was added dropwise in 5 min, and the reaction was allowed to proceed for 24 h at 45 \u00b0C under continuous mechanical stirring. The resultant products (denoted as pea-like core\u2013shell Fe3O4@SiO2@TiO2) were separated with a magnet and washed with deionized water and ethanol for 3 times, respectively.The as-prepared Fe2.2.33O4@TiO2 nanoparticles were prepared through an alkaline hydrothermal etching assisted crystallization method. The above obtained Fe3O4@SiO2@TiO2 nanoparticles (1.0 g) were mixed with NaOH solution , then transferred into a Teflon-lined stainless-steel autoclave (100 mL in capacity). The autoclave was heated at 200 \u00b0C for 24 h and then cooled to room temperature. The products were collected by a magnet and added in HCl solution  for 15 min, washed with deionized water until pH value was around 7 and subsequently dried at 60 \u00b0C thoroughly in vacuum oven.The PLYS-Fe3O4@TiO2) were calcined 400 \u00b0C in N2 atmosphere for 2 h, then the PLYS-Fe3O4@TiO2 nanosheets were obtained.The resultant products (denoted as PLYS-Fe2.3SBET) using adsorption data in the relative pressure range P/P0 = 0.04\u20130.2. Using the Barrett\u2013Joyner\u2013Halenda (BJH) model, the pore size distributions were derived from the adsorption branches of the isotherms, and the total pore volumes (V) were estimated from the adsorbed amount at the relative pressure P/P0 = 0.995. Transmission electron microscopy (TEM) was carried out on a JEOL 2011 microscope (Japan) operated at 200 kV. For TEM measurements, the sample was suspended in ethanol and supported on a holey carbon film on a Cu grid. High-resolution transmission electron microscopy (HRTEM) observations were performed on JEM-2100F transmission electron microscope with an accelerating voltage of 200 kV equipped with a post-column Gatan imaging filter (GIF-Tri-dium). Scanning electron microscopy (SEM) images were taken using a Zeiss ultra 55 ultrahigh resolutions thermal FEG with an in-lens electron optic operating at 3 kV. The magnetization was measured using a Vibrating Sample Magnetometer  under a magnetic field of 10 kOe and a temperature of 24 \u00b0C.X-ray diffraction (XRD) patterns were recorded on a Bruker D8X-ray diffractometer with Ni-filtered Cu K\u03b1 radiation . Nitrogen sorption isotherms were measured at 77 K with a Micromeritics Tristar 3020 analyzer (USA). Prior to measurements, the samples were degassed in a vacuum at 180 \u00b0C for 6 h. The Brunauer\u2013Emmett\u2013Teller (BET) method was utilized to calculate the specific surface areas  with a shaking water bath to mix and maintain the temperature  and the light intensity in the centre of the BPA solution was 800 \u03bcW cm\u22122. The reaction suspension was prepared by adding appropriate amounts of catalyst and H2O2 into 30 mL BPA solution. The desired pH value was adjusted by HClO4 or NaOH and measured by a pH meter (Orion 3 Star). Prior to addition of H2O2, the mixture was mixed in dark for 30 min to reach the adsorption/desorption equilibrium between the catalyst and pollutants. Afterwards, 1.0 mL of the suspension was removed using a 2 mL syringe at given time intervals and filtered via a membrane with a pore size of \u223c0.45 mm. Furthermore, 10 \u03bcL 0.5 M n-butanol was added to the sample above to terminate the reaction and the BPA concentration in each sample was analysed on a high-performance liquid chromatography (Agilent 1260) with an Eclipse XDB C18 column  and a diode array UV detector . For the reusability test, the catalysts were collected by magnetic separation, washed with deionized water several times, dried in vacuum and used it for the next reaction under similar experimental conditions. Experiments were carried out 3 times and all results were expressed as a mean value. The total organic carbon (TOC) was determined with a laboratory TOC analyzer (SIEVERS 5310C). Leached iron ions were detected by an Inductively Coupled Plasma Atomic Emission Spectrometer .All experiments were conducted in cylindrical batch reactors , a further sol\u2013gel process was used to coat TiO2 shell on the silica layer using TIPO as the precursor. In step (3), after a hydrothermal etching method followed by calcination process, PLYS-Fe3O4@TiO2 nanosheets were formed.iii) salts with ethylene glycol in the presence of trisodium citrate. SEM images clearly reveal that the obtained Fe3O4 particles possess a uniform spherical shape with an average diameter of \u223c130 nm  pattern of PLYS-Fe3O4@TiO2  shows si2 sorption isothermal . The integrated energy dispersive X-ray spectroscopy (EDS) analysis of PLYS-Fe3O4@TiO2 , the typical sandwich sphere structure was converted to a pea-like yolk\u2013shell structure /H2O2, UV/H2O2/Fe3O4, system, respectively.As shown in C0 is the initial concentration, C is the concentration at time t, k is the apparent rate constant, respectively.Furthermore, the reaction kinetic constants were evaluated through fitting the experimental data with Langmuir\u2013Hinshelwood model to better compare the catalytic performance. And the degradation kinetic curves can be assumed as pseudo first-order kinetic 2O2/PLYS-Fe3O4@TiO2 is 24.2 \u00d7 10\u22123 min\u22121 higher than the summation of UV/PLYS-Fe3O4@TiO2 (3.4 \u00d7 10\u22123 min\u22121), UV/TiO2/H2O2 (11.5 \u00d7 10\u22123 min\u22121), UV/Fe3O4/H2O2 (2.9 \u00d7 10\u22123 min\u22121), UV/H2O2 (3.0 \u00d7 10\u22123 min\u22121) and H2O2/PLYS-Fe3O4@TiO2 (2.7 \u00d7 10\u22123 min\u22121).The kinetic constant value of UV/H3O4@TiO2 catalysts and the BPA oxidation capacity of H2O2 directly is weaker than hydroxyl radical. It also suggests that the synergetic effect between photocatalytic process and photo-Fenton process, which not only inhibits the recombination between electrons and holes, but also accelerates the reaction speed of Fe3+ to Fe2+ to increase the kinetic constant. This is similar to the value given in the The results indicate that the relatively poor adsorption efficiency of BPA on the PLYS-Fe3.2.22O2 dose on the degradation of BPA in the heterogeneous photocatalytic photo-Fenton process was investigated  was obtained with 1.5 g L\u22121, which is about 75.4% greater than the kinetic constant with 0.5 g L\u22121 (13.8 \u00d7 10\u22123 min\u22121). Furthermore, there is a decrease of kinetic constant which was 6.8 \u00d7 10\u22123 min\u22121 when the catalyst dose was 5.0 g L\u22121.The BPA concentration change with catalyst dose from 0.5 to 5.0 g L3O4 and TiO2, which cannot only be occupied by H2O2, but also enhance the light utilization to generate more hydroxyl radicals. The decrease of kinetic constant after 1.5 g L\u22121 might be due to three reasons, higher turbidity which can inhibit the further penetration of light into the reactor, the consuming of HO\u02d9 by excess Fe2+ and other radicals, such as (7)28 and catalyst agglomeration.29The increase of the degradation rate might be attributed to a number of active sites on the surface of both Fe3.2.4\u22123 min\u22121. As the pH was increased from pH 5 to pH 11, the reaction rate constants were decreased to 25.4 \u00d7 10\u22123, 14.5 \u00d7 10\u22123 and 9.9 \u00d7 10\u22123 min\u22121, respectively. This may be attributed to that pH affects TiO2 through the charge 42,43 and 13 Benefiting from the yolk\u2013shell and nanosheet structure, BPA molecules can easily permeate into the surface of Fe3O4 and degrade by hydroxyl radicals. Most importantly, the photo-induced electrons which generated from TiO2 can not only promote the recovery of Fe2+ from Fe3+, but also inhibit the recombination of electrons and holes.Based on all the information obtained above and previous studies by other researchers,3.443\u201345 In order to observe the stability, the catalyst was collected by magnetic separation after treatment, washed by deionized water and ethanol respectively, dried at 353 K and was evaluated by BPA degradation under the standard reaction conditions. As shown in \u22123, 21.1 \u00d7 10\u22123 and 20.3 \u00d7 10\u22123 min\u22121 for the first, second and third run, respectively. In addition, the Fe leaching . The BPA decomposition rate constant decreased with increasing pH. As a result of studying the rate of decomposition reaction according to the initial concentration of BPA, we found that there is an optimal BPA decomposition rate constant value at a certain concentration. Through the XRD analysis after the cycling experiment and before and after the reaction of the catalyst, the activity of the catalyst was still very stable, indicating that the catalyst had excellent stability and reusability. This study may provide useful information to further develop some effective heterogeneous photocatalytic photo-Fenton catalysts for degradation of organic pollutants.The rate constant of BPA degradation in PLYS-FeThere are no conflicts to declare.RA-009-C9RA04084F-s001"}
+{"text": "In response to the problems in the signal identification of radiation sources during the communication process, the bispectral quadratic feature model is applied to the identification algorithm for communication signals. According to the signal eigenvalues obtained from the bispectrum of the diagonal slices in the radiation source signals, the eigenvalues of the bispectrum diagonal slices can be extended from the frequency domain to the complex plane through the chirp-z operation in this paper, and the relevant data are obtained based on the bispectrum quadratic feature model of the signals by using the separation rules corresponding to the extended Babbitt distance. The bispectral quadratic feature model method is used to establish a sparse observation model, and the communication signal processing problem can be transformed into an estimation problem of signal motion parameters through the construction of a parametric database. At the same time, the high-resolution distance of communication signals is tested, and the communication signals are estimated by using the variational inference method. Finally, practical cases are analyzed, and the results indicate that the algorithm proposed in this paper can be used to identify different types of communication signals in accordance with simulated and measured data in the processing of communication signals in various environments, which has the certain anti-interference capacity to noise, can improve the identification rate of communication signals, and has verified the effectiveness and practicality of the algorithm proposed in this paper. The identification of communication signals is one of the key means of the communication industry at present, and it has been widely used in all walks of life and in the military field. As carriers of information data transmission, signals contain the features of the radiation source, which can be used to achieve accurate identification and detailed analysis of the communication signal, obtain the communication radiation source bispectrum features, and provide a basis for the subsequent communication signal radiation source effective identification , 2. At pThe bispectral quadratic feature model algorithm is applied to the issue of high-performance computing identification and analysis of communication signals in this paper by converting the estimation of motion parameters of communication signal identification into the evaluation problem of identification and analysis of reconstruction. This method focuses on the effective synthesis of low signal-to-noise ratio HR-RP based on the parameters of the communication signal identification motion signal in the search interval. In this way, it can quickly implement the estimation of the motion parameter impact under the premise of solving the HRRP reconstruction error under a low signal-to-noise ratio to identify the local optimal value according to the population update speed and finally accomplish the accurate processing of the communication signal motion parameters.N pulses, while M pulses (M\u2009<\u2009N) are extracted according to the sequence.In accordance with the setting of the carrier frequency sequence of the communication signal, it is possible to obtain the results of the bispectral quadratic feature model in accordance with the complete step FM signal within , 14. In fsm=fc+g(m)\u0394f, in which \u0394f stands for the pulse bandwidth of the signal and g stands for a subset in the interval [0:N\u2009\u2212\u20091], then the bandwidth of the bispectral quadratic feature model can be expressed as B=N\u0394f. Through the detailed analysis, it can be known that the corresponding correlation accumulation time is no greater than the whole communication signal step FM signal. Then the carrier frequency sequence of the communication signal can be set in accordance with the environmental information so that it possesses the enhanced anti-interference capability of itself.As the signal carrier frequency sequence of the bispectral quadratic feature model is set to k-th groups of sparse step frequency modulation (FM) signals, then the sparse step FM high-performance operation of the first group can be expressed as follows:u) stands for the corresponding rectangular window. If the value of tect(u) is 1 when |u| \u2264 1/2, then the value of tect(u) will become 0, in which \u03b3 indicates the modulation frequency of the communication signal and TP and TR stand for the pulse width of the communication signal and the number of pulse repetition periods in turn, respectively.It is assumed that the communication signal transmits a total of p scattering point can be expressed as \u03c3p, which constitutes the \u201cStop-Go\u201d model. The corresponding time delay value \u03c4p(t) of the scattering point p within the pulse of the communication signal can be left unchanged, and then there will be \u03c4p(t) \u2248 \u03c4p, tm,k=mTR+kNTR. At the same time, with regard to the scattering point of the communication signal, the m-th subpulse echo under the k-th group of sparse step FM high-performance operation can be expressed as follows:\u03c4p=2RP/c, RP stands for the instantaneous slope distance between the p-th scattering point and the communication signal emission signal, in which c is the speed of light and \u03b8m,k of the signal to be processed is reduced, RP=R+xpsin\u2009\u2009\u03b8m,k+yp, in which R stands for the instantaneous slope distance between the communication signal,  stands for the reference point by the instantaneous slope distance, and the scattering point p of the communication signal is denoted by the coordinate value in its plane. Thus, for a high-speed communication signal, the distance between the signal reference point and the communication signal at the time point tm,k can be obtained as R=\u03c4R+vRtm,k+1/2aRtm,k2, where rR stands for the distance between the communication signal and the communication signal at the initial time, vR stands for the radial velocity of the signal, and aR stands for the radial acceleration of the communication signal. The time delay of the communication signal can be expressed as \u03c4ref=2Rref/c, where vR and aR can be obtained in the tracking phase of the communication signal. Then, for the echoes to be solved by line frequency modulation, it can be assumed that R=xpsin\u2009\u2009\u03b8m,k+yp and \u03a6P and \u03a6B stand for the phase error due to interpulse translation of the communication signal and the phase error due to interpulse translation of the pulse string, respectively. Thus, \u03a6P and \u03a6B can be expressed as follows:vR stands for the residual velocity; aR stands for the residual acceleration; and 1 stands for the m quadratic phase term, which will generate the primary flap spreading in the phase of distance synthesis of the communication signal. \u03a62 stands for the tertiary phase term of m; the communication signal distance image is synthesized as an asymmetric paraflap; and then this term can usually be expressed in the order of 10\u22124. Hence, it can be neglected. \u03a63 contains the coupling terms of both m and k, which will lead to a distance image shift and cause the bending of the envelope. \u03a64 stands for the primary phase term of k, which causes an azimuthal shift in the image of the communication signal and can also be neglected. \u03a65 stands for the quadratic phase term of k. The azimuthal pulse pressure will cause the main flap spreading of the communication signal. For the purpose of constructing the reconstruction algorithm with sparse HRRP efficiently, the echoes can be converted into discrete form. If the number of pulse sampling points of each communication signal is set to Nr, which complies with k-th group of echoes can be expressed as sk=L1\u00d7T, in which sm,k=Nr1\u00d7, snr,m,k=exp(j4\u03c0/c(fsm+nr/Nr\u0394f))+\u03b5(nr).If the communication signal contains multiple scattering points, then the coefficient of the backward scattering of the n-th echo, L=N \u00b7 Nr, \u03b8k \u2208 \u2102L\u00d71 stands for the HRRP corresponding to the k-th echo, and n stands for the noise vector. Thus, dlk can be obtained as follows:fl stands for the l-th column of the database F, F=L\u00d7LT, The remaining transmission process parameters of the communication signal are introduced into the database, and then the sparse observation model of the communication signal can be expressed as follows:m-th subpulse of the signal, it can be known thatIn accordance with the As the signal reconstruction method based on the bispectral quadratic feature model can operate on the real matrix, it can be expressed as follows according to equation :(9)y=\u03a6\u03b8+\u03b5 is Gaussian white noise with 0 mean, then the probability of the signal echo y thus obtained is also Gaussian distributed, and the probability of \u03b5 distributed with y can be obtained as follows:\u03b1 stands for the noise accuracy. The Gamma-Gaussian prior is introduced to the sparse vector \u03b8, and the following can be obtained:\u03bb1,\u2026, \u03bbd) stands for the dimensional diagonal array, in which D\u2009=\u20092L. The accuracy parameters \u03bbd and \u03b1 comply with the Gamma distribution, and the following can be obtained:In accordance with expression , the proThe product of the distributions according to the probability model can be obtained as follows:p(y), and thus, the following can be obtained:On the other hand, the posterior distribution of the random variables can be expressed as the joint distribution of the variables divided by the marginal distribution It is highly difficult to calculate the posterior distribution based on the model directly. Thus, high-performance computing is carried out to resolve the approximate posterior distribution so as to enable the HRRP synthesis under the low signal-to-noise ratio (SNR) conditions.(1)in and at the same time the power received by the load after insertion is P\u2112, then the equation for insertion loss in dB can be obtained as follows:Insertion loss (IL): The loss of signal power due to the addition of a device in a transmission line or fiber is denoted by decibels (dB). When the power transmitted to the load before insertion is Pin| stands for the reflection coefficient, which can be expressed as follows in accordance with the connection between IL and \ud835\udcae21:where |\u0393\u2112, Pin, and \ud835\udcae21 in equation (The relationship between Pequation  is as fo(2)Bandwidth represents the selected range of the signal, that is, the width of the spectrum to be passed, the unit of the frequency range width, which is expressed in Hz, with the following equation:X is 3, 1, or 0.5, fH and fL stand for the frequencies on both sides of the passband when the reduction in the values of insertion loss on the left and right sides of the center frequency is X (dB).When the value of (3)In-band fluctuation refers to the size of the fluctuation in the response amplitude of the passband, that is, the difference between the maximum and minimum values of the response amplitude. In the design process of communication signals, the smaller the in-band fluctuation, the better the performance of the communication signals.(4)Return loss (RL) refers to the functional loss in the signal returned or reflected by the discontinuity in the transmission line. Discontinuity may not comply with the terminating load or the equipment inserted into the line, which is expressed in decibels (dB). The reflection system of the communication signal is also referred to as the reflection loss. The equation for return loss is as follows:When VSWR represents the voltage standing wave ratio, the magnitude of the return loss is related to the standing wave ratio (VSWR) and the reflection coefficient (\u0393). The increased return loss corresponds to a lower VSWR. Return loss is an index that measures how well the equipment matches the line. If the return loss is high, it means that the match is good. Generally, in the design of a communication signal, a high return loss and a low insertion loss mean that the performance of the communication signal is relatively good.(5)VSWR refers to the value of the impedance matching between the load and the transmission line or waveguide. A larger value of the VSWR indicates a higher degree of matching. The VSWR stands for the ratio of the amplitude of the partial standing wave at the wave web  to the amplitude of the node along the line .(6)Q stands for the ability of the communication signal to allow separation of the neighboring frequencies in the signal, and its equation is as follows:The quality factor fo represents the center frequency of the communication signal, BW stands for the 3\u2009dB bandwidth. A larger value of Q indicates that the device has higher working stability, stronger frequency selectivity, lower loss, but narrower band; on the contrary, a smaller value of Q value indicates that the device's operating stability is lower, the frequency selectivity is weaker, the loss is relatively large, but the frequency band is wider. When the value of Q is increased in the design, the higher the Q value is, the stronger the performance of the device is.When (7)Resonant frequency: With regard to the communication signals, there are multiple ways to resolve the resonant frequency. Thus, there are also various factors affecting the resonant frequency. Common factors affecting the resonant frequency include the structure of the communication signals, the shape and the resonant mode, and so on. In practice, four methods are often used to resolve the resonant frequency: the electro-nano method, the total set parameter method, the field solution method, and the phase method.In the research on the performance of communication signals, the parameters related to the communication signals are analyzed, with the main parameters as follows:The indicators mentioned above are used to measure the performance of a communication signal. In practice, it is not required to set the limit for all indicators, and some of the indicators can be optimized according to the actual demand.\u03b5 stands for the ripple coefficient, \u2131n(\u03a9) stands for the functional characteristic of the low-pass prototype, and \u03a9 stands for the frequency variable.The transfer function of a communication signal is a mathematical formula that expresses the frequency response features of the communication signal. With regard to the classical two-port communication signal, the transmission function equation is as follows:The features of communication signals are represented through the frequency response characteristics. With regard to the classification of communication signals as mentioned at the beginning of this paper, they can be divided into four types  according to the frequency response characteristics. The high pass, band pass, and band resistance can be obtained through the low-pass prototype of the frequency and components.In the communication process, radiation source noise is mainly generated by the transmitter noise. In general, it refers to the amplitude of the communication signal, frequency, and pulse width and repetition frequency together causing abnormal changes, that is, the stability of the communication process signal resulting in radiation source noise. Signal instability can be roughly divided into two categories: regular instability and random instability. Regular instability is mainly due to inadequate power filtering, mechanical jitter, and so on; random instability is the noise generated by the transmitter tube and modulation pulse random jitter generated.According to the amplitude frequency characteristics and phase frequency characteristics of the system, the cause of frequency-domain distortion can be analyzed.The top of the modulated pulse generates vibration; the top begins to fall with the power supply fluctuations of the transmitter due to the signal corresponding to the parasitic phase or the amplitude caused by the time-domain distortion phenomenon.The main control oscillator has insufficient frequency stability and phase stability. The different circuits or devices used for different communication transmitters result in different transmitter noises for various communications.At present, the gradual improvement of communication signal protocol will help to improve the stability of transmitter. Thus, with the large-scale use of the main vibration amplification type of transmitter, the noise generated can mainly be divided into the following three aspects:Hence, the uncoordinated modulation within the communication signal pulse is due to the transmitter noise. As the noise is generated due to various kinds of parasitic modulation. The communication signals present differences in signal features.The noise output from the communication transmitter indicates more non-Gaussian and non-linear features, and in the bispectral analysis, the signal amplitude and phase information are maintained. At the same time, the effect of Gaussian non-color noise on non-Gaussian signals bispectrum is completely suppressed, which can be used for the extraction of unconscious modulation features. Thus, the following concept of bispectrum is obtained accordingly.ckx is absolutely summable, that is, the following can be obtained:It is assumed that the high order cumulant k-th-order spectrum is defined as the (k\u2009\u2212\u20091)-order discrete Fourier transform of the k-th-order cumulants, that is, the following can be obtained:The Thus, the bispectrum, that is, the third-order spectrum, can be defined as follows:X(n)=s(n)+W(n), where w(n) represents the Gaussian white noise signal, s(n) includes the signal of non-Gaussian noise output from the transmitter, and w(n) and s (n) are independent of each other. If the cumulative quantity is solved three times for x(n), the following can be obtained:The bispectrum of the signal including phase noise can be obtained from the bispectrum estimation. The discrete noise signal obtained by Scout is represented by Equation  is expanAs long as the mean value of the signal and noise is zero, the following can be obtained:w(n) is a Gaussian noise signal, cw3 can be excluded from the calculation. Thus, it can be known that the communication letter can be used to eliminate the white noise after the third-order accumulation, then the bispectrum can be determined by cs3, that is, the following can be obtained:As In accordance with the above analysis, it can be observed that the evaluated bispectral features are mainly composed of the features of the signal itself and non-Gaussian noise. Hence, the bispectrum in the communication signal is assessed mainly based on the features specific to the signal itself, and it is also possible to obtain the features specific to various communications.The implementation of high-performance computing bispectrum is often a high-performance computation along each path. However, the secondary features obtained by this mode of computation are not consistent for the results to be recognized, and some of the bispectral points have relatively less effect on the results of the recognized targets and are subordinate to the ordinary bispectrum.If there is a cross-term in the initial observed signal, the high-order accumulation calculated by using the multi-correlation function will lead to the result that the cross-term becomes more complicated. As the cross-term is generated by a random distribution, it is impossible to eliminate the cross-term based on the determined calculation method.However, if the two-dimensional function can adopt the full-duplex spectrum as the signal feature to produce a two-dimensional template for matching, the number of operations can be excessively high, which will not comply with the high standard requirements for signal radiation source identification. The key to solving this issue lies in the introduction of high-performance computing bispectrum; in equation , the twoFor the purpose of extracting the secondary features of the bispectrum as the features of the bispectrum and eliminating or decreasing the defects that occur in the process of high-performance computation of the bispectrum, the characteristic parameters that are available for communication signals can be obtained with optimal separability in the secondary features of the bispectrum. As a result, it can effectively solve many problems such as cross-terms caused by ordinary bispectral points and high-performance computing.In order to make the communication signal meet the actual performance characteristics, it is necessary to design an appropriate chamfer size in the design process. When the ring size becomes larger, the upper and lower passband edges move along the transmission zero point, and the attenuation pole becomes smaller. The intermediate frequency of the communication signal tends to be a higher frequency, which changes the frequency characteristics of the communication signal. With the shortening of the distance between patch resonances and the smaller the passband bandwidth, the attenuation point will first become smaller and then become larger. Therefore, HFSS software is used to optimize the structural parameters of the communication signal, and finally, the structural dimensions of the communication signal are obtained, as shown in By selecting a vector analysis instrument to test the communication signal, X stands for the observed data and w stands for the set of random variables; then the expression of the approximate posterior distribution can be obtained as follows:\u03b8, \u03b1, and \u039b are solved for the sparse reconstruction by using the high-performance computing. In accordance with the mean field assumption, equation  \u00b7 [(y \u2212 \u03a6\u03b8)T(y \u2212 \u03a6\u03b8)], in which y. At this point, the expectation of \u03b1 can be obtained as follows:Update the variable equation , as showd-th element \u03bbd is the Gamma distribution, as shown in the following equation:ed\u2032=v1+1/2 and fd\u2032=v2+1/2Eq(\u03b8)[\u03b8d2]. Thus, the expectation of \u03bbd can be obtained as follows:Update the variable \u039b, and the approximate posterior distribution of the \u03b8, and its approximate posterior distribution is the Gaussian distribution, as shown in the following equation:Eq(\u039b)[\u039b]+Eq(\u03b1)[\u03b1]\u03a6T\u03a6)\u22121 and \u03bc\u2032=Eq(\u03b1)[\u03b1]\u03a3\u2032\u03a6Ty. At this time, the following can be obtained:Update the variable \u03b71.Repeat Steps 1 can lead to distance image spreading. Hence, it is required that the change in the coherent accumulation time \u03a61 of the burst should be less than \u03c0/2. Thus, the following can be obtained:In the following section, the accuracy requirement for equation . With re3, it is required that the envelope shift induced by the residual velocity and the residual acceleration during the processing observation time should not exceed the specified range. Thus, the following can be obtained:In practice, equation  can usuaIt should be noted that the linear phase of For the purpose of obtaining excellent azimuthal focus, it is required that the peak reduction of the azimuthal image caused by the residual acceleration during the processing observation time should be no more than 3\u2009dB; then the following can be obtained:Hence, azimuthal focusing requires higher accuracy of the acceleration estimation. Finally, for the purpose of obtaining a well-focused image, the signal residual velocity and the residual acceleration should comply with equations  and 37)37). The 2, respectively. The bispectral quadratic feature model contains a total of 128 bursts, and each burst contains 64 randomly selected pulses from 80 consecutive full-band pulses (waveform 1). Through the addition of the complex Gaussian white noise to the echo, the echo signal-to-noise ratio can be increased from 0\u2009dB to 15\u2009dB in 5\u2009dB steps. For each signal-to-noise ratio, 25 independent trials with different noise states are carried out with the number of genetic algorithm populations set to 40 and the number of genetic terminations set to 20. The algorithm proposed in this paper is compared with the PSO algorithm based on the parametric database . In the comparison, the distance image entropy is weighted with the average distance image entropy as a signal function in accordance with algorithm 1.In this paper, the effectiveness of the proposed parameter estimation algorithm in the transmission process and the high resolution processing algorithm is verified based on the simulation data. The echoes of the satellite scattering point model \u22122\u2009m/s anAs the estimation error of algorithm 1 at the signal-to-noise ratio of 0\u2009dB is excessively large that the focusing processing cannot be implemented, for the purpose of a fair comparison, the database is established here in accordance with the motion parameter estimates obtained based on the proposed algorithm; then the OMP, GD, and high-performance calculations are carried out to resolve equation ; and theFor the purpose of fully verifying the proposed algorithm put forward in this paper, the stability of the three algorithms is further compared. The signal identification effect in the presence of external interference is analyzed accordingly. In the process of high-performance computing identification of communication signals, it is impossible to ensure that the reception length and quality of the target signals and the computing under a certain number of conditions can present the stability of features, which is taken as an essential evaluation criterion for the practicality of the algorithm. Excellent methods for feature value extraction should not be constrained by the number of training, and such methods should have good robust performance when they are applied. After taking this point into full consideration, the identification accuracy of the training samples by using the target experimental signals is studied accordingly. The number of signal samples in each segment contains 500 points. For each class, the number of signal test samples is set to 100, and the number of training samples is adjusted to 50, 100, 150, and 200, respectively. The experiment is carried out 30 times. The mean value and variance of the identification rates obtained based on the three methods are recorded in turn, which are shown in Nr is increased from 50 to 200, the identification rate of the distance selected bispectrum of the BAS is improved by about 4%, while the ISIB and the method proposed in this paper show the improvement of about 3% and 2%, respectively. The results of the experiment indicate that the algorithm put forward in this paper has excellent stability, the identification results are less affected by the number of training samples, and the model thus established has good robustness, which suggests that the proposed algorithm has relatively strong practicality.In the empirical testing, different modulation methods are used to increase the number of training samples as shown in On the basis of the research on the fine features in the bispectral analysis of individual identification of radiation sources, the bispectral secondary features extracted are optimized by using the extended Babbitt distance criterion in accordance with the in-depth analysis of the bispectral features of communication signals diagonally sliced. This method can be used to implement the bounded deviation and identify the observation error values quickly and perform the complexity calculation of variational parameters effectively. The experiments indicate that the method is effective. The information on the original signals can also be recovered in a low signal noise background. The statistical features of communication scattering points and noise are used. Through practical analysis, the experimental results indicate that the method proposed in this paper has reduced the usage time. In addition, it can be applied to multiple types of communication signals. The bispectral features thus obtained always have excellent robustness at a low signal-to-noise ratio (SNR). When the signal-to-noise ratio is 0, the identification rate of high-performance computing of communication signals can achieve more than 90%. However, in general, a series of individual identification methods based on bispectral analysis have the common issue of a relatively low identification rate. Taking into account the subtle features in the other aspects of the signals, a feature vector is formed, which can further improve the practical effectiveness of the algorithm proposed in this paper."}
+{"text": "Laparoscopic cholecystectomy is the first line of treatment for patients with symptomatic gallstones. Surgical clips made of stainless steel are still used by surgeons during these laparoscopic procedures and are considered relatively safe. These surgical clips, however, have been linked to severe side effects, including weight loss, dermatitis, angioedema, sleep hyperhidrosis, and more. This report documents the case of a 71-year-old female who presented for the removal of surgical stainless steel clips post-cholecystectomy after 16 years of chronic manifestations, including rashes on the torso and breasts, hives, weight loss, chills, and neuropathy due to a previously undiagnosed nickel allergy. Laparoscopic cholecystectomy is one of the most performed procedures in the United States, partly because gallstone disease remains a prevalent condition. Over 20-25 million Americans are affected by gallstone disease yearly ,2. LaparMore recently, various cases have been reported documenting the occurrence of allergies to nickel, titanium, cobalt, and chromium caused by surgical clips following laparoscopic cholecystectomy -12. SurgBecause of its intrinsic and practical characteristics such as resistance to high temperatures and deterioration, good tensile strength, and low manufacturing cost, nickel has been commonly used in surgical tools such as clips, forceps, clamps, scissors, holders, and implants . HoweverThis case involves a 71-year-old female who presented with nonspecific symptoms involving multiple body systems due to nickel allergy for several years. The patient\u2019s past medical history is significant for gastritis, primary hyperthyroidism, marginal zone lymphoma, cataracts of both eyes, vertigo, bilateral leg weakness, hip pain, neuropathy, and osteopenia. Her past surgical history includes laparoscopic cholecystectomy in 2006, total hip arthroplasty in 2016, and parathyroidectomy in 2019. The patient is allergic to latex, gold, copper, manganese, and nickel, a fact that remained unknown until she underwent testing in 2021 using memory lymphocyte immunostimulation assay (MELISA). She is currently on albuterol 90 mcg, two puffs every six hours, Allegra one tablet daily, vitamin D3 1,000-unit one tablet daily, diazepam 5 mg half tablet as needed, and gabapentin 300 mg three times a day.In June of 2021, an autoimmune\u00a0workup came negative for Sjogren\u2019s and systemic lupus erythematosus, inflammatory arthropathy, vasculitis, autoinflammatory syndrome, and sarcoidosis. In July of 2021, initial metal patch testing did not show sensitivity to common metals\u00a0but did show sensitivity to chlorhexidine. She then underwent a lymphocyte transformation test, which showed sensitivity to nickel, and\u00a0it was confirmed afterward\u00a0that she is allergic to nickel by MELISA. Around that time, she began a low-nickel diet, improving her general symptoms. In August of 2021, repeat allergy testing came positive for palladium chloride, vanadium, and trichloride. In addition, a neurodiagnostic skin biopsy was performed to\u00a0rule out small fiber neuropathy, which was negative. The myositis panel also came back\u00a0negative.The patient had ongoing widespread symptoms, mainly fatigue, neuropathy, and a widespread rash, significantly impacting her function. She had improvement with symptoms on a nickel-free diet and Allegra after she learned that the clips used in her gallbladder surgery were made of stainless steel and contained nickel. Considering the high possibility that the nickel surgical clips placed during her cholecystectomy might be the cause of her symptoms, the patient decided to pursue the removal of the surgical clips after discussing it with her primary physician.After consent was obtained, the patient was taken to the operative suite, where she was prepped and draped after satisfactory induction of general anesthesia. The abdomen was explored, and then, epigastric, mid-epigastric, and right upper quadrant ports were placed percutaneously under direct vision. Fluoroscopy showed four clips in the gallbladder fossa Figure . The galThe patient tolerated the procedure well, and there were no postoperative complications. She recovered well, and three months post-surgery, the patient reports improvement in all symptoms she experienced: neuropathy has improved, she is not taking gabapentin anymore, there were no more rashes,\u00a0her fatigue significantly improved, and her weight is stable with no recent weight loss.In this report, we presented the case of a patient who underwent a laparoscopic cholecystectomy at 55 years of age and two years later developed nonspecific symptoms. Symptom severity worsened until, at 71 years of age, the possibility of delayed hypersensitivity to the metal used in the surgical clips was considered. She underwent allergy testing, which confirmed the suspicion, 16 years after her cholecystectomy. Our study adds to a growing body of literature demonstrating the occurrence of metal-induced delayed hypersensitivity as a cause of systemic symptoms, even a decade or more after laparoscopic cholecystectomy ,11,16 orIn a previous case reported by our group, a patient presented with delayed hypersensitivity type IV symptoms that included abdominal pain, fatigue, lethargy, arthralgia, and nausea after cholecystectomy . In the T cells mediate type IV hypersensitivities by triggering inflammatory reactions in response to the presence of antigens, either exogenous or endogenous. An initial local immune and inflammatory response attracts leukocytes. Macrophages and monocytes engulf these antigens and present them to T cells. The latter become sensitized and activated, releasing cytokines and chemokines, which may cause tissue damage and illnesses .Allergic reactions to antigens such as nickel, the metal present in the surgical clips removed from the patient presented in this report, are common in the general population. The prevalence of nickel allergy is approximately 17% in females and 3% in males . CurrentPost-surgery, symptoms resolved quite dramatically and led to a discontinuation of pain medication and the resolution of rashes that had been present for many years. The fact that the patient was placed on both a gluten-free diet and a nickel-free diet could be a confounding variable, but the dietary changes began months before the clip removal was performed, while the improvement of the symptoms was much more closely related to the removal of the nickel-containing surgical clips.This case report reiterates that delayed hypersensitivity reactions to metals should be part of the differential diagnosis in postsurgical complications even decades after a surgical procedure. This may be especially relevant in the elderly, where the allergic disease may be masked by age-related physiological function decline .\u00a0FurtherA possible metal allergy could be considered when a patient presents with nonspecific, possibly life-threatening complications, as seen with the patient in this case report. Potential allergens should be investigated before procedures involving surgical clips by obtaining detailed history with an emphasis on any allergy history and performing metal sensitivities and\u00a0patch tests when necessary. In addition, hypoallergenic clips or alternatives to metal clips, such as plastic clips or suture ligation of cystic duct and artery, should be considered\u00a0in patients undergoing gallbladder surgery\u00a0with known reactivity to various allergens."}
+{"text": "Background: The SARS-CoV-2 pandemic and climate change are two simultaneously occurring large scale environmental health crises. This provides an opportunity to compare the risk perception of both crises in the population. In particular, whether experiencing the acute pandemic sensitizes people to the risks of ongoing climate change. Methods: Panel participants answered a web-based questionnaire. The risk perception of SARS-CoV-2 and influencing factors were assessed. Differences of risk perception dimensions regarding SARS-CoV-2 and climate change were analyzed as well as associations between dimensions. Results: The results show that an economic impact by the pandemic is associated with more dimensions of SARS-CoV-2 risk perception than an experienced health impact. Moreover, dimensions of risk perception of the pandemic and climate change are perceived differently. Furthermore, the affective dimension of pandemic risk perception is significantly associated with all dimensions of climate change risk perception. Conclusions: Emotional-based coping with the risks of SARS-CoV-2 is associated with risk perception of climate change as well as various factors that shape the individuals\u2019 risk perception. It is currently necessary and will be increasingly necessary in the future to solve coexisting crises, not selectively, but in a common context within the framework of a social-ecological and economic transformation. Becausemeasures . Studiesmeasures . It has Currently, SARS-CoV-2 and climate change are globally simultaneous environmental risks that threaten the health of humankind, but endemics and pandemics will be more likely in the future, driven by multiple environmental factors, such as changing climate and ecosystems . MoreoveBoth the SARS-CoV-2 pandemic and climate change are very complex in terms of their origins and consequences. Therefore, solutions to problems from past emergency events are hardly transferable . AlthougSuccessful coping with environmental health crises such as the global SARS-CoV-2 pandemic and climate change can only be realized by protection and prevention measures supported by society as a whole. Therefore, it is essential that these (from a scientific perspective) predictable crises are recognized by the population as a health risk as well as an existential threat . There aHence, personal risk perception may be an important precursor to health-related and other behaviors that determines how individuals respond to potential hazards . For exaThe current study aimed to analyze on a population level (1) whether personal health or economic impacts increase the risk perception of the SARS-CoV-2 pandemic. In addition, we analyzed (2) whether risk perception dimensions of the pandemic and climate change are perceived differently and (3) if dimensions of risk perception of the SARS-CoV-2 pandemic are associated with dimensions of risk perception of the simultaneous crisis of climate change.Respondi . The panel operator sent invitation links to the participants and paid them a small financial incentive after completing the survey. The quota sampling was based on socioeconomic variables  according to the distribution in NRW [n = 1000 was planned and participants were recruited until all quota for the sample were fulfilled. The questionnaire was created by the authors of this study within the online survey tool LimeSurvey . A questionnaire with closed items was developed in German and assessed questions such as sociodemographic characteristics, health and economic impacts of SARS-CoV-2, and risk perceptions of SARS-CoV-2 and of climate change. The questionnaire was pre-tested with n = 100 participants. Data were analyzed anonymously. The study was approved by the Ethics Committee of Bielefeld University (No. 2020-205).This cross-sectional study was carried out in February 2021, marking the second lockdown in Germany to manage the spread of SARS-CoV-2. Participants were recruited in North-Rhine Westphalia (NRW) in cooperation with the Institute for Sociology and Communication (SOKO Institute) via an ISO-certified Access Panel of the company n in NRW . A sampln = 1160 participants. After excluding participants due to not fitting the target population , incomplete questionnaires (n = 49), and response time less than 6 min (2 s/item), which is considered insufficient effort responding [n = 25) the study sample comprised n = 1049. Additionally, for the analysis of risk perception, a test of consistency was conducted. Participants who disregarded reverse-coded questions on the risk perception of the SARS-CoV-2 pandemic or climate change, and thus provided inconsistent responses, were not included in the analyses of risk perception. As a result, the sample size was reduced to n = 918 (The raw data sample comprised sponding  (n = 25) n = 918 . This sano risk factor and one or more risk factor(s). Answers regarding risk factors were tested for plausibility.The first part of the questionnaire included questions about the socioeconomic status of the participants . Moreover, the presence of pre-defined risk factors  for the affected and not affected. Furthermore, participants were asked whether they were affected economically by the SARS-CoV-2 pandemic or worried about economic consequences. Questions were adapted from other surveys regarding the same topic [low economic impact and values above the mean indicated high economic impact.Health impact was measured as direct or indirect impact. A person was considered directly affected if he or she contracted SARS-CoV-2 before the survey. Additionally, the progression of COVID-19 was asked  admission). If participants stated they had persons close to them who have been infected with SARS-CoV-2, they were considered as having an indirect health impact. Additionally, they were asked how the disease proceeded. As the question was created with multiple choices, a plausibility analysis was conducted. For the analyses, direct and indirect health impacts were dichotomized into me topic ,29. The affect, probability, and consequences. Affect refers to the emotional handling of the risk (five items), probability to the assessment of how probable the risk affects oneself or the community (three items), and consequences to the perceived severity of a risk or hazard (two items). The validation conducted by Wilson et al. [Risk perception of the SARS-CoV-2 pandemic and climate change was measured according to the risk perception scale developed by Wilson et al. . The muln et al.  includedAll items were answered via 5-point-Likert-scales . Values for each dimension  were summarized by calculating means of the individual items. Higher values represent higher risk perception. The internal consistency  of the three scales for the SARS-CoV-2 pandemic  and climate change , respectively, was found to be good. One item of the probability dimension asking \u201cHow often do infections with SARS-CoV-2 occur at your place of residence?\u201d was excluded. Due to the high dynamics with steadily increasing infection numbers during the second lockdown in Germany, the question was not purposeful to depict a differentiated assessment of the (subjective) estimation of infection spread.To assess associations between health impact or economic impact and SARS-CoV-2 risk perception dimensions, individual linear regression models were conducted for each dimension . Differences in the dimensions of risk perceptions of SARS-CoV-2 and climate change were tested via paired t-tests.affect, probability, consequences) and all three dimensions of risk perception of SARS-CoV-2 as the explanatory variable. The models also controlled for the possible confounding variables of age (continuous), sex (dichotomized), education , and health risk factor for severe COVID-19 progression (dichotomized). Data cleaning and analysis were conducted with IBM SPSS Version 28 and the R Statistical Environment [p-value of up to 0.05.The association between risk perception of the SARS-CoV-2 pandemic and climate change was tested via three individual linear regression models. Each regression model had one dimension of risk perception of climate change as the outcome variable  is representative for North-Rhine Westphalia, a German state in Western Germany, due to the quota recruiting method  ages ranged from 16 to 85 years . Females with an average age of 44.2 years (SD = 17.9) were slightly younger than males with an average age of 52.7 years (SD = 15.6). Net household income was almost equally distributed between the categories, except for the income group \u2018less than 1000 \u20ac\u2019 (EUR 1000), which only accounts for 7.2% of the sample corresponding to the NRW quota of 7% (The composition of the study sample (g method . Particita of 7% .n = 560) stated having at least one risk factor for severe progression of COVID-19. One third of the participants  stated having pre-existing condition(s)  and a quarter stated being a smoker . The risk factors obesity and suppressed immune system were less common  in the sample. More than half of participants  stated that they had not been infected with SARS-CoV-2. The participants reporting an infection  had mostly no symptoms  or light symptoms . Only a few participants reported hospital  or ICU admission , while 11.2% (n = 10) reported severe symptoms without hospital admission.At the time of the survey (February 2021) most of the participants . Of those reporting an infection of close persons, 12.2% (n = 127) passed their infection without symptoms, and 17.8% (n = 185) with light symptoms. Less common were severe symptoms , hospital , or ICU admissions . Several participants  reported the death of a close person due to the COVID-19 disease. After dichotomization, the ratio of health impact resulted in 63.1% not affected and 36.9% affected participants in the study population.An indirect health impact , was reported by 37.7% of participants (n = 857) answered that there was no change. However, some participants reported that they were either temporary part-time , lost their jobs , were freelancer/self-employed and could not do their occupation as usual , or had a contractual reduction in working hours .The economic impact of the SARS-CoV-2 pandemic was evaluated, including whether changes in the occupation status occurred. The majority of participants  to moderately  concerned. Only 18.2% (n = 190) were not concerned at all, while 17.3% (n = 181) were rather concerned and 12.8% (n = 134) were very concerned.Participants answered regarding their concerns of personal economic impact heterogeneously. More than half reported being little  had no financial losses. Yet about 28% had very little  to little  financial losses. Large  to very large  financial losses after almost one year of the SARS-CoV-2 pandemic were less common. After dichotomization (low and high economic impact), the ratio in the study population was 51% low and 48% of a high impact.Most of the participants . Thus, participants who reported a health impact because of the pandemic, also perceived an infection with SARS-CoV-2 as a higher risk , significant differences between both regarded risks are noticeable  (p < 0.001).Regarding the mean values of the risk perception of the pandemic and climate change in the dimensions ticeable . In both < 0.05) . The peraffect dimension of SARS-CoV-2 with the corresponding outcome dimensions affect, probability, consequences of climate change risk perception. Moreover, an increase in the dimensions of probability and consequences of a SARS-CoV-2 infection is associated with an increase in the probability and consequence dimension of climate change risk perception, respectively  by the pandemic may influence the risk perception of SARS-CoV-2. The study reveals that economic impacts are associated with more dimensions of risk perception of the SARS-CoV-2 pandemic than health impacts. Secondly, it has been assessed if dimensions of risk perception of the pandemic are associated with dimensions of risk perception of the current climate crisis. We could show that especially the affective dimension of risk perception of the SARS-CoV-2 pandemic is associated with all dimensions of risk perception of the currently occurring climate change. The unique situation of the parallel occurrence of two environmental health crises offered also the opportunity to analyze whether the respective risks are perceived differently. Regarding affect and consequences dimensions, SARS-CoV-2 risk perception is higher than climate change, whereas the probability dimension is rated higher for climate change.The SARS-CoV-2 pandemic had a massive impact on the lives of many people in Germany, especially with regard to being affected by health or economic consequences due to the infection events and protection measures. The present study shows that the personal experience of health consequences due to the pandemic, i.e., their own infection or of people in their close social environment, contributes to the rating of the probability of becoming infected as higher. In contrast, no association was found between a personal health impact and how severe the consequences due to a SARS-CoV-2 infection are estimated to be. According to another study, the \u201cexperience with the virus\u201d, i.e., having tested positive for SARS-CoV-2, was identified not to be the primary but the third most important predictor for risk perception in various countries, after individualistic views and pro-sociality .probability may be related to the time point of measurement. Hetkamp et al. [The higher risk perception of SARS-CoV-2 in the dimension of p et al.  found inp et al. . Therefore, when estimating risk perception, the time point of measurement or the current context of threat is important for a proper interpretation, respectively. There are indications that the temporal nature of the consequences certainly impacts how people place weight on different risks . In the affect. Moreover, in accordance with health-affected people, economically affected participants also estimated the probability for getting infected or having contact with an infected person as higher than people with low economic impact. Overall, the results indicate that economic worries due the pandemic influence people more than health worries. This assumption is supported by the findings of a worldwide study that considered what kind of concerns due to SARS-CoV-2 are higher and predict self-protective behavior better [With regard to personal economic impacts due to the pandemic, we could reveal that a higher economic impact  was significantly associated with an emotional involvement concerning worries and fears related to the pandemic, operationalized by the risk perception dimension r better . Consistr better . The impr better . Therefor better  reportedAlthough it has also been stated that emotions promote risk perception often more effectively than knowledge and facts ,8, it isThus, to successfully manage an environmental health crisis, risk communication strategies and crisis management must consider the potential consequences  and perceptions of risk, as well as the necessary preventive behaviors.probability with regard to getting infected was lower than the estimated probability of being affected by climate change. It is conceivable that contact with an infected person is more likely to be considered avoidable to lower the probability to infect yourself, while the probability to be affected by climate change is less controllable and cannot be prevented by only one\u2019s own decision or behavior [Survey participants rated the emotional risk dimension and consequences of the pandemic higher than those of climate change, possibly because an immediate threat was perceived to be more present. Instead, the dimension behavior . affect, probability, and consequences of both crises are associated. In particular, the emotional risk perception of the SARS-CoV-2 pandemic (affect dimension) showed statistically significant associations with all dimensions of risk perception regarding climate change.Moreover, the analysis of the risk perception of the two parallel occurring health-related crises, the SARS-CoV-2 pandemic and climate change, showed that the risk perception dimensions This result indicates that being emotionally affected by a crisis such as the SARS-CoV-2 pandemic might have an impact on the risk perception of another crisis, such as climate change. In particular, a negative frame of information captures the attention of people and supports negative emotions that may sensitize people to otherwise neglected risks for themselves or others . Similarprobability and consequences of the pandemic risk perception are associated with the same dimensions of climate change. It has to be analyzed in further studies if this awareness also promotes preventive behavior.Risk perception of a current crisis is, in addition, an important determinant of planned and actual self-protective behavior . The thrAlso in line with the study results regarding the influence of being affected by the SARS-CoV-2 pandemic is a study by Kecinski et al. , which fThere is further evidence that the co-occurrence of another risk, or a higher risk perception of another hazard, leads to threats such as climate change being taken more seriously as a global environmental health risk , but, thFuture studies should examine more closely whether being affected by a crisis such as the pandemic will increase the awareness of the simultaneous climate change. In particular, whether being affected might promote a positive spillover effect, in the sense that experiencing one event has a positive impact, i.e., on attitudes concerning another event that is independent of it ,43. ThatThe present study supports the assumption that a multidimensional measurement of risk perception might be a suitable tool to map the facets of risk perception in a more differentiated way, because the assessment of risks is individual and influenced by many factors. Therefore, the development of appropriate risk communication strategies continues to be a challenge. With regard to coping with environmental health crises, this and other studies have shown that emotions, especially as a result of personal concern, can significantly increase risk perception ,38,44. FThe results of the present research are limited in some respects. Inherent to cross-sectional study designs, the possibility to draw causal inference is restricted. Moreover, the results rely on self-report measures, which could have caused the results to be contaminated by common method variance. While this is the usual practice for most of the examined constructs, concerns about the validity of self-reports cannot be excluded . The preWherever available, we used validated measures. However, due to the novelty nature of the pandemic, it was not possible to use validated instruments at all points; therefore, a precise measurement of certain study constructs might be limited. Accordingly, future research should aim to verify the results of this study, as well as validate, and incorporate further measurements. probability (\u201cHow often do infections of SARS-CoV-2 occur where you live?\u201d) can only be answered with minor differentiation, and thus only allow an imperfect assessment of the risk perception.In the study, the instrument developed by Wilson et al.  was usedConsidering this, follow-up studies are recommended that incorporate further informants and other methods of data collection , in-depth qualitative methods) to obtain a more comprehensive picture. In particular, longitudinal studies should be carried out to further substantiate the evidence for the large effects found. Additionally, different approaches of sample recruitment to ensure a broader representation of the population might be considered in future research. Emotional-based coping  with the risks of SARS-CoV-2 is associated with risk perception of climate change as well as various factors  that shape the individuals\u2019 risk perception. Hence, successful climate protection is an economic as well as an ecological and social challenge. It is currently necessary, and will be increasingly necessary in the future, to solve coexisting crises, as well as pandemics, not selectively, but in a common context within the framework of a social-ecological and economic transformation. This requires a better understanding not only of what people know about crises such as climate change and the SARS-CoV-2 pandemic, but also of the experiential, social, and cultural factors that shape risk perceptions and their potential role in motivating preventive behavior. Better insight will help policymakers and other stakeholders develop evidence-based risk communication strategies tailored to societal subgroups."}
+{"text": "Ehrlichia, and Rocky Mountain spotted fever (RMSF) were negative. Parvovirus DNA and chikungunya IgG, antinuclear antibody (ANA), and antineutrophil cytoplasmic antibody (ANCA) screens were negative. IgG for mycoplasma, dengue, and herpes simplex virus 1 (HSV1) were positive. During all this time, the patient did not show clinical improvement in spite of being on antibiotics. In fact, her oxygen saturation dropped, and she required oxygen through the nasal cannula. A lung tissue biopsy taken on bronchoscopy showed chronic inflammation and organizing pneumonia. To note, mycoplasma DNA PCR and HSV culture from bronchoalveolar lavage (BAL) were negative.Since its emergence in December 2019, coronavirus disease 2019 (COVID-19) has been detrimental worldwide. Although COVID-19 infection is primarily known for its respiratory manifestations, extrapulmonary features are increasingly being reported. Among these, cutaneous manifestations are apparent but have a high likelihood of not being attributed to COVID-19. We present the case of\u00a0a 63-year-old female unvaccinated against COVID-19. She presented with fever, cough, shortness of breath, and rash. The symptoms were present for four days and appeared after contact with a confirmed symptomatic COVID-19-positive family member. The rash started first on the chest and then spread to the face and whole body including the palms and soles. It was erythematous and maculopapular and is associated with ulceration and swelling of the lips. In places, it was confluent and had a target-like appearance. On admission, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR) was negative. As she was septic with initial suspicion of tick-borne infections, she was started on doxycycline. Given her symptoms on presentation, the suspicion of COVID-19 was very high, and the SARS-CoV-2 nasal swab PCR was repeated, which was negative yet again. With the index of suspicion being very high, her presentation was speculated to be atypical, especially in the setting of a target-like rash involving the palms and soles. The antibody was checked. IgG antibodies for SARS-CoV-2 were positive. All other\u00a0antibodies for mycoplasma, Lyme disease, The patient was started on intravenous steroids. A confirmatory skin biopsy was done, and it showed perivascular, interstitial, and spongiotic dermatitis related to a viral infection.\u00a0While on steroids, the patient improved dramatically.\u00a0Her skin rash also improved, and she was discharged. On outpatient follow-up, she was doing exceptionally well with ambulatory oxygen saturation of 100%.This patient who was COVID-19 PCR-negative twice could have been easily deemed as not having COVID-19. However, the fact that she was unvaccinated, had positive sick contact with imaging concern\u00a0for COVID-19 pneumonia, and COVID-19 antibody being positive\u00a0and no other test being positive\u00a0clearly attributes her manifestations to the virus. The presence of a rash could easily be misleading. Awareness of the fact that a rash like erythema multiforme (EM) could be a sign of underlying COVID-19 is extremely prudent and is an addition to the ever-expanding knowledge of this virus. Since the time it first emerged in December 2019, coronavirus disease 2019 (COVID-19) has been detrimental worldwide. With constant undergoing research, it still is an ocean to be explored in-depth. Although COVID-19 infection is primarily known for its respiratory manifestations, extrapulmonary features including cutaneous manifestations are increasingly being reported [A 63-year-old female unvaccinated against COVID-19 was admitted with complaints of fatigue, cough, mild shortness of breath, fever, and a rash. The symptoms started after the patient had contact with her daughter who was a confirmed symptomatic COVID-19 patient. As per the patient, the rash started four days prior to her presentation to the hospital. It started first on the chest and then spread to the face and involved the whole body including the palms and soles. On examination, the rash was erythematous and maculopapular, diffused all over the body, and was associated with ulceration and swelling of the lips , which were negative, as well as antinuclear antibody (ANA) and antineutrophil cytoplasmic antibody (ANCA) screens. Parvovirus DNA and chikungunya IgG were also negative, while IgG for mycoplasma, dengue, and HSV1 were positive. The working diagnosis at this time was COVID-19 pneumonia versus mycoplasma. The patient was given levofloxacin and vancomycin, which were changed to linezolid as there were no signs of improvement in the patient\u2019s status. Despite being on multiple antibiotics, the patient did not show any improvement;\u00a0in fact, her oxygen saturation dropped to the point that she required oxygen supplementation of 2-3 L with a nasal cannula. At this time, a bronchoscopy was done, and a sample for\u00a0bronchoalveolar lavage (BAL)\u00a0was taken. Mycoplasma DNA PCR and HSV culture from BAL was negative. A lung tissue biopsy showed chronic inflammation and organizing pneumonia.In places, it was confluent and had a target-like appearance consistent with a rash similar to erythema multiforme (EM). At the time\u00a0she was admitted, she was found to be tachycardic with a heart rate of 121 bpm\u00a0but not tachypneic and saturating above 95% on room air. Basic bloodwork showed leukocytosis and lactic acidosis. As the patient was in sepsis, she was started on IV fluids and antibiotics, ceftriaxone, and also doxycycline\u00a0to cover for possible tick-borne infections. The rest of the admission laboratory results showed elevated inflammatory markers including erythrocyte sedimentation rate, C-reactive protein, and D-dimer . Hence, a computed tomography pulmonary angiogram (CTPA) was done that did not show pulmonary embolism. However, it showed bilateral peripheral opacities consistent with multifocal pneumonia with concern for COVID-19 pneumonia. Ironically, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nasal swab polymerase chain reaction (PCR) done on admission was negative. As the patient had a history of recent close COVID-19 contact and CTPA concerning COVID-19 pneumonia, the suspicion of COVID-19 was high. A repeat SARS-CoV-2 testing was done, and it was negative again. It should\u00a0also be noted here that the patient showed no clinical improvement with the antibiotics. Due to consistently high suspicion of COVID-19 organizing pneumonia, the antibody was checked. IgG antibodies for SARS-CoV-2 were positive. The other antibodies checked were mycoplasma, Lyme disease, Hence, the patient was started on intravenous steroids. While on steroids, the patient showed dramatic improvement. The requirement for oxygen supplementation decreased, and eventually, the patient was on room air. Clinically, she felt better. The EM-like rash\u00a0improved on initiation of steroids and resolved completely before the patient was discharged\u00a0Figure .A skin biopsy was done from one of the sites of rash, and it showed perivascular, interstitial, and spongiotic dermatitis related to a viral infection. After discharge, the patient was followed as an outpatient, and she was doing exceptionally well with ambulatory oxygen saturation of 100%.Mycoplasma pneumoniae\u00a0are the main infectious agents, but other viruses have been reported, such as adenovirus, coxsackieviruses, and parvovirus B19.Erythema multiforme is an immune-mediated mucocutaneous condition. The lesions are typically targetoid-like with concentric variation in color. Various causative factors are attributed to the development of EM, which include\u00a0mainly infections, medications, immunization, underlying malignancy, autoimmune disease, radiation, and menstruation ,3. HerpeNew information, especially extrapulmonary manifestations, including cutaneous presentations of COVID-19, is emerging every day ,5. We suErythema multiforme in COVID-19 patients is attributed to a delayed hypersensitivity reaction. It has been seen usually in patients with age less than 30 years or older patients with age more than 55 years, or it could be a drug-induced reaction .Our patient, who did not have typical COVID-19 pneumonia symptoms initially and was SARS-CoV-2 PCR-negative twice, could have been easily deemed as not having COVID-19. Her only presentation to the hospital was fever and rash. However, the fact that she was unvaccinated and had positive sick contact with imaging concern of COVID-19 pneumonia and COVID-19 antibody being positive\u00a0clearly attributes her manifestations to the virus. The other tests done to look for a causative factor were negative. She had not received any medications before she started having the EM-like rash that her symptoms could be attributed to. Our patient had both cutaneous and oral mucosa with lesions. Hence, this EM-like rash is highly likely to be\u00a0erythema multiforme major.COVID-19 is still an ocean to be explored. The focus is not only on pulmonary signs and symptoms but also on multisystemic features taken all together that this virus can present. The fact that a rash like EM could be associated with COVID-19 is extremely prudent. Not many cases have been reported, and further studies are required to establish a confirmed relationship between EM and COVID-19. The future effects of COVID-19 are still unknown. EM being attributed as a delayed hypersensitivity reaction could open new doors to the study of various other immunological reactions that the virus could bring about. It could give us more insight into the pathophysiology of the disease and eventually help enhance the current pharmacological interventions.In addition to the pulmonary and cutaneous features, a\u00a0high level of vigilance is required to catch other extrapulmonary manifestations of the COVID-19 disease as well. The ultimate goal is to collaborate data obtained worldwide to build up solid literature on this virus to be prepared for the future effects, if any, of the COVID-19."}
+{"text": "Arborophila rufipectus.Intestinal microbiota composition plays a crucial role in modulating the health of the host. This evaluation indicator is very sensitive and profoundly impacts the protection of endangered species. Currently, information on the gut microbiota of wild birds remains scarce. Therefore, this study aimed to describe the gut microbial community structure and potentially, the pathogen composition of wild A. rufipectus and Lophura nycthemera in their habitats for two quarters. The 16S rRNA gene was then sequenced using high-throughput sequencing technology to examine the intestinal core microbiota, microbial diversity, and potential pathogens with the aim of determining if the composition of the intestinal microflora varies seasonally.To guarantee comprehensive data analysis, we collected fecal samples from wild A. rufipectus and L. nycthemera primarily comprised four phyla: Proteobacteria (45.98%), Firmicutes (35.65%), Bacteroidetes (11.77%), and Actinobacteria (3.48%), which accounted for 96.88% of the total microbial composition in all samples. At the genus level, core microorganisms were found, including Shigella (10.38%), Clostridium (6.16%), Pseudomonas (3.03%), and Rickettsiella (1.99%). In these genera, certain microbial species have been shown to be pathogenic. This study provides important indicators for analyzing the health status of A. rufipectus and formulating protective measures.The gut microbiota of  Arborophila rufipectus is a mountain partridge that is a member of the pheasant family. It is a medium-sized partridge with rich colors and a body length of approximately 30\u2009cm. Currently, it is only distributed in southwest China  and A. rufipectus (group B) between April 2021 and June 2021 (summer) and labeled them as group A/B . Another 10 fresh fecal samples from A. rufipectus were collected between July 2021 and September 2021 (autumn) and were labeled as group C (C1\u2013C10). The feces were stored in sterile centrifuge tubes, avoiding ground contact during collection and using separate tools for each sample. Samples were collected at least 5\u2009m apart to ensure that feces were excreted from different individuals. During the fieldwork, all samples were frozen in a portable freezer at \u221220\u00b0C. Samples were stored in liquid nitrogen and sent to the laboratory for subsequent processing. The detailed data collected for each sample are shown in Lophura nycthemera\u2019s range of activities increased in autumn, and no stool samples that met the requirements were found within the stool collection range we set.Samples were collected from the Sichuan Laojunshan Mountain National Nature Reserve, China . We collected 10 fresh fecal samples from 2O. The cycling conditions were as follows: initial denaturation at 94\u00b0C for 10\u2009min; 30\u2009cycles run at 94\u00b0C for 30\u2009s, 55\u00b0C for 30\u2009s, and 72\u00b0C for 2\u2009min; and final extension at 72\u00b0C for 5\u2009min. PCR products were sent to Sangon Biotech  for sequencing, and NCBI BLASTFeces were inoculated on MacConkey (MAC) agar, Salmonella Shigella (SS) agar, and blood agar mediums using a disposable aseptic inoculation ring. All plates were incubated in an incubator (35\u00b0C), and the bacterial growth was monitored at 12-h intervals. We selected bacterial colonies of different shapes and colors for isolation until a single colony was obtained by purification. We used the TIANamp Bacteria DNA Kit  to extract bacterial DNA. PCR amplification of the V1-V9 region was performed using universal primers: 27f (5\u2032-TACGGYTACCTTGT-TACGACTT-3\u2032) and 1492R (5\u2032-AGAGTTTGATCMTGGCTCAG-3\u2032). The PCR mixture consisted of 2.0\u2009\u03bcl DNA template, 9.0\u2009\u03bcl 2\u2009\u00d7\u2009Taq PCR Super Master Mix and 2.0\u2009\u03bcl forward and reverse primers, respectively, and 10\u2009\u03bcl ddHThe CTAB method was selected to extract total DNA from fecal samples, and agarose gel electrophoresis was used to confirm the quality of DNA extraction . FinallySpecific primers , the quality filtering of the raw data is filtering under specific filtering conditions to obtain high-quality clean tags. Preconditioning the raw readings was done by using the following procedure: Cutadapt (v1.9), FLASH (v1.2.8), fqtrim, and Vsearch (v2.3.4) were used for data stitching and filtering . DADA2 wenoising . We obtaenoising . Picrustenoising . BugbaseAfter quality control, the samples yielded 1,376,048 sequences. The mean number of all sequences in all samples was 68,802. The sequencing quality was calculated using a rarefaction curve. All curves were flat, and the number of operational taxonomic units (OTUs) was close to saturation . The rarMultiple algorithms, such as observed_otus and Chao1, were used to estimate alpha diversity . The resA. rufipectus and L. nycthemera belong to Phasianidae. In contrast, the time interval between groups B and C was shorter. The UPGMA cluster number results also showed that most samples in the same group did not have allied branches (The PCoA results showed that the three groups of stool samples did not cluster . This mabranches .Shigella (10.38%), Lysinibacillus (6.04%), Bacteroidales_unclassified (4.95%), Enterococcus (4.19%), and Clostridium (6.15%), with 16 core genera constituting 50.13% of the microbial composition identified in all samples , Firmicutes (35.65%), Bacteroidetes (11.77%), and Actinobacteria (3.48%), accounting for 96.88% of the total microbial composition in all samples. Moreover, the core genera included  samples . At the  samples .Shigella (12.19%), Lysinibacillus (8.65%), Acinetobacter (7.88%), Bacteroidales_unclassified (7.2%), Pseudomonas (6.37%), Clostridium (4.53%), Barnesiella (3.33%), Escherichia (2.96%), Escherichia-Shigella (1.83%), and Rickettsiella (1.82%) were the predominant taxa (>1%) in group A. Lysinibacillus (7.37%), Shigella (5.28%), Clostridium (7.12%), Enterococcuswere (6.57%), Bacteroidales_unclassified (4.47%), Escherichia-Shigella (1.8%), Barnesiella (1.28%), and Paenibacillus (1.19%) were the predominant taxa (>1%) in group B. Shigella (13.67%), Paenibacillus (7.1%), Clostridium (6.83%), Enterococcus (5.46%), Lactobacillus (5.08%), Rickettsiella (4.15%) Bacteroidales_unclassified (3.2%), Pseudomonas (2.53%), Lysinibacillus (2.11%), Citrobacter (2.06%), Escherichia-Shigella (1.8%), and Barnesiella (1.28%) the predominant taxa (>1%) in group C . Due to the limitations of traditional bacterial culture techniques, Clostridium, Rickettsiella, and Chlamydiales could not be isolated. However, this also confirmed the existence of Shigella in the feces of these two species of wild birds. The evolution tree of bacteria is presented in After the Community-Composition Analysis demonstrated that the dominant genus of A cladogram of the LEfSe analysis is shown in Pantoea, Dickeya, Candidatus_Saccharibacteria_unclassified, Pediococcus, Anaerotruncus, and Proteobacteria in the A group; Chlamydiales_unclassified, Catellicoccus, Ruminococcaceae_unclassified, Labrys, llsosphaeraceae_unclassified, Roseiarcus, and Verrucomicrobia in the B group; Atopobium, Citrobacter, Cronobacter, Enterobacteriaceae_unclassified, and Raoultella in the group C.There were significant differences at the phylum level: Candidatus Saccharibacteria and Proteobacteria in the A group; Verrucomicrobla and Chlamydiae in the B group. There were also significant differences at the genus level: Shigella, Clostridium, Pseudomonas, and Chlamydiales_unclassified, were detected using community-composition analysis and LEfSe analysis. These bacteria can cause bird diseases  and Kyoto Encyclopedia of Genes database. There was little difference among the three groups, and membrane transport, carbohydrate metabolism, amino acid metabolism, and replication and repair accounted for the largest proportion. This is also consistent with the results of the principal coordinate analysis. However, genes related to cell growth and death were detected, which is generally related to pathogenic bacterial infection, indicating that groups A, B, and C may have been infected with pathogenic bacteria . AccordiArborophila rufipectus is one of the rarest Arborophila species worldwide, with approximately 2,200 individuals distributed in China. China has implemented many conservation strategies, such as establishing nature reserves and captive breeding, to protect the habitat and populations of A. rufipectus. Nonetheless, these strategies have overlooked the effect of the gut microbiome in wild A. rufipectus. In this study, we analyzed the gut microbiota of A. rufipectus for two consecutive seasons by analyzing the 16S rRNA gene sequences.The rarefaction curve precisely reflects the justifiability of the sequencing data and indirectly reflects the enrichment of species in the sample. When the curve tends to be flat, the quantity of sequencing data is gradually reasonable , which dp\u2009<\u20090.05) between the B and C groups. Regarding seasons, there was a significant difference in the Chao1, observed_otus, and indices (p\u2009<\u20090.05) between groups A and B. The abundance of group B was the highest, indicating that the species richness and diversity of group B were the highest among the three groups. It can also be seen that the OTU of group B is the highest in the Venn diagram  can be found in the article/The animal study was reviewed and approved by Sichuan Agricultural University Animal Ethics Committee and Sichuan Laojunshan Mountain National Nature Reserve (permit number DYYS20210329).XM, JL, BC, XL, ZL, SF, and SC carried out the sample collections, conceived the study, and drafted the manuscript. JL, SC, ZZu, JD, XH, DC, YWa, and QZ participated in the data analysis. ZZh, YWe, GP, YJ, and YG participated in the study design and coordination and helped draft the manuscript. All authors contributed to the article and approved the submitted version.Arborophila rufipectus of Laojun Mountain National Nature Reserve (CDXZ-2021-0307-1).This study was supported by the Investigation on the pathogen of 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": "There is a paucity of knowledge about cosmetic vaginal tightening procedures; therefore, the present study aimed to describe the clinical effects of a novel combination technique of human acellular dermal matrix (HADM) and enriched platelet therapy (EPT) for the treatment of vaginal laxity.This single-arm, observational study was conducted on 52 patients with grade II to III vaginal relaxation. HADM biological band (U-shaped) was implanted in these patients by submucosal puncture in vagina under anesthesia. This was followed by thrice administration of EPT injection, once at the time surgery followed by each dose at a time interval of one month. Patients were followed up for a period of 6 months based on Female Sexual Function Index (FSFI) and Vaginal Health Index (VHI) scores. Patient satisfaction was measured using Visual Analogue Score (VAS).p value: <0.001). The mean VHI score also increased significantly after 6 months of treatment . The mean VAS after surgery was 1.61\u2009\u00b1\u20090.31. About 96% of the patients did not feel any pain after treatment at 6-month follow-up. No adverse effects were reported in this study.About 52 women with median age of 39 years were included in the study. The average time reported to complete HADM surgery was reported as 27 minutes. Following implantation, it was found that labia minora was significantly closed and perineal length was increased from 1.5 to 2.2 cm. Moreover, there was improvement in elasticity, contractility and lubricity of vaginal mucosa. The sexual function scores from pre- to post-surgery were significantly increased (7.95 vs. 30.09; These findings supported that combination treatment with HADM and EPT was safe and associated with both improved vaginal laxity and sexual function. These results may provide a novel surgical technique for this prevalent and undertreated condition.www.springer.com/00266.: Therapeutic Study This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors  Sexual dysfunction is a common complex multifactorial issue faced by many married women . VaginalHuman acellular dermal matrix (HADM), an extracellular matrix of three-dimensional cell scaffold structure had been used widely for wound repair, tissue regeneration and plastic surgery with good histocompatibility. The antigenic components of HADM that might cause immune rejections are generally removed by physical or chemical decellularization process. HADM is known for low absorption rate after implantation and inducing angiogenesis in host tissue. HADM is used for urethral reconstruction, congenital classic bladder exstrophy, penile skin defect and hidradenitis suppurativa . ConsideThis non-randomized, single-arm, observational study conducted between January 2021 and June 2021 enrolled 52 patients in the clinical observation group. Patients were included based on the following criteria: women aged \u2265 18 years with grade II~III vaginal relaxation, 3\u20136 months post-partum, physical examination must have vaginal cavity and mouth relaxed to varying degrees, flat vaginal mucosa, collapsed perineal area, and shortened linear distance between vagina and anus, inner diameter line of vagina (\u2265 3 transverse fingers), loose lateral wall with weak vaginal contraction. Patients with menstruation, pregnancy, severe perineal laceration and contraindications were excluded. The study protocol was approved by Ethics Committee  of Yunnan Wushi Jiamei Cosmetic Hospital, Yunnan, China (YWJC008956C) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all the patients before participation in the study.Surgery was conducted from 3~5 days after the end of menstruation to 10 days before start of the next menstrual cycle. Moreover, patients must abstain from sexual activity for 3 days before surgery.Surgery was conducted under routine intravenous anesthesia so that patient can comfortably lie down in the bladder lithotomy position. Disinfect the perineum and vagina using single-use disposable\u00a0towels\u00a0impregnated with a\u00a0disinfectant (0.5% iodophor). In order to increase the vaginal rectal space, 40 ml of swelling solution comprising 0.5% lidocaine (20 ml), ropivacaine and normal saline (10 ml each) and dexamethasone (10 mg) was injected into the lateral and posterior walls of vagina and perineum. HADM biological strap (1.0 cm\u00d715cm) procured from Beijing Jayyalife Biotechnology Co. Ltd. was rinsed thrice with normal saline (0.9% sodium chloride solution) and set aside.About 0.5-cm-long incisions at 3-5 points and at 7-9 points were given inside and outside the hymen mark, respectively Fig. . Submuco9/L. The average red blood cells, white blood cells, hemoglobin and hematocrit counts were 5.6 \u00d7 1012/L, 5.6 \u00d7 109/L, 121g/L, and 42%, respectively. PRP was stored under normal light condition at 24\u201328\u00a0\u00b0C. Livsraft EPT  and centrifugal equipment Fig. A procureFor every 40 ml of withdrawn blood, 15 ml of PRP (high concentration) and 5 ml of very high concentration PRP was obtained, which was injected using 30g sharp needle to surround multi-point injection into vaginal mucosa. Using the same needle, inject 2ml of EPT into point G, 1ml in point A area of point U and 1ml in clitoris. The G-spot is located at about 5cm away from urethral orifice, a bean-shaped region. The A point is located between G-spot and cervix. It can receive direct stimulation or can be stimulated by rubbing the vaginal wall, while U point is located at about 5 cm away from urethral orifice and the patient feels the urge to urinate on stimulation. Multiple studies have agreed on the existence of G-spot, but there was no agreement on the exact location, size, or nature . HoweverThe primary endpoint was to assess Female Sexual Function Index (FSFI) in patients when they were followed up for 3-6 months which included parameters such as female sexual function, vaginal tightness and vaginal moisture. The FSFI was validated by Rosen et.al for the assessment of key dimensions of female sexual function. The evaln\u2009=\u200935), cesarean section (n\u2009=\u20095), secondary delivery (n\u2009=\u200912), vaginal spontaneous delivery and cesarean section (n\u2009=\u20091 each) and abortion (n\u2009=\u200923) was reported. The average time reported to complete HADM biological band tightening surgery was 27 minutes, and the time from preparation to injection of EPT was about 30 minutes. Blood loss estimation was performed by gauze weighing method which revealed a loss of 5 and 0 ml after 24 hours of surgery with HADM and EPT injection, respectively. Following surgery, bleeding stopped after 3 days and < 24h with HADM and EPT injection, respectively.A total of 52 patients were included in this study with an age range of 21 to 52 years (median age: 39 years). A history of vaginal delivery . The vagina was accommodating two transverse fingers before surgery Fig.  which wap value: <0.001) . The elasticity and vaginal secretions had increased significantly at 6 months after treatment  (P\u2009<\u20090.0001). There was a gradual decrease in the vaginal pH from baseline to 6 months post-surgery . The moisture rate increased by twofold compared to before treatment  (P < 0.0001). The epithelial integrity also increased at 6 months compared to before treatment  (P < 0.01)  is a new concept related to excess looseness of the vaginal walls, and rejuvenation techniques are gaining popularity among women to correct this condition . It may Surgical methods, namely vaginoplasty and perineoplasty, have been used traditionally for wound repair after childbirth. Recently, these techniques have been increasingly used for vaginal laxity and aesthetic treatment . FibrillOur results show that the injection of EPT after HADM implantation could improve the vaginal laxity and sexual function in patients experiencing dryness post-HADM implantation. The VHI score was significantly increased post-3 months of treatment. These improvements were maintained for the 6 months of study. At the same time, there was improvement in the quality of patients\u2019 sex lives after treatment with enhanced vaginal trophicity. Our results were consistent with a study by Hersant et al.  which rePRP is rich in a variety of growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor (TGF-B), insulin-like growth factor (IGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) . These gThe present study utilized new generation Livsraft EPT device with a unique design of centrifuge tube. This device is highly efficient in preparing large and absolute sterile doses of PRP. High concentrations of platelet plasma EPT are obtained which are 5-10 times more concentrated as compared to conventional centrifugal devices. This enables injecting high concentrations of PRP into the entire vaginal mucosa in one centrifugal cycle . There aTo conclude, EPT injection supplemented the collagen of mucous membrane, thereby increasing the secretory function, and lubrication of vagina. Therefore, HADM implantation followed by EPT was safe and associated with both improved vaginal laxity and sexual function. These results may provide a novel surgical technique for this less recognized and undertreated condition."}
+{"text": "Interstitial lung disease (ILD) is a progressive fibrotic disease associated with rheumatoid arthritis (RA); real-world data for evaluating RA\u2013associated ILD (RA\u2013ILD) are limited. We evaluated prevalence, time to onset, clinical characteristics and prognostic factors in patients diagnosed with RA (n\u2009=\u20098963) in the Discus Analytics JointMan database (2009\u20132019) with and without ILD. ILD prevalence was 4.1% ;\u2009>\u200990% had an ILD diagnosis after RA diagnosis (mean time to onset 3.3\u00a0years). At baseline, a higher proportion of patients with RA\u2013ILD were older (>\u200965\u00a0years), male, with history of chronic obstructive pulmonary disease (COPD) compared with patients in the RA cohort. Patients in the RA\u2013ILD cohort were likely to have more severe RA characteristics and joint evaluation compared with patients without ILD, at baseline and before/after ILD diagnosis. In this large, real-world database patients with (vs without) ILD had a higher burden of RA characteristics. Previously established risk factors for RA\u2013ILD were confirmed ; thus, recognition of these factors and tracking routine disease activity metrics may help identify patients at higher risk of RA complications and lead to improved diagnosis and earlier treatment. RA is associated with many comorbidities and several extra-articular manifestations, including the most prevalent lung manifestation, interstitial lung disease (ILD). ILD is a progressive fibrotic disease of the lung and is associated with increased morbidity, mortality, and healthcare resource utilization4.Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting nearly 1.3 million people in the United States, and can severely impact patient quality of life9. Clinically significant ILD presents in approximately 10% of patients with RA10, and may be defined by the presence of respiratory symptoms, such as shortness of breath and coughing9. Pre-clinical ILD may be present in 33\u201360% of patients with RA, measurable by high-resolution computed tomography or pulmonary function tests, with no respiratory symptoms11. While patients with RA may lack clinical symptoms of ILD, they may be at high risk for developing this comorbidity12; thus, further studies are warranted in order to better understand the prevalence and time-to-onset of RA\u2013ILD. The 10-, 20-, and 30-year cumulative incidence rates of ILD among patients with RA have been estimated as 4%, 6%, and 8%, respectively, and are significantly higher than those among patients without RA 13. With an estimated 5-year mortality rate of approximately 36\u201339%, a survival time of\u2009\u2264\u200910 years14, and delays in diagnosis potentially increasing the mortality risk15, prompt diagnosis and identification of patients with RA at high risk for development of ILD is crucial.The prevalence of ILD among patients with RA has shown great variability in prior studies, ranging from 1 to 58% depending on the methodology and definitions used 17. Nevertheless, given the increased incidence and mortality associated with RA\u2013ILD, these risk factors are insufficient, and thus emphasize the need to identify additional risk factors that could lead to earlier diagnosis, and for collaboration between rheumatologists and pulmonologists. For example, two multi-centre, prospective, early RA inception cohorts (the Early RA Study and the Early RA Network) found that a higher risk of RA\u2013ILD may be associated with factors such as rheumatoid nodules, higher baseline erythrocyte sedimentation rate (ESR), and a longer time from first RA symptoms to first outpatient visit18. Other potential risk factors include the presence of erosions or destructive joint changes13.Well-established risk factors for RA\u2013associated ILD (RA\u2013ILD) have been identified from observational and medical records database studies  and/or anti-cyclic citrullinated peptide (anti-CCP) antibodiesThere are limited real-world data available for evaluating ILD among patients with RA, and further studies are needed to better understand the prevalence of and risk factors for ILD, including how ILD impacts RA disease activity, use of biologic treatments, and rheumatologist encounters.The objectives of this analysis of real-world data were to evaluate the prevalence and time to onset of ILD in patients with RA. Exploratory objectives included a comparison of baseline clinical characteristics of patients with RA versus patients with RA\u2013ILD and the evaluation of risk factors for RA\u2013ILD. Further analyses were conducted with a subset of the population in order to compare RA disease activity, rheumatologist encounters, and treatments in a cohort of patients with RA versus a cohort of patients with RA\u2013ILD, using data collected in the periods before and after the earliest recorded ILD diagnosis date.Patient demographics and disease characteristics were retrospectively analyzed following data extraction from the Discus Analytics JointMan database, a large US electronic medical records-based dataset initiated in March 2009. The JointMan database includes\u2009>\u200917,000 rheumatology patients covered by commercial, Medicare, or Medicaid insurance health plans. Practices across the following eight states are included: Washington, New York, Oregon, Florida, Georgia, California, Wisconsin, and Kentucky. Patient data were collected at rheumatology centers and were de-identified prior to analysis. In addition to electronic medical record data, the JointMan user interface collects clinical outcomes recorded by physicians at the time of the encounter.Patients were included if they were aged\u2009\u2265\u200918\u00a0years at the initial visit with a rheumatologist participating in the JointMan network, had a provider-selected diagnosis of RA between January 1, 2009 and September 20, 2019, and had\u2009\u2265\u20091 visit after the initial visit date. Patients were excluded if their initial encounter occurred after RA diagnosis or if they experienced a drug-induced ILD diagnosis  at any time during the study period. Patients were assigned to either the RA cohort (patients with confirmed RA but no diagnosis of ILD during the study period) or the RA\u2013ILD cohort . RA index date was defined as the first RA diagnosis date recorded in the JointMan database (provided by the rheumatologist).The overall study population was comprised of patients who were followed from the day after the RA index date to the last patient encounter date or the end of the study , whichever occurred first. RA was diagnosed according to the ICD, Ninth Revision, CM (ICD-9-CM) code 714.0 and ICD-10-CM codes M05 and M06. ILD was identified by ICD diagnosis codes  or by provider indication.A subanalysis was conducted in a set of patients grouped based on ILD diagnosis. For the subanalysis population, the ILD diagnosis index was defined as the first date of ILD diagnosis recorded in the JointMan database (for patients in the RA\u2013ILD cohort), and patient characteristics were described for the 90-day periods before and after the ILD diagnosis index. For patients without ILD, the index date was based on distribution of the number of days from RA diagnosis to ILD diagnosis in the RA\u2013ILD cohort; characteristics were described for the 90-day periods before and after the index date  remission score was defined as\u2009\u2264\u20093.3; SDAI low, moderate, and high disease activity scores were defined as\u2009>\u20093.3 to 11,\u2009>\u200911 to 26, and\u2009>\u200926, respectively19. Disease Activity Score in 28 joints using CRP (DAS28 [CRP]) remission score was defined as\u2009\u2264\u20092.3; DAS28 (CRP) low, moderate, and high disease activity scores were defined as\u2009>\u20092.3 to 2.7,\u2009>\u20092.7 to\u2009<\u20094.1, and\u2009\u2265\u20094.1, respectively20. DAS28 (ESR) remission score was defined as\u2009<\u20092.6; DAS28 (ESR) low, moderate, and high disease activity scores were defined as 2.6 to <\u20093.2, 3.2\u20135.1, and\u2009>\u20095.1, respectively.19 Routine Assessment of Patient Index Data 3 (RAPID3) remission score was defined as\u2009\u2264\u20093; RAPID3 low, moderate, and high disease activity scores were defined as\u2009>\u20093 to 6,\u2009>\u20096 to 12, and\u2009>\u200912, respectively21. Variables were assessed as potential predictors of RA\u2013ILD.Exploratory endpoints, assessed in the exploratory analysis population, included baseline demographics, comorbidities, RA characteristics, and overall RA disease activity in the RA cohort compared with the RA\u2013ILD cohort. RA characteristics included joint stiffness, erosions, extra-articular disease, anti-CCP antibodies, joint swelling, ESR, C-reactive protein (CRP), and Clinical Disease Activity Index (CDAI). CDAI remission score was defined as\u2009\u2264\u20092.8; CDAI low, moderate, and high disease activity scores were defined as\u2009>\u20092.8\u201310,\u2009>\u200910\u201322, and\u2009>\u200922, respectively22.For patients included in the subanalysis population, CDAI and RAPID3 scores, swollen and swollen28 joint counts, the number of rheumatologist encounters, and treatment utilization pre- and post-ILD diagnosis index were also assessed. The swollen and swollen28 joint counts are components of the DAS/DAS28 score: the swollen joint count is an assessment of 28 or more (up to 44) joints, while the swollen28 joint count is an assessment of only 28 pre-selected jointst-test and percentages for categorical and binary baseline variables were compared using the Chi-square test.The prevalence  of the first observed ILD diagnosis during follow-up was calculated. The time to ILD diagnosis was examined using unadjusted Kaplan\u2013Meier survival curves. Descriptive statistics for continuous baseline variables were compared using Student\u2019s p values were provided for each covariate. Statistical significance for model inclusion was set at p\u2009<\u20090.05.Potential predictors of RA\u2013ILD were analyzed by a Cox regression model. Patient demographic data and comorbidities were collected at baseline and were controlled for in the Cox model. RA characteristics were identified during and after the initial RA diagnosis and were controlled for as time-varying covariates in the Cox model. The final covariate lists were based on clinical rationale and model fitting; hazard ratios, 95% confidence intervals, and p\u2009<\u20090.05.The number and percentage of patients with rheumatologist visits, treatment utilization, and each disease activity score in the pre- and post-index periods were calculated. P values for disease activity score category compared pre- and post-index periods and correspond to Fisher\u2019s exact test or Chi-square test with statistical significance set at 23. The study protocol was reviewed by the internal BMS Observational Protocol Review Committee (OPRC). No identifiable protected health information was extracted or accessed from the database during the study, therefore the BMS OPRC confirmed that this analysis did not require ethical oversight. Additionally, the study did not involve the collection, use, or transmittal of individually identifiable data, and data were collected in the setting for the usual care of the patient. Informed consent from the study participants was not required because the dataset used in this observational study consisted of de-identified secondary data released for research purposes.This study was conducted in accordance with the International Society for Pharmacoepidemiology Guidelines for Good Pharmacoepidemiology Practices and applicable regulatory requirementsIn the overall study population, a total of 8963 patients with RA were identified during the period of January 1, 2009 to September 20, 2019. The prevalence (95% CI) of ILD in the overall population of patients with RA was 4.1% (3.7\u20134.5%).Of the patients in the RA\u2013ILD cohort, 91.8% (n\u2009=\u2009337/367) had their first ILD diagnosis after their RA diagnosis. The mean time to onset of ILD after RA diagnosis was 3.3\u00a0years  had RA and no comorbid ILD diagnosis (RA cohort) and 3.5% (n\u2009=\u2009205) had RA\u2013ILD (RA\u2013ILD cohort). Compared with the RA cohort, a significantly higher proportion of patients in the RA\u2013ILD cohort were older, male, white, had Medicare as their primary insurance category, and had a history of chronic obstructive pulmonary disease (COPD) . In addition, baseline ESR level was significantly higher in the RA\u2013ILD cohort Table . PatientPotential predictors of RA\u2013ILD diagnosis were assessed in the exploratory analysis population (patients with 6\u00a0months of follow-up). Older age (\u2265\u200965\u00a0years old) and a history of COPD at baseline were shown to be risk factors for developing ILD Fig.\u00a0. SeveralIn order to evaluate RA disease activity, rheumatologist encounters, and treatments in patients in the RA\u2013ILD versus RA cohort, data from the 90-day periods before and after the earliest recorded ILD diagnosis date were compared. In total, there were 7150 patients with RA only and 240 patients with RA\u2013ILD who had data in both the 90\u00a0days prior to and 90\u00a0days after the ILD diagnosis index.For both patient cohorts, disease severity measure missingness was lower in the post-index period compared with the pre-index period  versus 68.2% (n\u2009=\u20094877/7150), respectively. However, for patients in the RA\u2013ILD cohort, there was an increase in the number of rheumatologist visits in the post-ILD diagnosis index period; pre- versus post-ILD diagnosis index periods: 74.2% (n\u2009=\u2009178/240) versus 99.6% (n\u2009=\u2009239/240), respectively.For both the pre- and post-index periods, a greater proportion of patients in the RA\u2013ILD cohort used glucocorticosteroids/disease-modifying antirheumatic drugs (DMARDs) and biologics compared with patients in the RA cohort. For patients in the RA\u2013ILD cohort, a similar proportion of patients in the post-ILD versus pre-ILD diagnosis index periods used glucocorticosteroids/DMARDs (82% vs. 83%) and biologics (48% vs. 45%). However, for patients in the RA cohort, a lower proportion of patients used glucocorticoids/DMARDs (58% vs. 74%) and biologics (31% vs. 35%) in the post-ILD diagnosis index period compared with the pre-ILD diagnosis index period.3, which is supported by our results showing that patients with RA\u2013ILD had more active RA at baseline and after ILD diagnosis. Consequently, patients with RA\u2013ILD may require more clinical consultation.In this large, real-world study, using data from the United States-based Discus Analytics JointMan database, the prevalence of RA\u2013ILD was 4.1% and the mean time to onset of ILD after RA diagnosis was 3.3\u00a0years. We identified several risk factors for RA\u2013ILD: age (\u2265\u200965\u00a0years), COPD at baseline, anti-CCP positivity, CRP\u2009>\u20095\u00a0mg/L, and a moderate-to-high CDAI score. Patients with RA\u2013ILD have increased morbidity compared with patients with RA without ILD9. A recent United States-based cohort study using Medicare claims data from\u2009>\u2009500,000 patients between 2008 and 2017 estimated the baseline prevalence of RA\u2013ILD to be 2.0% and overall prevalence  to be approximately 5.0%, which is in line with our results24. A study, similar to that reported here, using the United States-based Truven Health MarketScan Commercial and Medicare Supplemental health insurance databases, showed the prevalence of RA\u2013ILD in the US was 3.2 to 6.0 cases per 100,000 people4. A retrospective review of patient data in Jordan found prevalence of RA\u2013ILD among 210 patients to be 3.7%25. It is important to note that the study reporting an RA\u2013ILD prevalence at the higher end of the range of 58% was a small analysis of 36 patients with early RA (duration\u2009<\u20092\u00a0years); the prevalence estimate included both patients with \u201cclinically significant ILD\u201d and with \u201cabnormalities compatible with ILD but no clinically significant ILD\u201d9. As previously noted, in our study, patients were only classified as having RA\u2013ILD if a diagnosis of ILD was definitive.The prevalence of RA\u2013ILD ascertained from our study (4.1%) falls towards the lower end of the range previously reported; however, those studies had differing methodology and ILD definitions29, we confirmed baseline COPD30, and baseline moderate-to-high CDAI score, and CRP\u2009>\u20095\u00a0mg/L as risk factors. Although smoking is an established risk factor for RA\u2013ILD31, in our analysis, differences in baseline smoking prevalence were not significant based on statistical testing. However, it should be noted that identification of smoking exposures in patient data is limited by missingness, and there may have been a large proportion of false negatives, which would limit reliability. It should further be noted that although COPD and ILD have distinct, separate pathophysiologies, they share overlapping risk factors, and so may develop either simultaneously or successively32.In this study, assessment of the clinical characteristics of patients in the RA and RA\u2013ILD cohorts showed that patients with ILD were more likely to be older, male, have a history of COPD, and have more prominent RA disease characteristics . A higher proportion of patients with RA\u2013ILD had Medicare insurance when compared with the RA cohort; this can be at least partially explained by the age difference, as a larger proportion of patients with RA\u2013ILD were over the age of 65 when compared with the RA cohort. Potential risk factors for RA\u2013ILD were further analyzed by a Cox regression model and, in addition to older age and seropositivity, which are already established risk factors33 or CDAI34 as the measure. A retrospective analysis of data from patients (n\u2009=\u20091419) with early/mild or severe interstitial lung abnormalities in the Brigham and Women\u2019s RA Sequential Study revealed that those with high or moderate disease activity (defined by DAS28) had an increased risk of developing RA\u2013ILD (compared with patients in remission or with low disease activity)33. A smaller (n\u2009=\u2009118) case\u2013control study showed that a CDAI score\u2009>\u200928 was associated with the presence of RA\u2013ILD34. Previous studies have also identified baseline CRP level as a risk factor for RA\u2013ILD: CRP\u2009>\u200910\u00a0mg/L or \u201chigher\u201d baseline levels36. Our analysis refines these further by identifying baseline CRP\u2009>\u20095\u00a0mg/L to be predictive of RA\u2013ILD. The identification of new risk factors for RA\u2013ILD may help physicians diagnose and treat patients earlier in the course of the disease.Disease activity has previously been identified as a risk factor for RA\u2013ILD, using DAS28Our subanalysis of outcomes before versus after ILD diagnosis provides some insight into RA disease severity and healthcare utilization  for patients with RA who develop ILD. Based on swollen joint counts, patients with RA\u2013ILD appeared to have worse RA symptoms after ILD diagnosis compared with patients who did not develop ILD. It should be noted that more patients in the RA cohort had missing disease severity data, which may be an artifact of scheduling routine assessments 1\u20132 times per year. Missing data may also be accounted for by patients with low disease activity or those in remission being less likely to consult their physician as frequently as patients with medium/high disease activity. Thus, more complete disease activity data may highlight a greater disparity in RA symptom control between patients with RA who develop ILD and those who do not develop ILD. Our descriptive subanalyses suggest that this disparity contributes to greater use of glucocorticoids/DMARDs, biologics, and rheumatologist encounters in patients who develop ILD compared with patients with RA alone.37.This was a large analysis of real-world data collected by rheumatologists across several regions of the United States. The comprehensiveness of the JointMan database, which incorporates rheumatology encounters, rheumatology-specific laboratory results, clinical evaluations, and prescriptions within the JointMan network for patients covered by commercial, Medicare, and Medicaid insurance plans, allows for longitudinal analysis of RA and related treatments and conditions. Other strengths are the integration of live patient electronic records allowing for continuous coverage, and being part of a rheumatology network which suggests the clinicians are knowledgeable on disease surveillance practice. Compared with randomized clinical trials, real-world studies are important to provide evidence that is generalizable to different populations and are useful for assessing specific characteristics of patient populations, risk factors on a pre-defined outcome, and comparative effectiveness38. Additionally, encounters outside of the JointMan network such as inpatient visits, emergency department visits, and visits with non-rheumatology physicians are not captured. The use of the JointMan database also varied between sites and over time. Although data were collected across many regions of the United States, the JointMan database population was limited to eight states, with most of the population located in Washington. As mentioned, our dataset also had different levels of missing data for swollen joint counts and disease severity scores for patients in the RA and RA\u2013ILD cohorts. Missing data may have been driven by lower disease activity, especially for patients in the RA cohort. Furthermore, as this study covers patients from 2009 to 2019, clinical assessment of disease activity scores may have become more common since the beginning of the study period, which may contribute to missing data.Despite the above strengths, there are naturally some limitations to the analysis. Coding errors may have occurred in the patient data, and in some instances, diagnostic codes may have been entered as rule-out criteria and not actual disease. Due to the nature of the study design, the symptoms and tests used to reach diagnosis were not captured in this study. Specific validation studies assessing the codes for RA are lacking, however the validity of ICD-9-CM and ICD-10-CM versus chart review data have been shown to be comparable for rheumatic diseaseIn conclusion, this work further describes the disease and natural history of patients with the debilitating conditions of RA and ILD. The prevalence of RA\u2013ILD in this large, real-world study using data from the United States-based JointMan database was 4.1%. This study provides insight into the increased burden of disease among patients with RA\u2013ILD versus RA without ILD; RA disease activity may be worse after ILD diagnosis compared with the pre-ILD diagnosis index period and compared with patients with RA alone. Several previously established risk factors for developing ILD were confirmed, including older age, COPD at baseline, anti-CCP positivity, CRP\u2009>\u20095\u00a0mg/L, and a moderate-to-high CDAI score. Recording and tracking routine clinical disease activity metrics may help identify patients at higher risk of RA complications. Recognition of the risk factors underscored here may lead to early diagnosis of RA\u2013ILD and quicker treatment initiation, leading to better clinical outcomes for these patients.Supplementary Figure S1."}
+{"text": "The variables associated with RA-ILD in the Cox regression analysis were disease activity (DAS28)  and high levels of ACPA , IL-18 in pg/mL , MCP-1/CCL2 in pg/mL , and SDF-1 in pg/mL . The only variable associated with the progression of ILD was IL-18 in pg/mL . Our data support that the inflammatory activity was higher in patients with RA-ILD than RA patients without ILD. Some cytokines were associated with both diagnosis and poorer prognosis in patients with RA-ILD.This study aimed to identify inflammatory factors and soluble cytokines that act as biomarkers in the diagnosis and prognosis of rheumatoid arthritis-associated interstitial lung disease (RA-ILD). We performed a nested prospective observational case\u2013control study of patients with RA-ILD matched by sex, age, and time since the diagnosis of RA. All participants underwent pulmonary function testing and high-resolution computed tomography. ILD was defined according to the criteria of the American Thoracic Society/European Respiratory Society; the progression of lung disease was defined as the worsening of FVC > 10% or DLCO > 15%. Inflammation-related variables included the inflammatory activity measured using the DAS28-ESR and a multiplex cytokine assay. Two Cox regression models were run to identify factors associated with ILD and the progression of ILD. The study population comprised 70 patients: 35 patients with RA-ILD (cases) and 35 RA patients without ILD (controls). A greater percentage of cases had higher DAS28-ESR ( Rheumatoid arthritis (AR) is a chronic inflammatory disease that mainly affects the joints. Without treatment, patients with RA experience progressive joint deterioration, which, in turn, can lead to disability and reduced quality of life. RA often affects organs and systems outside the musculoskeletal system, including the lungs . RA-assoNo useful serum biomarkers are currently available for the diagnosis of RA-ILD, although various candidates have been evaluated. The protein Krebs von den Lungen 6 (KL-6) in serum has been investigated for the diagnosis of ILD and has been associated with systemic inflammatory diseases . The higRA was characterized by the dysregulation of innate and adaptive immune systems, in which cytokines play a major role. Given that cytokines contribute to chronic inflammation and joint destruction, there are many reasons why they should be assessed in patients with RA-ILD. First, they can provide valuable information on all phases of the pathogenesis of RA through their role in promoting autoimmunity, maintaining chronic synovitis, and leading to the destruction of neighboring joint tissue ,16,17,18While interest in ILD has grown in the last 10 years, and studies are being performed to gain a greater insight into the clinical characteristics and risk factors associated with RA-ILD, our knowledge remains very limited ,11. GiveThe study population comprised 70 patients: 35 patients with RA-ILD (cases) and 35 RA patients without ILD (controls) . Somewhap = 0.040) and anti-TNF agents (p = 0.041) and more frequently took hydroxychloroquine (p = 0.010) and abatacept (p = 0.004). Only 4 patients with RA-ILD received mycophenolate mofetil (p = 0.032), and 1 patient took antifibrotic medication combined with mycophenolate mofetil and abatacept.All the participants received a DMARD . While tAccording to the protocol, the HRCT scan revealed lung involvement in all the cases and none of the controls. The most common histopathologic patterns visible on the HRCT were UIP in 29 patients (82.9%) and NSIP in 6 (17.1%). As was to be expected, the cases had lower mean PFT values than the controls . The PFTp = 0.032), a higher number of swollen joints (p = 0.040), and poorer physical function according to the HAQ (p = 0.003). As for cytokines, the cases generally had higher values of IL-1 alpha, IL-6, IL-18, MCP-1/CCL2, MIP1beta (CCL4), and SDF-1 alpha than the controls (controls . In paticontrols .p = 0.017) and high levels of ACPA , IL-18 in pg/mL , MCP-1/CCL2 in pg/mL , and SDF-1 alpha in pg/mL .p = 0.001), DLCO , and FEV1  (Thirteen patients (37.1%) fulfilled the criteria for the progression of lung disease, 20 (57.1%) of these were for stabilization, and 2 (5.7%) were for improvement, with a mean (SD) follow-up of ILD for 66.1 (47.2) months. At the cut-off, the mean PFT values had fallen significantly compared with the date of the diagnosis of RA-ILD for FVC  .p = 0.041). While progression was more common in men and with the UIP pattern, these differences were not significant (p = 0.004) (As can be seen in nificant . As for nificant . Supplem= 0.004) .Several biomarkers that could prove potentially useful in the diagnosis and follow-up of RA-ILD have been identified. However, the role of cytokines in RA-ILD has received scant attention. The present study compared cytokine levels and inflammatory activity in patients with RA-ILD and patients with RA but not ILD. In addition to the determination of cytokines, all patients underwent complete PFT. Given that patients with RA-ILD underwent complete PFT since the diagnosis of ILD, we were also able to evaluate the association between cytokines and the progression of lung disease.In line with this approach, our findings show that inflammatory activity according to the DAS28-ESR is more pronounced in patients with RA-ILD, and these patients more frequently had high ACPA titers. They also showed differences in the overexpression of cytokines, specifically MCP-1/CCL2, SDF-1 alpha, and IL-18. In this sense, other studies have also found that patients with RA-ILD have more pronounced joint disease activity than RA patients without ILD ,28. In fAs for the cytokines we found to be associated with ILD, MCP-1/CCL2 was involved both in RA and in ILD. On the one hand, in patients with RA, the signaling pathway for chemokines (CCL4/CCR5/c-Jun and c-Fos/CCL2) was involved in the expression of CCL2, which could lead to the chronic inflammation associated with RA . On the In the present study, we also found that IL-18 values were higher in cases than in the controls. Similarly, this was the only cytokine associated with the progression of RA-ILD. IL-18 is a member of the IL-1 cytokine superfamily and is produced predominantly by macrophages. Data from animal studies show that IL-18 can lead to pulmonary inflammation ; in humaOur study is subject to a series of limitations. First, the analysis of inflammation and cytokines was performed ad hoc, thus preventing us from confirming a causal relationship with other variables. However, a key strength of our study was that both groups of patients were studied using HRCT and PFT, thus enabling us to clearly identify cases and the controls and assess the differences between them. Furthermore, the prospective follow-up of the RA-ILD cohort enabled us to study the progression of lung disease and its association with cytokines and inflammation. It is also noteworthy for all the participants who received DMARDs that these drugs directly affected cytokines and re-established the inflammatory process. Nevertheless, since both the cases and the controls were treated with DMARDs, this effect applied to all the participants included. Furthermore, we did not include a healthy control group without RA since the main objective was to identify soluble cytokine biomarkers in patients with RA-ILD and the progression of lung disease; performing HCRT in healthy controls could be more controversial. Lastly, our sample may have been too small to detect more robust differences and more clinical or laboratory factors. However, cases differed from the controls in inflammatory parameters, and some of the cytokines studied.CasesWe performed an observational case\u2013control study nested in a single-center prospective cohort of patients with RA from Hospital Regional Universitario de M\u00e1laga (HRUM), Malaga, Spain. The study was approved by the Research Ethics Committee of HRUM (2627-N-21). All patients gave their written informed consent before participating.ControlsThe case group comprised patients with established RA from the initial cohort of HRUM  who wereThe control group comprised patients with clinically significant RA without ILD who were consecutively selected from all other patients with RA in the prospective RA cohort of HRUM and followed the same procedure as the cases in . Each caAll participants were managed according to a pre-established protocol for data collection after signing an informed consent document. A blood sample was taken to measure the levels of cytokines and other inflammatory parameters from all patients at inclusion. All patients with RA-ILD underwent systematic PFT and HRCT at a diagnosis of ILD, at years 2 and 5 if they remained stable, and at any other visit if clinically necessary. At inclusion, all participants underwent PFT and HRCT, with this date being considered the cut-off. The approach for reading the HRCT scan and the methodology have been published elsewhere ,50,51. CThe 2 main outcomes were  the presAs for the progression of lung disease, we defined 3 stages:  progressThe secondary outcome was the radiologic progression on HRCT, defined as an increase of 20% or more in the presence and extension of ground-glass opacities, reticulation, honeycombing, low attenuation, centrilobular nodules, other nodules, emphysema, or consolidation compared with the baseline HRCT scan.Inflammation-related variables included data on inflammatory activity based on the multiplex cytokine assay and clinical activity indices. Clinical inflammatory activity was evaluated for all participants using the 28-joint Disease Activity Score with an erythrocyte sedimentation rate (DAS28-ESR) . The results of the DAS28-ESR were stratified as follows:  high act2]), history of smoking , and socioeconomic and educational level. As for treatment, we recorded conventional synthetic DMARDs (csDMARDs), biologic DMARDs (bDMARDs), other immunosuppressants, corticosteroids at the cut-off, and previous treatment.Severity variables included the presence of autoantibodies and their titers. The rheumatoid factor (RF) was considered positive if >10 IU/mL, ACPA was considered positive if >20 IU/mL, and high ACPA titer if >340 IU/mL. The presence of radiologic erosions and the Health Assessment Questionnaire (HAQ) score were also taken into account . The dur2 test and t test or Mann\u2013Whitney test were used to compare the main characteristics between cases and controls, as well as between cases with RA-ILD with and without the progression of lung disease at the cut-off. A paired t-test or Wilcoxon test, as applicable, was performed to compare PFT findings at the onset of RA-ILD and at the cut-off in patients with RA-ILD. Finally, we ran 2 stepwise Cox regression analyses , one to identify the severity and inflammatory factors associated with RA-ILD adjusted for the time since diagnosis of RA and another to determine the factors associated with the progression of RA-ILD when adjusted for the time since the diagnosis of RA-ILD. The factors that we included in this model were those that were significant in the univariate analysis. The multicollinearity of the independent variables was checked using the Pearson correlation coefficient. The analysis was performed with IBM-SPSS Statistics, Version 28.We performed a descriptive analysis of the main variables. Qualitative variables were expressed as the absolute number and percentage, and quantitative variables as the mean and standard deviation (SD) or as the median and interquartile range (IQR) depending on the normality of the distribution (Kolmogorov\u2013Smirnov test). The \u03c7In conclusion, values for inflammatory activity and ACPA were higher in patients with RA-ILD than RA patients without ILD. Some cytokines, for example, MCP-1/CCL2 and SDF-1 alpha were associated with the diagnosis of RA-ILD, and IL-18 levels were associated with a diagnosis of RA-ILD and a more marked progression of lung disease. If validated, these cytokines could be potential diagnostic and prognostic markers for RA-ILD disease and, therefore, could contribute to the early identification of patients with high morbidity and mortality. Further studies are necessary to validate these results."}
+{"text": "During the first year of the population based colorectal cancer (CRC) screening program on Cura\u00e7ao, about 20% of invitees participated. This study explored the target population\u2019s perceptions and awareness on CRC (screening), beliefs on the program provision, their preferences and information needs for informed decision-making.Semi-structured interviews with 23 individuals, who were not yet invited for CRC screening, were recorded, transcribed, coded and analyzed.CRC (screening) was discussed in the context of personal health, where own responsibility and food were important. Cancer was perceived as an unpredictable disease that causes suffering and leads to death and was also associated with fear. Despite being aware of the program, most respondents were not familiar with the screening procedure. Provision of the screening program was regarded positively and as an opportunity to contribute to health improvement. This seemed related to the expressed trust in the Caribbean Prevention Center (program organizer). Respondents preferred to make independent decisions about CRC screening participation. A personal approach, visual aids and media were the preferred sources of information.The results of our interviews suggest that it may be beneficial to provide information on CRC screening in Cura\u00e7ao within the context of personal health. While including sensitivity to fears and respect for the autonomy of the target population. Finally, electronic media maybe useful in supporting informed decision-making.The online version contains supplementary material available at 10.1186/s12889-023-16335-x. Colorectal cancer (CRC) is the third most common type of cancer and the most common form of gastro-intestinal cancer globally . ThroughCura\u00e7ao is an autonomous nation within the Kingdom of the Netherlands, located in the Caribbean. The population is multi-ethnic and multi-cultural, with most being Afro-Caribbean. There is also significant immigration from Latin America and Europe. More than 80% of the population speaks the local language of Papiamentu. Other languages make up the rest of the fraction, including Dutch, Spanish and English to name the most prevalent. Although the island is considered to have a high-income status for the Caribbean, the unemployment rate is relatively high at 19% , 6.During the first year, approximately 20% of the population that was invited by postal mail completed a FIT . For scrThe novel setting on Cura\u00e7ao and low participation rate raise questions on how the target population possibly makes decisions on participating in CRC screening. Therefore, this study aims to gain insight into: (i) the target population\u2019s awareness and perceptions of CRC (screening), (ii) their beliefs on the provision of the CRC screening program, (iii) their preferences regarding decision-making on CRC screening and (iv) what information they need to make an informed decision. The outcomes can be used to improve communication strategies and support informed decision-making in CRC screening in Cura\u00e7ao and may also be relevant for other studies to build on.We conducted a qualitative study based on the grounded theory approach. Individual semi-structured interviews were carried out between September 2021 and February 2022. All interviews were scheduled and carried out by SB , a female PhD-student, who is fluent in English, Dutch and Papiamentu.To minimize the possibility of the shared opinions being influenced by personal experience with the screening program, those not yet invited for CRC screening  were identified in the FP database (Supplementary file 1). The \u201cset.seed\u201d function in R statistical programming was used to generate a random sample of 300, to ensure a large enough pool based on anticipated non-response and possible incomplete records. The FP database contains contact information. Of the 300 records, 164 did not include a phone number. Ultimately, phone calls were made to numbers belonging to 72 unique records. Snowball sampling was also used for additional interviews. During the initial phone call for invitation, the study aims and the interview procedure were explained and the status of program attendance was confirmed. If the individual agreed to be interviewed, an appointment was made at the FP main office, the respondent\u2019s home or, if desired, by phone. Informed consent was obtained prior to each interview.The interview topic-guide was developed by the research team (Supplementary file 2). The topic-guide consisted of five main domains and reflected the research goals of the study: CRC perception and awareness, perception and awareness of CRC-screening, CRC-screening provision, decision-making and information needs. All interviews lasted between 30\u00a0min to one hour and were recorded and transcribed verbatim in the original interview language. Subsequently, all non-English transcripts were translated into English. The English version of all transcripts were coded for analysis. During the interview period the research team met regularly to discuss the findings in order to ensure the process of reflexivity.All transcripts were coded with MAXQDA software. First, transcripts were read and re-read by SB to become familiar with the data. Then a selection of transcripts were open coded by SB and MF, independently. Subsequently, themes were classified into code trees per research question. The final code trees were discussed and agreed upon by SB and MF. To ensure trustworthiness, each interview was then uniformly coded with the code trees by SB and TV, independently. No major discrepancies emerged from the comparison.A total of 20 consecutive interviews resulted from the recruitment phone calls. An additional three interviews were carried out based on snowball sampling, resulting in a total of 23 interviews. The last five interviews did not introduce any new topics and therefore data saturation was observed.In total 13 men and 10 women were interviewed . Most respondents did not know that the FIT is the primary screening method and that the colonoscopy is only performed in case of a positive FIT. Furthermore, colonoscopy was also described as potentially painful Table\u00a0, Q03.In general there seemed to be no apparent rationale behind the decision to participate in CRC screening. The respondents did not state any clear motivations or mentioned positive beliefs when asked whether or not they would participate Table\u00a0, Q01.In some cases the presence of physical symptoms related to the intestines was perceived as a prompt to participate in CRC screening. This thought process indicated that the decision was not always completely unconscious.Having autonomy was described as paramount to several respondents, as individuals ultimately want to make their own choices. The respondents clearly indicated that they really did not want to be influenced by others when making a decision related to their physical health, because participating should be a personal choice.Although the ultimate choice was made alone, it was noted that the experiences and opinions of family members and peers were also taken into consideration. Consulting with family members and peers was seen as valid when weighing options Table\u00a0, Q02-05.The use of media  were mentioned as the preferred way to receive information and also seen as an effective method to inform others.Respondents also mentioned that a multi-facetted approach, repetition and representation can be important tools when attempting to inform the public about CRC screening.A personal approach was suggested as support to media coverage. For example a conversation with a FP employee, a phone call or text message. The use of lectures and visual tools were also suggested to be potentially helpful. Lastly, it was highlighted that information should be short, simple and phased Table\u00a0, Q01-03.Most respondents indicated that providing the information in plain and intelligible language was of the utmost importance. This mainly concerns background information about what CRC entails, CRC epidemiology, risk factors for CRC and symptoms of the disease. In addition, the respondents indicated that an explanation about the screening process would also be useful. For example by making it clear that the first step of the CRC-screening program is the FIT and not the colonoscopy Table\u00a0, Q04.Our interviews revealed that perceptions of cancer, CRC and CRC screening are embedded in the larger context of personal health and prevention, where food plays an important role. Health maintenance is considered as a personal responsibility. Fear of being sick, the potential social implications of cancer (shame and taboo) and machismo among men seem to be important barriers to screening. Beliefs on the provision of CRC screening are positive and related to trust in the FP. Autonomy in decision-making is highly valued, whereby advice from others is appreciated. The need for information mainly relates to lack of knowledge on screening procedure. A personal approach, visual aids and  media were reported to be the preferred means of receiving information.The perceived connection between cancer, CRC (screening) and personal health maintenance is interesting. A systematic review on barriers and facilitators of CRC screening reported that the desire to stay healthy and have peace of mind are motivators for screening . Our resFood was seen as a true determinant of overall health, both capable of healing and the contrary, illness induction. Cultural beliefs surrounding food may play a role. Interviews about perceptions on CRC (screening) with 147 Puerto Ricans and Dominicans living in Rhode Island also revealed that the thought that consuming \u201cbad food\u201d potentially caused CRC was common . In a smThe fear of being diagnosed with cancer and of the unknown mentioned during the interviews seem to be primarily caused by negative beliefs on the consequences of cancer, such as death and the unpredictability when it comes to treatment. A systematic review and meta-synthesis on CRC screening participation described such negative beliefs as barriers to screening . In a laTaboo and shame were brought up in relation to how others may perceive a cancer diagnosis. This points to the anticipation of social stigma. Perceived stigma is a widely discussed topic in cancer literature, and has been related to depression and decreased quality of life , 23. ConIn our study, machismo seemed to be a barrier to screening for men. This is in line with previous findings based on interviews with Mexican Americans . The potAs for the offer of CRC screening, the sentiment was generally positive and participation was seen as an opportunity to improve health. A positive view on cancer screening has also been observed in other studies that focused on the perceptions of cancer screening target populations. Mainly because early detection was considered to offer a better chance of recovery and would usually require less treatment \u201338. DespThe positive beliefs also seem related to the trust that respondents had in the FP. The FP has history with the community. It is the only screening organization on the island. The organization has provided free screening for breast and cervical cancer since 2010 and 2014, respectively . The truHowever, trust in health care organizations is not self-evident, especially not in Caribbean populations. An evaluation of the health care system performance on vector borne diseases in Cura\u00e7ao found that lack of coordination between the government and non-governmental organizations had negative effects . This maThe expressed desire to make decisions independently may also be related to the past colonialism. This manifests itself in a need for control over one\u2019s own body and freedom of choice in making decisions about health. The need for autonomy may also be related to fears about cancer, its treatment and cancer detection discussed above.To make autonomous informed decisions certain skills are necessary. Namely, the ability to find, understand and apply information. These are called health literacy skills , 46. To In line with our findings, a Jamaican survey study exploring knowledge and attitudes on the concept of CRC screening of 324 people found that only 14% of the subjects had not heard of CRC screening, but 69% stated that they did not know enough about it, with 32% not being familiar with any screening test . In a DuA distinct strength of our qualitative semi-structured interviews is the unfiltered insight into the thought process of these members of the target population. Additionally, similar numbers of men and women were interviewed, coming from various social backgrounds, thereby offering variability within the group.Because interviews were only conducted with people who explicitly consented after being informed of the purpose, the study results may be biased in favor of those who are more health-conscious, have a more positive attitude towards CRC screening and trust in the FP than those who did not consent. In addition to a positive attitude, it seemed that our respondents did not perceive any downsides to CRC screening. However, to reach an informed decision the pros and cons should be weighed. Furthermore, an interview with a medical doctor (SB) may have been a source of response bias where participants gave socially acceptable answers due to who was asking the questions, while knowing that a scientific research paper would be written. Respondents were encouraged to speak freely. They were informed that the goal was to explore their perceptions, awareness and possible insights on why the target population would (not) participate in CRC screening.Some aspects of our study were dually strengths and weaknesses. The fact that we interviewed individuals who had not yet participated in screening meant that some of the answers were hypothetical, which is a possible limitation. Simultaneously, it offered the opportunity to hear perspectives unbiased by experience with the program, which is also a strength. Respondents often voiced their observations through a generalist lens, commenting primarily on the behaviors and perceived thought processes of others within society. The strength is the insight on how they perceived themselves in relation to others, as it suggests that they may feel disconnected from their peers. Overwhelmingly, the opinions of respondents on others were not positive, suggesting that respondents saw themselves as somewhat better. The limitation in this form of communication is that it may leave room for interpretation. The opinion of someone else could purely be a reflection of how they perceive others or their own disguised opinion about an issue. The interviewer usually tackled this by asking directly what made the interviewee different from others. Furthermore, in our results we limited the inclusion of respondent opinions on others. We did however, include these perceptions when relevant.Personal health maintenance in relation to screening can be covered in information materials, while addressing possible fears. For example by highlighting favorable survival outcomes when CRC is detected early . It is iThe current study has provided useful insights, but has also made clear that there are still areas to be explored. Further insight is needed on how masculine ideals may influence CRC screening participation. It would also be interesting to investigate how health literacy plays a role in CRC screening on Cura\u00e7ao. Additionally, quantitative research to investigate how and to what extent the identified factors play a role in decision-making in CRC screening maybe useful.The insights gained about decision-making in our study could also be beneficial to other Caribbean islands, where they have not yet implemented organized CRC screening. The population of the Caribbean totals about 44\u00a0million . In addiTo effectively inform the target population and assist informed decision-making, CRC screening should be framed within the broader perspective of health on Cura\u00e7ao with sensitivity to possible fears and the expressed preference for independence in decision-making. Social media could be applied to disseminate targeted information about the CRC screening program and support invitees in their decision-making process.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5"}
+{"text": "The artillery firing process will instantly produce high-temperature and high-pressure gunpowder gas, this process will produce shock waves. The gunpowder gas has a limited effect on the projectile during the firing and ballistic after-effects period, however, it has a very obvious effect on the stability of the gun body, and the reduction of the stability of the gun body directly affects the firing accuracy and the safety of the firing personnel. Based on the method of Computational Fluid Dynamics (CFD), numerical simulation is carried out, and the structure and flow parameters of the muzzle flow field are obtained by using three-dimensional Euler's control equation, gas equation of state, and k-epsilon model, as well as dynamic mesh technology. By comparing the flow parameters of the brake before and after optimization, and analyzing the results obtained from the 8-round firing experiments, the efficiency of the optimized brake is increased by 8.2%, and the deviation between the experimental data and the simulation results is only 10.5%, which not only verifies the accuracy of the numerical simulation calculations but also verifies the optimized brake's good retracting effect. The results of the study can provide a reference for the optimization and design of the double-chamber brake. Generally speaking, the role of the muzzle brake has two main points one is to reduce the recoil kinetic energy. When the recoil part of the mass and recoil length is certain, it can reduce the force on the gun frame when firing, thus reducing the longitudinal size of the gun frame and reducing the weight of the gun; or when the recoil resistance is certain, then shorten the recoil length. The second is to use a uniform gun mount. To install the gun body with different power on the same gun mount, from the mechanical point of view, it is only necessary to ensure that the free recoil kinetic energy is equal.5 are obvious, and the recoil impulse can reach more than 20% of the overall recoil impulse of the gun. By distributing the flow of gunpowder gas in the after-effect period10 hanging the direction of air velocity to produce an overall forward impulse, this impulse can provide a recoil force to the gun body and reduce the recoil impulse generated by the corresponding combined force of the gun bore to reduce the firing load to achieve the recoil effect, which effectively improves the firing accuracy18.However, the aftereffects of the gunpowder gas on the gun body during the after-effects period21 to reveal the flow field changes caused by the muzzle retractor. In the last decade or so, computational fluid dynamics (hereafter referred to as \"CFD\")28 has developed greatly, replacing some approximate computational and graphical methods in classical fluid dynamics. All problems involving fluid flow30, heat exchange, molecular transport31, and other phenomena can be analyzed and simulated almost by means of computational fluid dynamics. CFD is being used to solve a lot of difficult real-world problems34, and is playing an important role as a research tool in aeronautics, nano-materials36, national defense, etc.38.Due to the complexity of the structure of the gun itself and the difficulty in mastering hydrodynamics, as well as the large amount of human and material resources required to perform weapon testing in the near-flow field and far-flow length, the validation cost is very high. There are very few calculations and simulations on double-chambered brakes and fewer experimentsUsing the software Ansys Fluent in Computational Fluid Dynamics, with the help of Ansys Fluent, you can create advanced physical models and analyze a variety of fluid phenomena so that all the work is done in the same customizable intuitive space. Currently, complex simulation tasks such as downscaling, multiphase flow, fluid\u2013solid coupling, etc. are accomplished in this software, which has been widely recognized.In this paper, taking the double chamber brake as the research object, numerical simulation of artillery firing with or without muzzle brake is carried out by establishing a mathematical-physical model based on the method of fluid computational mechanics. According to the simulation results to optimize the design, and finally,the optimized numerical simulation results are compared with the experimental results to verify the accurate availability of the numerical simulation simulation calculation.Figure\u00a0As shown in Fig.\u00a0The adjustment of the angle between the side aperture and the bore axis of the optimized type of brake makes the expanded powder gas more efficiently discharged to the side aperture, reduces the backward axial reaction force and further increases the forward lateral reaction force, which is a reference value for the study of the improvement of the efficiency of the brake.A model of the computational grid area is shown in Fig.\u00a0As in Fig.\u00a0The flow is quasi-constant.The flow is one-dimensional, even if the cross-sectional area of the flow varies, the radial fractional velocity of the flow and the effect of the inhomogeneity of the flow parameters in the cross-section are corrected by the corresponding coefficients.The flow is isentropic, i.e., the heat exchange between the airflow and the chamber wall is neglected, and the friction caused by the viscosity of the gas is neglected. The resulting errors are also corrected by the coefficients.The gunpowder gas is regarded as a complete gas  and the mass force is neglected.In order to simplify the problem of airflow in the gun chamber during the after-effect period, the following assumptions are introduced.Under the above assumptions, the three-dimensional constant isentropic flow theory in gas dynamics can be applied to study the flow-void problem of gunpowder gas in the post-effect period.According to the above assumptions, the Euler equation for three-dimensional non-constant compressible flow in the gun bore can be established under the premise of continuous medium mechanics.Among them:\u03c1 is the gas density; E is the total energy of the gas; u, v, w is the velocity component of the gas along the x, y, z direction, respectively; p is the gas flow pressure; \u03b3 is the specific heat ratio; \u03b3 is the enthalpy of the gas flow; c is the local speed of sound.The equation of state and auxiliary equations are:In this paper, the coordinate system is established with the projectile launch direction as the positive direction of the coordinate axis and the moment of projectile movement to the rifling port as the initial time, i.e., T\u2009=\u20090.The equation of the variation law of gas density inside the gas conduction chamber:Pressure change equation of gas chamber:Equation of state:The meaning of each symbol in the formula is:Gas pressure calculation mainly requires solving five unknown quantities of pressure, temperature, density,velocity of the piston and displacement, here we establish a mathematical model by gas motion and piston motion for solving. In this paper, we take time T as the independent variable and the dependent variable as the previous five unknown quantities and establish the equation for the joint solution. The expressions are as follows:Under the assumption of continuous medium mechanics, airflow can be described by the three-dimensional Euler equation with the gas equation of state. In calculating flow under hypersonic conditions, the Reynolds number is so high that the viscosity of the gas can usually be neglected. The flow of fluids is governed by physical conservation laws, and the above control equations are mathematical descriptions of these conservation laws. Computational mechanics of fluids is based on the above control equations to accurately calculate the flow of fluids.Shape function:Combustion velocity equation:Projectile equation of motion:\u03c8:Equation of motion expressed in terms of average pressure and secondary work factor Basic equations of internal ballistics:This simulated gun uses a single charge, and the set of ballistic equations within the single charge can be summarized as follows:In this paper, the rifling end face is set as the entrance boundary, and the initial conditions and flow parameters of the powder gas are obtained by solving the internal ballistic program. The projectile trajectory equations are given by the UDF preparation. The exit boundary is set as a pressure exit and the rest is given as a solid wall boundary. The internal ballistic time velocity curve and time rifling pressure curve are shown in Figs. Combining the results of the internal ballistic calculation above and the model of the computational grid area in Fig.\u00a0Numerical calculations based on the computational fluid dynamics method are carried out to obtain the bore flow parameters and the bore side aperture and central bullet aperture airflow parameters based on the computational fluid dynamics method to calculate the bore efficiency in the after-effect period of different schemes.As shown in Fig.\u00a0Figure\u00a0Figure\u00a0As shown in Fig.\u00a0As shown in Fig.\u00a0As shown in Fig.\u00a0As shown in Fig.\u00a0As shown in Fig.\u00a0From Fig.\u00a0From Fig.\u00a0As can be seen from Fig.\u00a0As shown in Fig.\u00a0The experimental apparatus available to the public for this shooting experiment is shown in Fig.\u00a0A certain type of cannon was used in this shooting, with a maximum range of 15650\u00a0m.The maximum range was 15,650\u00a0m, with a high and low firing angle of -7\u00b0 to\u2009+\u200935\u00b0, an average firing rate of 15\u201320 rounds/min, a full marching weight of 1725\u00a0kg, a 54\u00b0 directional firing range, and a gun crew of 8.A schematic diagram of this shooting experiment is shown in Fig.\u00a0The experimental method was to clamp the gun launcher on a special fixed stand and use a pull cord to fire. The laser vibrometer was used to simultaneously measure the recoil speed, initial velocity, and fall speed of the recoil body. High-speed photography of the airflow and body tube movement was also conducted.Table As Fig.\u00a0Figure\u00a0From the momentum theorem, the free recoil body momentum during the internal ballistic period is obtained as:The free recoil impulse during the after-effect period is:Then the total momentum of the recoil body free recoil is:There are two methods of calculating the efficiency of the braking brake: the energy method and the impulse method. For small-caliber weapons, the influence of the braking brake quality factor is greater, and the impulse method is mostly used for calculation. The momentum method is used to calculate the efficiency of the brake as followsIn this paper, the momentum method is used to calculate the efficiency of the brake for each scheme.Calculate the decommissioning efficiency of the conventional type decommissioner as:Calculate the decommissioning efficiency of the optimized decommissioner as:According to Table The optimized side aperture exit cross-section pressure is significantly lower than the optimized exit cross-section pressure, and the reduction of wall pressure after the improvement indicates that the optimized recoil-making efficiency is effectively improved.The optimized type of recession maker makes the recoil impulse of the recession maker effectively reduced compared with that before the improvement, and the recession maker efficiency is significantly better than that of the conventional double-cavity recession maker.The pressure of the first side aperture of the double-chamber brake is much greater than that of the second side aperture, and the impact on the first side aperture is significantly greater during the firing process.The recoil velocity and displacement obtained from the experimental apparatus under good meteorological conditions, four rounds of firing experiments were conducted on each of the guns equipped with the conventional and optimized type of recoilers, and it is obvious that the optimized type of recoilers reduces the recoil displacement and the recoil velocity significantly, which effectively reduces the recoil impulse.The difference between the numerical simulation efficiency and the experimental efficiency of the conventional type recoil brake is 6.3%, while the difference between the numerical simulation efficiency and the experimental efficiency of the optimized type recoil brake is 10.5%, which is generally in line with the expected assumption.After the gun is fitted with the double chamber brake, the maximum recoil velocity is effectively reduced, and the resulting recoil impulse and overall recoil impulse. The forward impulse generated by the recoiling body provides a restraining force on the gun body, so that the recoil impulse generated by the combined force of the gun bore is reduced, thus effectively reducing the firing load and gun recoil kinetic energy acting on the gun frame.Through the use of experimental equipment in the case of good meteorological conditions, assembled with the traditional and optimized brake in each of the four rounds of shooting experiments, the recoil velocity and displacement can be intuitively seen in the optimized brake so that the recoil body displacement is reduced, the recoil velocity is significantly reduced, and consequently effectively reduce the recoil impulse.In this paper, the air velocity and cross-sectional pressure of the first and second side orifices of the double-cavity brake were monitored and plotted numerically. After the shape optimization, the same numerical simulations were performed and compared with the pre-optimization one by one. After that, the recoil generator before and after optimization was subjected to a four-round firing experiment using a high-speed camera and a laser vibrometer. The recoil velocity and displacement obtained from the experiments were plotted separately for visual comparison, and the efficiency was finally calculated by the momentum method. The following conclusions were obtained:In this study, numerical simulations and experiments of a double-chambered brake were conducted based on fluid computational mechanics. At present, there are fewer domestic and foreign studies on the flow parameters and optimization scheme of the double-chamber brake, and the ones that can be tested are even more valuable. In this paper, the use of computational fluid dynamics in the dynamic grid technology research, all grids using structural mesh, the efficiency and accuracy of the entire numerical simulation has a greater significance. The test shot after the numerical simulation has a positive significance on the design and optimization of the subsequent brake \u2018\u2019structure. And this study follows a more scientific and rigorous program in the writing process, the research method can provide a reference for the subsequent research on the retreating brake.The purpose of this study is to numerically simulate and test a double-chamber muzzle brake based on fluid computational mechanics. However, because the numerical simulation does not allow for a complete reduction of the model during meshing, the model is partially simplified, which can lead to an idealization of a portion of the data from the numerical simulation. Because of the difficulty of the tests, only eight shots were fired in this study, and it is hoped that in future research, more shots can be fired under more ideal conditions to make the test results more convincing. It is also hoped that future work will be directed toward the multi-component combustion of gunpowder and fluid\u2013solid coupling, which can make the research more cutting-edge and practical."}
+{"text": "To simplify sample preparation and monitor the DNC rapidly and accurately, a comparable icELISA and lateral flow immunoassay (LFIA) was developed in this study. Briefly, the reaction parameters were explored for improving the sensitivity of icELISA and LFIA. Under the optimal conditions, methanol was selected as the extracting solvent for DNC in chicken, and 20- and 10-fold dilutions of sample extraction eliminated the matrix effect for icELISA and LFIA, separately. After sample pretreatment, the analysis properties of icELISA and LFIA were compared. The limit of detection of icELISA for DNC was 0.8 \u03bcg/kg, and the visual and quantitative limits of detection of LFIA were 8 and 2.5 \u03bcg/kg. Compared with icELISA, LFIA showed lower sensitivity but obvious advantages in terms of matrix tolerance and detection time (within 15 min). The sensitivity, specificity, and accuracy of the developed assays satisfied the detection requirement even if using simple sample pretreatment. This comparable icELISA and LFIA provided mutual verifiability methods for the accurate detection of DNC in chicken. Coccidiosis is a common parasitic disease in poultry, which has negative impacts on the absorption of nutrients and feed conversion, and leads to loss of weight and even death of poultry animals . At presAt present, the analytical methods for DNC in chicken, liver, and eggs mainly depend on instrumental analysis methods, including high-performance liquid chromatography, liquid chromatography\u2013tandem mass spectrometry, and gas chromatography . AlthougImmunoassays, based on the specific recognition of antibodies and antigens, are consistently considered the traditional rapid detection methods with high specificity and satisfactory accuracy . CurrentIn this study, we explored the influence mechanism by which reaction parameters  affect the sensitivity of the analysis method. We further evaluated the matrix effect of chicken and developed a sample pretreatment method for DNC using extraction and dilution procedures, which reduced the analysis time. Under optimal conditions, icELISA and LFIA were established with improved sensitivity and could satisfy the detection requirements of the maximum residue limit. Meanwhile, we compared the detection parameters  between icELISA and LFIA. This study provides two comparable analysis methods for the rapid, sensitive, and accurate detection of DNC in food samples.2CO3, methanol, acetonitrile, and other chemical reagents were provided by Sinopharm Chemical Reagents Co., Ltd. . The chicken sample was purchased from Darunfa Market  and was verified to be negative by liquid chromatography\u2013mass spectrometry.Bovine serum albumin (BSA), tetramethylbenzidine, and horseradish peroxidase were obtained from Merck . Goat anti-mouse IgG was purchased from Shanghai Kinbio Tech. Co., Ltd. . Nicarbazin was obtained from Biovet JSC . Chloramphenicol, trimethoprim, and sulphanilamide were purchased from J&K Scientific Ltd. . Coating antigen and monoclonal antibody 14D2 were prepared and provided by the Xiya Zhang research team at Henan Agricultural University. Trisodium citrate, aurichlorohydric acid, KNitrocellulose membrane (Millipore 135) was obtained from Millipore AG . A 96-well microplate was purchased from Yunpeng Technology Development Co., Ltd. . Blood filtration membrane, polyvinyl chloride plates, and absorbent paper were purchased from Shanghai Kinbio Tech. Co., Ltd. . A Milli-Q Ultrapure water meter was obtained from Millipore . An HQ-6-\u2161 vortex mixer was bought from Beijing North Tongzheng Biotechnology Development Co., Ltd. .2SO4  was added to stop the reaction, and the OD values at 450 nm were measured. The schematic illustration of icELISA for DNC detection is shown in The ELISA plates were first coated with coating antigen (100 \u03bcL/well) which was diluted with carbonate buffer solution (pH 9.6) and then incubated at 37 \u00b0C for 2 h. A washing solution (280 \u03bcL/well) was added, and the ELISA plates were washed 3 times. Subsequently, a blocking buffer (150 \u03bcL/well) was added and incubated for 1 h at 37 \u00b0C. The ELISA plates were washed by washing buffer three times and stored in the refrigerator before use. The standard solutions of DNC  and mAb (50 \u03bcL/well) were added to the ELISA plates and incubated for 30 min at 37 \u00b0C. After three washings, horseradish peroxidase-labeled IgG (HRP-IgG) was added to the ELISA plates, incubating for 30 min at 37 \u00b0C. Then, tetramethylbenzidine that acted as the substrate (100 \u03bcL/well) of horseradish peroxidase was added into wells and incubated for 15 min at 37 \u00b0C after washing. Finally, HTo improve the sensitivity of icELISA, we optimized several crucial factors during the specific recognition of the antibody and antigen, including antigen and antibody concentrations, pH values and ion strength of buffer solution, and organic solvent tolerance.A checkboard assay was preliminarily adopted to evaluate the antigen and antibody concentrations. The antigen concentrations were set as 0.4, 0.2, 0.1, and 0.05 \u03bcg/mL, and the monoclonal antibody (mAb) 14D2 ones were 6.25, 12.5, 25, 50, 100, and 200 ng/mL, respectively. The DNC concentrations were 0 and 0.5 ng/mL when exploring antigen and antibody pairs at different concentrations.Phosphate buffer solutions (PBS) at different pH values  and at diverse ion strengths  were chosen as the dilution buffers of the antigen and antibody. Simultaneously, the DNC concentrations were prepared to yield standard curves, with the eight dilution gradients of 0, 0.01, 0.03, 0.09, 0.27, 0.81, 2.43, and 7.29 ng/mL.50) of ELISA was calculated.Two commonly used organic solvents (methanol and acetonitrile) were used as the dilution buffer of the DNC, then the standard curves were constructed. The concentrations of methanol and acetonitrile were 0%, 5%, 10%, 15%, and 20% . Under the optimal conditions, the standard curve of DNC was fitted in the buffer solution using a four-parameter equation (Origin Software 2019). The maximal inhibition concentration (IC4\u00b74H2O (w/v) was added under a magnetic stirrer until heated to 100 \u00b0C. Then, 50 mL trisodium citrate solution  was added into the mixture solution and heated for 30 min, until the color of the mixture turned to claret. Finally, the AuNPs were cooled and stored at room temperature. AuNPs were characterized by UV\u2013visible spectroscopy, transmission electron microscopy, and dynamic light scattering.AuNPs were synthesized according to previous research . Briefly2CO3 (0.1 M) for adjusting the pH values of AuNPs. The mAb 14D2, diluted with pure water, was conjugated to AuNPs via electrostatic adsorption [AuNPs (1 mL) were placed into a centrifuge tube (1.5 mL), followed by adding Ksorption . After iFirst, goat anti-mouse IgG and coating antigen were coated onto the nitrocellulose membrane as the control (C) line and test (T) line, respectively. Then, the nitrocellulose membrane was incubated in an oven at 45 \u00b0C for 2 h. Subsequently, the nitrocellulose membrane, sample pad, and absorbent paper were successively pasted onto the polyvinyl chloride plates to assemble the test strips. The polyvinyl chloride plates were cut into test strips with a width of 3.18 mm by a cutting machine.The detection process for the lateral flow immunoassay was as follows: AuNPs\u2013mAb conjugates (4 \u00b5L) were placed onto the microplate, and then DNC solution  was added and mixed thoroughly for 5 min. The test strip was inserted into the mixture solution, and then, the test strip was observed by the naked eye and measured by Image-J software after 5 min of incubation. The schematic illustration of LFIA for DNC detection is shown in 2CO3 solution (0.1 M) at different volumes  was added to adjust the pH values of AuNPs. The concentrations of mAb 14D2 were set as 3, 5, 7, and 9 \u00b5g/mL, and then AuNPs were labeled with the antibody. The mAb 14D2 was diluted with three buffers, PBS , Tris-HCl buffer , and 1% BSA buffer . Then, the volumes of AuNPs\u2013mAb conjugate were 2, 3, 4, and 5 \u00b5L, respectively. Finally, the antigen at different concentrations  was explored. These parameters were optimized using monofactor analysis [Several key parameters for LFIA were optimized, such as pH values of AuNPs, antibody and antigen concentrations, dilution buffer types of antibodies, and the amount of AuNPs\u2013mAb conjugate. In detail, Kanalysis . Under oDNC-negative chicken (1 g) was homogenized and extracted with methanol (2 mL). The mixture was vortexed for 10 min as well as centrifuged at 9168 g for 10 min, and the supernatant was collected. Subsequently, the extract solution was diluted to different folds with optimal buffer solution and was then used to generate matrix standard curves.10 of the matrix standard curve that could eliminate the matrix effect was calculated as the limit of detection for the ELISA [10 of the matrix standard curve acted as the quantitative limit of detection.The IChe ELISA . For LFINon-target veterinary medicines were spiked into negative chicken with a concentration of 1000 \u03bcg/kg. After the sample pretreatment, the extract solution was measured by ELISA and LFIA.DNC at different concentrations was spiked into negative chicken. The recovery ratio was calculated according to Formula (1).2SO4. Thus, the substrate solution changed from dark yellow to light yellow with the increased concentration of DNC , lower IC50 value, higher inhibition ratio, and Amax/IC50 values were considered the optimal conditions.The parameters for optimizing icELISA were mainly evaluated by optical density (OD) values, inhibition ratio, ICmax) was lower than 1.2. We abandoned the above concentrations as the optimal condition because of the poor stability of the OD value. By contrast, the satisfied inhibition ratio and OD value were observed at the concentrations of 12.5 ng/mL and 0.05 \u03bcg/mL for the mAb and coating antigen.Concentrations of coating antigen and mAb were closely related to the sensitivity of icELISA, and thus, these parameters were primarily analyzed in the conventional icELISA . The OD 50 was the lowest (0.05 ng/mL) at the pH value of 6.0; however, the Amax and Amax/IC50 were 0.6 and 12.5, respectively. Actually, it was not enough to comprehensively express the sensitivity of icELISA only by IC50 value. The Amax value was 1.33, and the IC50 value was low, which generated the largest Amax/IC50 value at the pH of 7.0. Different concentrations of ionic strength  were prepared, ranging from 0.07 to 1.00 mol/L. A significant decrease in Amax was observed when ionic strength varied from 0.14 to 1.00 mol/L  was 0.02 ng/mL in the buffer solution.Under the above optimal conditions, a standard curve in the buffer solution was generated with the increase of DNC concentrations a. A fourThe maximum absorption peak of AuNPs was 530 nm measured by the UV\u2013visible spectra a, which In our study, several parameters were optimized to explore the sensitivity of LFIA by the single-factor variable method. The optimal parameters were determined by evaluating the peak area of the T line of the test strip and calculating the inhibition by a two-point (or three-point) competitive format. Generally, the higher peak area and inhibition ratio indicates superior parameters. But there are concrete analyses of concrete conditions.2CO3 (0.1 M). The color intensity of the T line gradually turned darker and then lighter with the increase in the K2CO3 amounts  of mAb might be not suitable for the labeling procedure of LFIA. Hence, we compared the effects of three different buffer types on the color intensity and sensitivity for LFIA. As shown in With the increase in the AuNPs\u2013mAb amount, the color intensity of the T line for negative control became gradually deeper . The colThe concentration of coating antigen also played a key role in the establishment of LFIA, and its evaluation method was similar to the above analysis method. It was observed that the higher the coating antigen concentration, the deeper the color intensity of the T line, and this led to a decrease in sensitivity g. A dynaWe adopt a simple sample pretreatment method to save testing time in this study. In detail, the chicken was extracted with methanol, and the extraction was diluted with a buffer solution, aiming to eliminate the matrix effect. Generally, the standard curve in diluted extraction matches well with that in the buffer solution, which means the matrix effect could be eliminated .As shown in With the increase in the dilution fold of extraction, the color intensity of the T line showed almost the same when the DNC concentration was 0 ng/mL c; howeveWe systematically investigated the sample pretreatment of icELISA and LFIA. It was observed that a 10-fold dilution of sample extract solution could eliminate the matrix effect of chicken for LFIA; however, a double dilution fold was required for icELISA. Although the same mAb and coating antigen were applied for both icELISA and LFIA, the LFIA exhibited higher matrix tolerance to the sample extract of chicken. We considered that the difference in mAb and coating antigen concentrations and the activity of horseradish peroxidase influenced the matrix effect. The higher concentration of mAb and coating antigen might improve the matrix tolerance of LFIA. Despite possessing higher sensitivity for icELISA, the trace concentration of mAb and coating antigen was more likely to be interfered with by the matrix factor. Moreover, the optical density of icELISA was produced by horseradish peroxidase catalyzing tetramethylbenzidine. The activity of horseradish peroxidase was significantly interfered with by the matrix factor , and a hTo decrease the matrix effect of samples, some researchers have tried different sample pretreatments . Xu et aAs reported, the immunoassay usually has the advantage of high specificity, because of the precise recognition of mAb and antigen . Here, tIn summary, the comparable icELISA and LFIA were developed as efficient and straightforward tools for DNC screening in chicken. Owing to the signal amplification of horseradish peroxidase-catalyzed tetramethylbenzidine, the icELISA exhibited relatively high sensitivity (at least eightfold) compared to LFIA; however, it also displayed low tolerance of the chicken matrix using the same sample pretreatment procedure. After sample pretreatment, the limit of detection of the icELISA for DNC was calculated to be 0.8 \u03bcg/kg with an average recovery of 68.5\u201387.4% and a coefficient of variation of less than 12.3%. The visual and quantitative limits of detection of the LFIA for DNC were 8 and 2.5 \u03bcg/kg, which could be detected within 15 min. Meanwhile, the average recovery of the LFIA ranged from 74.2% to 93.2%, and the coefficient of variation was less than 9.3%. Overall, the established icELISA and LFIA met the requirement of rapid detection in terms of sensitivity, specificity, and accuracy. They also provided mutually verifiable assays for the detection of DNC in samples, which could improve the accuracy of the detection results."}
+{"text": "A considerable number of patients with colorectal cancer (CRC) do not have elevated serum carcinoembryonic antigen (CEA) levels before undergoing radical surgery for colorectal cancer, but they may still have a poor prognosis. It is imperative to find sensitive and dependable prognostic markers to quickly identify high-risk populations who are susceptible to adverse outcomes in order to offer timely interventions to improve prognosis. In this real-world study of more than 1000 samples, a nomogram based on serum tumor markers and other clinicopathological features was used to accurately forecast the 3-year and 5-year overall survival rates of stage I\u2013III CRC patients after radical resection with normal preoperative CEA, and its predictive power and clinical applicability markedly exceed those of the AJCC 8th TNM stage. In addition, we found the potential prognostic values of carbohydrate antigen 125 (CA125) and carbohydrate antigen 242 (CA242) as supplementary tumor markers in CRC patients who have normal preoperative CEA.We aimed to develop a clinical predictive model for predicting the overall survival (OS) in stage I\u2013III CRC patients after radical resection with normal preoperative CEA. This study included 1082 consecutive patients. They were further divided into a training set (70%) and a validation set (30%). The selection of variables for the model was informed by the Akaike information criterion. After that, the clinical predictive model was constructed, evaluated, and validated. The net reclassification index (NRI) and integrated discrimination improvement (IDI) were employed to compare the models. Age, histologic type, pT stage, pN stage, carbohydrate antigen 242 (CA242), and carbohydrate antigen 125 (CA125) were selected to establish a clinical prediction model for OS. The concordance index (C-index)  indicated that the nomogram had good discrimination ability. The decision curve analysis highlighted that the model has superior efficiency in clinical decision-making. NRI and IDI showed that the established nomogram markedly outperformed the TNM stage. The new clinical prediction model was notably superior to the AJCC 8th TNM stage, and it can be used to accurately assess the OS of stage I\u2013III CRC patients undergoing radical resection with normal preoperative CEA. Colorectal cancer (CRC) stands as a predominant gastrointestinal malignancy, ranking third in global incidence and second in global mortality . In receIn recent years, research prospects have seen a growing interest in the prognostic potential of serum tumor markers other than CEA. A recent study  found thTNM staging has been widely used for prognostic evaluation. This staging system, which takes into account the depth of invasion, the extent of lymph node metastasis, and so on, serves as a predictive tool for the prognosis of CRC patients. However, other possible prognostic factors, such as age, histologic type, serum tumor markers, and so on, were not considered in this staging system, which may limit the accuracy of prediction to some extent ,25. The In our research, we retrospectively analyzed the association of 15 clinicopathological features, including 7 serum tumor markers, and the prognosis of stage I\u2013III CRC patients after radical resection with normal preoperative CEA. Finally, six variables  were chosen to develop the clinical prediction model, and its predictive value was further compared with that of the TNM stage. The results show that the nomogram exhibited a robust predictive capacity. Furthermore, the superiority of the model was further demonstrated by comparing the TNM stage using NRI, IDI and decision curve analysis (DCA).n = 758) and a validation set of 30% (n = 324). All patients were staged according to the latest NCCN guidelines. All patients included in the study underwent radical (R0) resection of the primary tumor. Adjuvant chemotherapy was administered according to NCCN guidelines to patients who met the criteria for postoperative adjuvant chemotherapy.This study encompassed consecutive patients who underwent radical resection of colorectal cancer in the Department of Colorectal and Anal Surgery, Xinhua Hospital Shanghai Jiao Tong University School of Medicine from January 2010 to August 2017 . The excPreoperative tumor markers were measured 1 week before radical surgery for CRC. The threshold values for marker positivity were set as follows: CA242 at 20 U/mL, CEA at 10 ng/mL, AFP at 7 ng/mL, NSE at 16.3 ng/mL, CA125 at 35 U/mL, and CA19-9 at 39 U/mL. For CA211, the cutoff was 3.3 ng/mL, and for CA724, it was 6.9 U/mL.Patients underwent quarterly follow-ups for the initial two years, biannually for the subsequent 3\u20135 years, and annually thereafter. Follow-up evaluations included physical examinations, a serum CEA and CA19-9 test, a chest CT scan, and an abdominal and pelvic MRI or CT scan. A colonoscopy was performed every year. The definition of overall survival (OS) was the period from radical resection to either any-cause mortality or the last follow-up. The follow-up evaluation of this study ended on 1 August 2022.2 test or Fisher exact test. Continuous variables have been presented as mean \u00b1 standard deviation or median (interquartile range (IQR)), and they were compared using the independent sample t-test or the Mann\u2013Whitney U test. Patients included in the study were randomly divided into a training set (70%) and a validation set (30%). The Kaplan\u2013Meier method and log-rank test were employed to evaluate and compare the OS curves of each group. Prognostic factors associated with OS were selected by Cox proportional hazards analysis. Significant variables with p < 0.050 in univariate analysis were included under the multivariate Cox regression analysis. We selected variables for inclusion in the nomogram by utilizing stepwise regression according to Akaike\u2019s information criterion (AIC). The probability of 3- and 5-year OS was predicted using the nomogram. Internal validation of the nomogram was conducted based on the validation set. The discrimination ability was evaluated by the concordance index (C-index) and the receiver operating characteristic (ROC) curve. The calibration plot was employed to assess the calibration capacity. NRI and IDI are two mutually complementary validation methods. NRI primarily serves the purpose of comparing the predictive capacity of the old model with the new one. IDI is mainly utilized to examine the overall improvements in the model, assessing its overall performance enhancement. NRI(IDI) > 0\u2014the new model has a better predictive ability compared to the old model; NRI(IDI) < 0\u2014the new model has a poorer predictive ability compared to the old model; and NRI(IDI) = 0\u2014the new model does not provide any predictive improvement compared to the old model. DCA serves as a tool to evaluate a model\u2019s clinical utility, quantifying the net benefit at different threshold probabilities. The curves representing full patient treatment, denoting maximum clinical benefits, and representing no treatment, denoting zero clinical benefit, were employed as benchmarks. Risk stratification was performed based on the median of the risk group scores in the training set to test the differentiation ability of the nomogram.Categorical variables were compared using the \u03c7http://www.R-project.org/ accessed on 26 November 2023). The R packages used in our study are shown in the p < 0.050.All statistical tests were performed using SPSS  and R for Windows . The median age at operation of CRC patients with normal and elevated preoperative CEA stood at 65 years (interquartile range (IQR): 57\u201375) and 65 years (IQR: 59\u201376), respectively. CRC patients with normal preoperative CEA were randomly divided into a training set (758 cases) and a validation set (324 cases). p = 0.016). Except for the pT stage, all variables displayed no notable disparities between the two sets (p > 0.050).Of the 1359 patients who underwent radical surgery for CRC, 1082 had normal preoperative CEA, while 277 presented with elevated preoperative CEA. The CEA-negative group boasted a 5-year OS rate of 80.04%, markedly surpassing the 59.80% in the CEA-positive group (p > 0.050). The 5-year OS rate of the negative groups of the other five serum tumor markers  was better than that of the positive groups , histologic type , pT stage , pN stage , CA242 , and CA125  served as independent determinants for OS  were correlated with OS  . For thep < 0.010). These were also confirmed in the validation set , significantly surpassing that of the TNM stage (p < 0.001). The risk score grouping showed that the prediction model had good discrimination ability. In addition, the DCA revealed that our nomogram outperformed the TNM stage in clinical decision-making efficacy. The calibration curves for both groups demonstrated a strong alignment between the predicted and observed outcomes.Although the TNM staging system is important for prognosis, it may not be a comprehensive predictor of patient outcomes . To furtNRI and IDI were initially introduced to gauge the enhancement in precision upon the incorporation of new biomarkers into regression models to predict binary outcomes. Recently, these two indices have been extended from binary outcomes to multi-class and survival outcomes . In thisThe clinical prediction model we constructed showed the following advantages: At first, the proposed predictive model was applicable to stage I\u2013III CRC patients after radical resection with normal preoperative CEA and could reflect the prognosis of this population accurately. Secondly, the clinical prediction model we constructed was superior to the TNM stage, and it showed better prediction efficiency and clinical decision-making abilities. Thirdly, seven serum tumor markers were incorporated into this research, which fully assessed their predictive value for patients. Finally, serum tumor markers and other clinicopathological features are easily accessible indicators in clinical practice, and a nomogram based on these indicators might hold promising clinical applicability. However, our study has limitations. Firstly, due to the lack of multicenter study data, the nomogram was constructed and validated based on a single database. Therefore, the study is limited by single-institution experience and a lack of external validation. Secondly, the C-index of the nomogram in the validation set was 0.702, which can be used to assess the prognosis of patients with preoperative normal CEA and is smaller than that in the training set. This may be due to the different proportions of pT stage between the training and the validation sets. Thirdly, some factors that may be related to prognosis  were not taken into account. Finally, considering the difference in treatment methods between stage I\u2013III patients and stage IV patients, we did not include stage IV patients in the study, which limits the application of this nomogram in stage IV CRC patients.Compared with the AJCC 8th TNM stage, our clinical prediction model based on serum tumor markers and other clinicopathological features had more accurate predictive power and better clinical applicability, and it served as a potent tool for forecasting the postoperative overall survival outcomes of stage I\u2013III CRC patients after radical resection with normal preoperative CEA. This research offers a theoretical basis for the detection of other serum tumor markers in CRC patients, such as CA125 and CA242."
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