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+{"text": "The sperm penetration assay (SPA) is used to predict the fertilizing capacity of sperm. Thus, some programs rely on SPA scores to formulate insemination plans in conjunction with in-vitro fertilization (IVF) cycles. The purpose of this study was to evaluate if a relationship exists between SPA scores and polyspermy rates during conventional IVF cycles.A total of 1350 consecutive IVF patients using conventional IVF insemination were evaluated in the study. Oocytes were inseminated three hours post-retrieval by the addition of 150,000 to 300,000 progressively motile sperm. Approximately 18 hours after insemination, the oocytes were evaluated for fertilization by the visualization of pronuclei. The presence of three or more pronuclei was indicative of polyspermy. Polyspermy rates, fertilization success, embryo quality, and pregnancy rates were analyzed retrospectively to evaluate their relationship with SPA score, count, motility, number of progressively motile sperm inseminated, oocyte pre-insemination incubation time, patient age, and diagnosis.s = 0.10, p < 0.05). Patients with a normal SPA score had significantly higher polyspermy rates than those with abnormal SPA scores . Fertilization percentage was significantly lower in the group with severely abnormal SPA scores versus all other SPA groups . Although embryo quality was not affected, both clinical pregnancy and implantation rates improved slightly as SPA score increased. In addition, there was a decrease in the rate of spontaneous abortion as SPA score increased.A significant positive relationship was observed between SPA score and polyspermy rate (rThese data indicate SPA score is positively correlated with polyspermy rates and IVF fertilization percentage. Additionally, there is a slight increase in clinical pregnancy rates, and embryo implantation rates with increased SPA. Furthermore, there is a slight decrease in spontaneous abortions rates related to increased SPA. The sperm penetration assay (SPA) is used to evaluate the fertilizing capacity of human spermatozoa . Some inOver the past decades success rates have steadily improved for human IVF embryo transfer programs. Recent advances in ovulation induction, micromanipulation, culturing conditions, and media formulations have fostered a technological revolution for IVF leading into the 21'st century . However, many common problems still remain.One common problem associated with conventional IVF insemination is polyspermic fertilization when more than one sperm successfully penetrates and fertilizes the oocyte. These pre-embryos are considered abnormal and typically discarded. Thus, increased rates of polyspermy ultimately result in a reduced number of embryos.Studies have been conducted that evaluate the factors associated with human IVF polyspermy -5. HowevThe purpose of this study was to evaluate if a relationship exists between SPA scores and polyspermy rates during IVF cycles utilizing conventional insemination. We also evaluated the relationship between SPA score and fertilization rate, embryo quality, and pregnancy rate.After Institutional Review Board approval, we conducted a retrospective study of 1350 consecutive IVF patients using conventional IVF insemination. Intracytoplasmic sperm injection (ICSI) patients were excluded from the study. The fertilizing capacity of each patient was assessed with the SPA. The SPA was performed on patients undergoing IVF using techniques previously described . The SPADuring the IVF cycle ovarian stimulation was performed using standard techniques of gonadotrophin-releasing hormone (GnRH) agonist down-regulation combined with controlled ovarian stimulation using a combination of recombinant follicle stimulating hormone (rFSH) and urinary-derived gonadotropin stimulation. Ovarian follicles were aspirated using a trans-vaginal ultrasound-guided needle.Oocytes were inseminated three-hours post-retrieval by the addition of 150,000 to 300,000 progressively motile sperm. Approximately 18-hours post-insemination, the oocytes were evaluated for fertilization by visualization of pronuclei. The presence of three or more pronuclei was indicative of polyspermy in which case the fertilized oocyte was discarded. Embryos were cultured in HTF medium and transferred 72-hours post-retrieval. Embryo quality was assessed using a previously reported embryo scoring system that took into account the number of cells present and the level of cellular fragmentation [Polyspermy rates, fertilization success, embryo quality, and pregnancy rates were analyzed retrospectively with respect to their relationship with SPA score. The correlation between SPA score and polyspermy was conducted using Spearman's correlation coefficient. Additionally, polyspermy rates, fertilization percentage, and embryo quality for the different SPA groups were analyzed for statistical difference using Kruskal-Wallis analysis. Lastly, pregnancy rates in the different groups were evaluated with a Chi-square analysis.s = 0.10, p < 0.05). Patients with normal SPA scores (Group 3) had significantly elevated polyspermy rates over those with severely abnormal SPA scores  compared with the studies of van der Ven 06 sperm . Our resPoly-pronuclear formation in in-vitro fertilized eggs usually arises from polyspermic fertilization but may also be a product of second polar body retention . Thus, oConsistent with other reports, the SPA was correlated with fertilization percentage ,6. FurthDone correctly, the SPA provides a reliable assessment of the fertilizing ability of human spermatozoa with very low false negative rates (< 0.03%) and serves a valuable tool for clinicians to treat infertility patients with the appropriate modality . The SPAVWA designed the study, carried out statistical evaluation, and was the primary author of the manuscript. CMP, KPJ, HHH, MG, and IH were involved in management of the IVF cases and collection of the clinical data. DTC was responsible for the implementation of the study, administrative requirements including IRB approval, statistical evaluation of the data, and preparation of the manuscript.Table 1. Relationship between SPA and IVF outcome measuresClick here for file"}
+{"text": "Anvi-DAB1) in the little greenbul (Andropadus virens) from four localities in Cameroon and one in Ivory Coast, West Africa. Previous microsatellite and mitochondrial DNA analyses had revealed little or no genetic differentiation among Cameroon localities but significant differentiation between localities in Cameroon and Ivory Coast.We investigated genetic variation of a major histcompatibility complex (MHC) pseudogene (Levels of genetic variation, heterozygosity, and allelic diversity were high for the MHC pseudogene in Cameroon. Nucleotide diversity of the MHC pseudogene in Cameroon and Ivory Coast was comparable to levels observed in other avian species that have been studied for variation in nuclear genes. An excess of rare variants for the MHC pseudogene was found in the Cameroon population, but this excess was not statistically significant. Pairwise measures of population differentiation revealed high divergence between Cameroon and Ivory Coast for microsatellites and the MHC locus, although for the latter distance measures were much higher than the comparable microsatellite distances.We provide the first ever comparison of variation in a putative MHC pseudogene to variation in neutral loci in a passerine bird. Our results are consistence with the action of neutral processes on the pseudogene and suggest they can provide an independent perspective on demographic history and population substructure. Portrayed as the paradigm of neutral evolution , pseudogLevels of population differentiation and variability depend on the type of molecular marker used. Modes of inheritance , mutatioAndropadus virens) is a small passerine that inhabits rainforests in Sub-Saharan Africa ). In contrast, the mean FST for all pairwise comparisons within Cameroon is lower for Anvi-DAB1 allelic (0.004 +/- 0.03 [s.d.]) than for microsatellite data  whereas sequence data has the highest levels of FST (0.026 +/- 0.01 [s.d.]). The FST measures from microsatellites were not significantly different from Anvi-DAB1 allelic  and Anvi-DAB1 sequence FST . However, all pairwise values within Cameroon are low suggesting high rates of gene flow. The results of the linkage disequilibrium (LD) test for recent divergence versus ongoing gene flow are all positive, indicating that gene flow is most likely occurring among the sampled sites within Cameroon (Table 2 = 0.11).Pairwise measures of population divergence based on nt Table . For micns Table . All pairo Table . Likewisro Table . HoweverST for allelic and sequence Anvi-DAB1 information were highly correlated (r2 = 0.944) and both statistics were correlated with values of FST for microsatellite loci . However, none of these relationships were significantly based on the Mantel's test likely reflecting the small number of matrix entries (n = 4). The Mantel's test was also preformed omitting the pairwise measures from Lamto, and a non-significant positive correlation was still found .Pairwise values of FS with Anvi-DAB1 sequence data  is homozygous in three individuals, nearly equal rates of synomonous and nonsynomonous substitutions, absence from a survey of transcribed genes in the little greenbul, high divergence in sequence type when compared to classical transcribed MHC sequences, and a lack of conserved MHC class II vertebrate amino acid residues . Pseudogenes are rarely used in studies of natural populations, yet they may be valuable tool for quantifying genetic variation and differentiation. For example, polymorphism at the psGBA pseudogene in humans was found to be concordant with previous studies of neutral genes .The nuclear pseudogene used was the w). SSCP  was usedSCP [32P  and thesSCP [32P , 1 mM dNmplified . PCR proAnvi-DAB1 and the microsatellite loci were calculated using GENETIX [Observed and expected heterozygosity for  GENETIX . Deviati GENETIX . We also GENETIX  and Fu a GENETIX  for each GENETIX . Both Ta GENETIX .ST or \u03b8) was calculated from allelic data [Anvi-DAB1 and for the six microsatellite loci. Significance of pairwise FST measures was assessed with 500 bootstrap replicates in GENETIX. We calculated FST from sequence data using the method of Hudson et al. [ST (for Anvi-DAB1 and microsatellite loci) was assessed with a Mantel's test (5000 permutations) using GENETIX. Allelic richness, a measure of allelic variation that takes into account differences in sample sizes among populations, was estimated with the rarefaction method [Pairwise population differentiation   calculatThis work started out of a collaborative effort between the laboratories of TBS and RKW. AA designed the study, carried out the laboratory work and statistical analyses, and drafted the manuscript. TBS collected samples and TBS and RKW participated in the design and drafting of the manuscript. All authors read and approved the final manuscript."}
+{"text": "Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia. However, the errors implicit in HPV gene quantification by PCR are not well documented. We have undertaken the first rigorous evaluation of the errors that can be expected when using SYBR green qPCR for quantification of HPV type 16 gene copy numbers. We assessed a modified method, in which external calibration curves were generated from a single construct containing HPV16 E2, HPV16 E6 and the host gene hydroxymethylbilane synthase in a 1:1:1 ratio.When testing dilutions of mixed HPV/host DNA in replicate runs, we observed errors in quantifying E2 and E6 amplicons of 5\u201340%, with greatest error at the lowest DNA template concentration (3 ng/\u03bcl). Errors in determining viral copy numbers per diploid genome were 13\u201353%. Nevertheless, in cervical keratinocyte cell lines we observed reasonable agreement between viral loads determined by qPCR and Southern blotting. The mean E2/E6 ratio in episome-only cells was 1.04, but with a range of 0.76\u20131.32. In three integrant-only lines the mean E2/E6 ratios were 0.20, 0.72 and 2.61 . When E2/E6 ratios in fourteen HPV16-positive cervical carcinomas were analysed, conclusions regarding viral physical state could only be made in three cases, where the E2/E6 ratio was \u2264 0.06.Run-to-run variation in SYBR green qPCR produces unavoidable inaccuracies that should be allowed for when quantifying HPV gene copy number. While E6 copy numbers can be considered to provide a useable indication of viral loads, the E2/E6 ratio is of limited value. Previous studies may have overestimated the frequency of mixed episomal/integrant HPV infections. Cervical carcinoma is the second most common malignancy affecting women . Most caPersistent infection by high risk human papillomavirus (HR-HPV) represents the most significant risk factor in development of cervical carcinoma ,3, with Various PCR based assays have been devised to measure HPV copy number and physical state in cell lines and clinical samples -13, withBoth TaqMan and SYBR green strategies have been developed for HPV gene PCR, with the latter approach offering advantages of simplicity and low cost. Existing methods adjust for sample cellularity, generally determining HPV gene copy numbers per unit mass of genomic DNA (gDNA), per copy of an independent host calibrator gene, or with reference to a cell line -8,11-14.Despite these concerns, and the potential use of quantitative PCR (qPCR) data in clinical decision-making, remarkably few previous studies have undertaken detailed technical evaluation of the performance of PCR methods for assessing HPV load and physical state. The existing reports have typically been carried out using DNA mixtures or cell lines, but not both, and have only rarely been performed to a good standard ,11. WherAs the literature contains no adequate evaluation of the errors inherent in SYBR green qPCR analysis of HPV gene copy number, the present study sought to rectify this omission. We undertook detailed assessment of a modified SYBR green based qPCR strategy for absolute quantification of the copy number of HPV16 E2 and E6 genes, compared to a host diploid genome. We produced a single clone, NA6, containing the amplicons HPV16 E2, HPV16 E6 and host gene hydroxymethylbilane synthase  in a 1:1:1 ratio, for use in generating external calibration curves. In principle, this system may offer advantages of simplicity and comparability. Firstly, all external calibration curves are generated from one template dilution series, rather than several series required with independent calibration constructs , thereby reducing the amount of manual handling needed. Secondly, the amount of host gDNA in each test sample can be quantified directly with reference to the HMBS standard curve, rather than relying on an estimation of gDNA concentration by spectrophotometry. Thirdly, a standardised calibration construct such as NA6 may reduce inter-laboratory variation in HPV gene quantification.We have undertaken rigorous evaluation of the performance of HPV16 SYBR green qPCR and present data documenting the potential sources of error, even in the 'best case scenario' of cell line analysis. We first used mixtures of HPV16 DNA and host gDNA, then cervical keratinocyte cell lines in which the HPV16 copy number and physical state were determined by Southern blot and densitometry, and finally HPV16-positive SCC tissue samples. We demonstrate the error range that should be anticipated when quantifying HR-HPV gene copy number by SYBR green qPCR. We also show that E2/E6 ratios are of limited use in assessing HR-HPV physical state, being of value only for identifying the subset of integrant-containing cells in which the E2/E6 ratio is near zero.The HPV16 positive cervical SCC cell lines SiHa, and CaSki and the HPV negative cervical SCC line C33A  were obtained from the American Type Culture Collection (ATCC). We also used early and late passages from one long-term culture series of the W12 cell line . W12 was established from a cervical low grade SIL and in long term culture recapitulates cervical neoplastic progression genetically and phenotypically -20. All Genomic DNA (gDNA) was prepared from pelleted cells by overnight digestion with proteinase K, brief phenol chloroform extraction, ethanol precipitation, and removal of contaminating RNA by RNAse A. The purified gDNA was quantified by spectrophotometry.\u00ae2.1 vector using the original TA cloning system (Invitrogen). Briefly, fragments of the E2 hinge region (81 bp), and E6 gene (80 bp) were amplified from the pSP64-HPV16 construct , while 6 Da.In order to construct a single clone containing each amplicon in a 1:1:1 ratio, the following cloning strategy was adopted. The E2 and E6 clones were linearised with XbaI, and the products ligated with T4 DNA ligase. Using the ligated product as template, PCR with E2 forward and E6 reverse primers generated a single E2-E6 product for subsequent TA cloning. Following selection, a single E2-E6 clone and a separate HMBS clone were digested with HindIII, and similarly ligated together. PCR with E2 forward and HMBS reverse primers generated a single product that following TA cloning, selection and sequencing, was shown to contain a single copy of the E2, E6 and HMBS amplicons , 2 ng of NA6 equates to 4.176 \u00d7 10All qPCR was carried out using an Opticon I thermal cycler (MJ Research) with SYBR Green JumpStart qPCR Kits . Reactions comprised 1\u00d7 SYBR Green mix, 500 nM primer pairs, and 2 \u03bcl of template. In initial optimisation experiments we investigated the effects of primer concentration on crossing point (Cp) determination. We found that a primer concentration of 500 nM was optimal, and that a reduction to 300 nM resulted in PCR artefacts at low template concentrations. It was found that a template volume of 2 \u03bcl could be dispensed more accurately and reproducibly than 1 \u03bcl volumes (data not shown).The following cycling conditions were employed: initial denaturation 94\u00b0C 2 min, followed by 40 cycles of 94\u00b0C 20 seconds, 58\u00b0C 20 seconds, 72\u00b0C 20 seconds, 76\u00b0C 15 seconds, plate read. A final extension of 72\u00b0C 10 minutes, and melting curve of 65\u00b0C to 90\u00b0C, 1\u00b0C/second transition were incorporated. Opticon raw data was exported to Microsoft Excel for analysis.Absolute quantification strategies require that the fluorescence threshold used to derive the calibration curve is also applied to the sample data. Hence it may be anticipated that little effect would be experienced by varying the fluorescence threshold (Ft), provided that the Ft passes through the centre of the log transformed reaction curve data. However, we found that gross changes in absolute quantification were seen for relatively small changes in Ft following theoretical evaluations (data not shown). To avoid issues related to placement of Ft values, an automated derivation of optimum Ft assignment was implemented. Fluorescence thresholds were calculated for each of the four calibration curves according to the cycle before second derivative maxima method, and then averaged during the process of producing the calibration curve. The E2, E6 and HMBS Ft values were then applied to all sample data in order to calculate crossing points.Calibration curves for E2, E6 and HMBS were constructed by plotting crossing points (Cp) versus the log of template copy number. For copy number determination in test samples, the fluorescence threshold (Ft), primer efficiency (E) and numbers of molecules at fluorescence threshold (Nt) were taken to be constants and were determined as the means of the four standard curve replicates for each amplicon. Hence, for an unknown sample, number of copies is given by equation 1.Calibration curve primer efficiencies were determined by equation 2, and numbers of molecules at threshold by equation 3.The coefficient of variation between data obtained in replicate calibration curves represented the ratios of standard deviations over the mean, multiplied by 100%.Gene copy numbers in test samples were obtained by comparing the Cp value to those in the relevant external calibration curve. A Microsoft Excel template was prepared to calculate viral loads, and E2/E6 ratios  cloned into pcr2.1 TA cloning vector, or from full length HPV16 (7.9 kbp) excised from pSP64-HPV16 using BamH1, and labelled using RediPrime labelling kit. Hybridisation was for 16 hr, followed by stringency washing of membranes in 2 \u00d7 SSC/0.1% SDS and 0.1 \u00d7 SSC/0.1% SDS twice for 15 minutes, and then autoradiography.In order to validate the qPCR method, comparisons were made with HPV16 copy numbers determined in cervical keratinocyte cell lines by Southern blotting. 5 \u03bcg of gDNA was digested at 37\u00b0C overnight with either BamH1, PstI or HindIII, and electrophoresed on a 0.8% agarose gel. Agarose gels were depurinated in 0.25 M HCl, denatured in 0.5 M NaOH/1.5 M NaCl and neutralised in 0.5 M tris HCl/3 M NaCl before transfer to Hybond N membrane. UV cross-linked membranes were prehybridised in 20 ml of hybridisation buffer for 4 hrs at 65\u00b0C prior to introduction of Cervical squamous cell carcinoma samples were kindly provided by Dr Geetashree Mukherjee from the archives of the Kidwai Memorial Institute of Oncology, Bangalore, India. All tissue samples were obtained with informed consent, anonymised, and used with approval from the Kidwai Local Research Ethics Committee (reference: PER/CAB-I/D-I/13/01).gDNA was prepared from 14 snap frozen HPV16-positive cervical SCC samples, as described elsewhere . The meaAF125673), were located in the part of the E2 open reading frame (ORF) that is most often deleted on HPV16 integration [In generating the NA6 construct and preparing the calibration curves , PCR primers were chosen to give amplicons of similar length Table . The E2 The SYBR green qPCR method used was developed following numerous optimisation experiments to determine ideal primer concentration, template gDNA concentration and cycling parameters . We analysed melting curves of reaction products to verify specificity of primer binding and thereby circumvent any issues of non-specific SYBR green fluorescence. Following four replicate qPCR runs, seven point external calibration curves were generated for E2, E6 and HMBS. Figure 2 = 0.996). For each amplicon, the mean Cp for each titration point 1 ng/\u03bcl to 1 fg/\u03bcl was plotted to generate the final calibration curves, which are shown in Figure Tight correlations were observed between the values obtained for each amplicon in the replicate qPCR runs, as illustrated for the E2 amplicon in Figure Despite the apparent concordance between replicate runs, when we calculated viral gene copy number using calibration curves generated from individual qPCR runs, we observed inter-assay differences in the copy number values generated. Each of the four replicate calibration curves (Replicate 1 to Replicate 4) for E2, E6 and HMBS amplicons, as well as the final mean calibration curve derived from the quadruplicate runs, were used to calculate gene copy number according to a series of theoretical crossing points , ranging from a Cp of 10 to 30  was fouBy Southern blotting using full length HPV16, W12.Ser1p57 (known to be near tetraploid ) was epiSiHa cervical SCC cells (which are near triploid ) were shCaSki cervical SCC cells (which are near tetraploid ) were shThe data from the detailed cell line analysis confirms that errors in SYBR green qPCR-based HPV16 gene quantification should be allowed for, especially at low copy number. Nevertheless, the viral loads determined from mean E6 copy numbers were reasonably close to the values shown by Southern blotting and can be considered to provide a useable indication of actual loads. The E2/E6 ratios also showed considerable variation, which again was greatest at low copy number. Moreover, when taking mean ratio values, our data shows that while integrated HPV16 in the absence of episomes may produce a low E2/E6 ratio, the presence of a high E2/E6 ratio, even one greater than 1.0, does not exclude the presence of integrated HPV16 only.The SYBR green qPCR method was also used to assess copy number of HPV16 genes in frozen tissue samples from fourteen HPV16-positive cervical SCCs Table . Viral lE2/E6 ratios were also determined for the SCCs. In three samples , E2/E6 ratios were 0.06 or less, indicative of fully integrated HPV16 only  in a set of fourteen HPV16 positive cervical SCC clinical samples are likely to be broadly accurate, albeit subject to the errors that we demonstrated using HPV16/gDNA mixtures. Taken together, our detailed evaluation supports the use of SYBR green HPV16 qPCR in studies attempting to correlate viral copy number with clinical outcome using tissue specimens, at least where the degree of sampling of abnormal tissue is known.We observed a wide range of E2/E6 ratios in cell lines and clinical samples. In integrant only SiHa cells the E2/E6 ratio was substantially greater than 0, at 0.2 (0.11\u20130.24), while in integrant only CaSki cells it was 2.61 (1.37\u20133.46), as a result of multiple integrants with greater representation of E2 than E6. These values (and their ranges) are attributable to the errors implicit in HPV gene quantification by qPCR, as well as to the retention of E2 sequences in some integrants. The latter may be full-length E2, as in CaSki, or alternatively fragments of E2 retaining the hinge region that is amplified by the most frequently used gene quantification primers (including those in the present study). While the hinge is the region of E2 that is most commonly deleted in HPV16 integration, it may also be retained; as is the case in the integrant in W12.Ser1p57 . It shouThe E2/E6 ratios in episome-containing W12Ser1p16 cells ranged from 0.76\u20131.32 in triplicate assays. Nagao et al observed E2/E6 ratios of 0.61\u20131.13 in cervical carcinomas that were confirmed to contain HPV16 episomes only . This grIn our opinion, only very low E2/E6 ratios are likely to be good indicators of the integrant only state. We propose an E2/E6 ratio of 0.10 or less, as values greater than this are unlikely to discriminate reliably between integrant only samples and samples where episomes are present. A previous study showed that E2/E6 ratios under 0.10 were only seen where integrated HPV16 DNA was in 10 fold or greater excess over episomal HPV16 DNA . HoweverE2/E6 ratios less than 0.10 are likely to be encountered rarely. Indeed, in our study we observed such values in only three of 14 HPV16 positive cervical SCCs. In the other cases, we consider that no reliable conclusions regarding viral state could be drawn from the E2/E6 ratio, indicating its limited usefulness when applied to clinical samples and uncharacterized cell lines. Based on these findings, we suggest that data from previous studies using the E2/E6 ratio to examine the physical state of HPV16 in clinical samples may not be accurate, and, in particular may have overestimated the frequency of mixed integrant and episomal infections in cervical neoplasia ,13.IR devised the experiments, prepared the NA6 calibrator clone, and carried out accuracy tests and assessment of cell lines by qPCR. GN undertook assessment of clinical samples by qPCR and NF prepared calibration curves. W12 cell line establishment, propagation and gDNA preparations were made by MS and MRP. MTH prepared other cell line gDNA and contributed to the development of the qPCR method. AT provided statistical input and advised on qPCR mathematics. IR and NC wrote the manuscript. This work was funded by MRC and CRUK programme grants held by NC.1: NA6 Standard Curves. The crossing point values used in generation of external calibration curves for absolute quantification of viral E2, viral E6 and host HMBS genes are presented. For each gene, four runs of a seven point NA6 titration series were conducted in triplicate. 2: Accuracy Test. The crossing point values used to determine viral load and physical state of a three point serial dilution of a mixture of test gDNA and HPV16 plasmid DNA are presented. Three runs were undertaken at each titration point in triplicate. 3: Assessment of Cell Lines. The crossing point values used to derive viral load and physical state of five cervical carcinoma cell lines are presented. Each cell line was assessed in three separate runs, and each reaction was performed in triplicate. 4: Assessment of Clinical Samples. The crossing point values used to derive viral load and physical state of 14 squamous cell cervical carcinoma samples are presented. One run was undertaken for each sample, and all reactions were performed in triplicate.Crossing point values used in critical evaluation of HPV16 gene copy number quantification by SYBR green PCR. The Microsoft Excel workbook of Additional file Click here for file"}
+{"text": "Statistical analysis was carried out using SPSS software , values were expressed as mean \u00b1 SEM, and P value <0.01 was considered significant. Effect of Sal B on expression of NF-kB p65 and IkB\u03b1 was studied and OD values of densitometry of western blots were taken. MPO activity was also studied. It was observed that treatment of Sal B significantly reduced the expression of both compounds in Sal B treated group as compared to control group after 28 days of treatment.In this study effect of salvianolic acid B was observed on motor function recovery of rats with spinal cord injury. 50 rats were selected and after inducing SCI their recovery under controlled conditions was studied using Sal B and PBS (as control). Both compounds were introduced intraperitoneally in respective groups of traumatic rats at the same time intervals for 28 days. It was observed that Sal B introduced at 5\u2009\u2009mg/kg/day resulted in better motor function recovery. BBB score was recorded which increased significantly along with the reduction in cavity area observed by bright field microscopy of tissues, that is, from 1 to 10 and from 0.20 \u00b1 0.05\u2009mm Salvia miltiorrhiza has been responsible for exhibiting characteristics like anti-inflammatory and neuroprotective both in vivo and in vitro [\u03b2 protein (A\u03b2) induced cytotoxicity [\u03b1 injury in human aortic vascular endothelial cells [Traditionally used in Chinese medicine the bioactive compound extracted fromin vitro , 2. The in vitro . Liu et in vitro , showed in vitro . Other ptoxicity  and protal cells . That isal cells . In the al cells \u201311.In traumatic injury of spinal cord a number of cellular and molecular events occur that can be included in primary and secondary injury pathways. The pathology of SCI can be increased significantly by secondary injury in association with the primary injury , 13. A m\u03b1 and NF-kB p65 was carried out by selecting two experimental groups of rats with traumatic SCI where Sal B and PBS (control) groups were tested. The study of functional recovery of locomotor function in rats using Basso-Beattie-Bresnahan (BBB) scale was also performed and MPO activity was studied in both groups to establish the effect that treatment of Sal B might have on reducing inflammation after injury.In this study we have made an effort to explore the use of Sal B as a potential inhibitor of IKK/NF-kB pathway and evaluation of expression of IkB36H30O16, molecular weight: 746, purity: 98.5%, Green-Valley, Shanghai, China) was used in the experiment while phosphate buffered saline (PBS) was used as a control. A total of 40 adult SD female rats (weight: 200\u2013230\u2009g) were obtained and divided into two main groups of Sal B and PBS (20 rats per group). In each group there were four subgroups having 5 rats each (n = 5). Four subgroups were monitored till 28 days where in each week one subgroup was used. Sal B was injected intraperitoneally in all the subgroups of Sal B group from 0, 1 to 28 days and PBS was injected in all the corresponding subgroups of PBS group like Sal B in the same amount after injury at similar time interval for the same duration of 0, 1, 2 to 28 days. In every assigned group the treatment was stopped at the end of the week subsequently while in the other subgroups the treatment continued and potential recovery of SCI was studied. At T9-T10 laminectomy was performed after administering 10% chloral hydrate anesthesia. A 10\u2009g NYU impact rod centered above T9 was dropped from 12.5\u2009mm height and a consistent partial, incomplete SCI was induced and then the postinjury care was taken out according to the previous reports and bladder was emptied manually twice a day. For Sal B and PBS groups the rats were given Sal B 5\u2009mg/kg per day intraperitoneally and in PBS group PBS was given only as a control.Sal B  antibody . The reactive protein bands were visualized using horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies  and an ECL western blotting kit , which was performed according the manufacturer's instructions. The membranes were exposed to X-ray film for 10\u2009s to 1\u2009min. A polyclonal rabbit anti-actin antibody  was used to detect actin in the samples as a loading control. The protein bands were scanned and digitized, and the optical density (OD) of each band was determined using the Gel-Pro analyzer 4.0 software.Then transcardial perfusion was made using 4% paraformaldehyde after animals were anaesthetized with 10% chloral hydrate. T9-T10 portion of spinal cord was removed and then immersed in the same fixative for 24\u2009hrs. Tissue was taken and sectioned sagitally after embedding in paraffin for 24 hours. For all the subgroups of Sal B and PBS groups sections were collected on microscopic slides and after the removal of paraffin graded ethanol was used to rehydrate the slides and then all sections were stained with hematoxylin eosin (HE) for general purpose histology. Behavior testing of rats was done using Basso-Beattie-Bresnahan (BBB) scale at different points before and after injury  in both Sal B and PBS control groups. Here a score of 0 represents absence of locomotion while 21 shows normal locomotor function and subsequent points show the improvement in function up till 21 . StatistStudies have shown that in case of spinal cord injury inflammatory responses are triggered because inflammatory cells at the site of injury release neurotoxins and other inflammatory mediators that can result in the generation of reactive oxygen and nitrogen species resulting in cellular damage . In orde2 to 0.10 \u00b1 0.03\u2009mm2 compared with the PBS group   after 28 days of treatment (Figures In order to observe the effect of Sal B on IKK/NF-kB pathway, the expression of NF-kB p65 in spinal cord tissues was observed using western blotting where after 24 hours of SCI the increase in NF-kB p65 protein was observed in both groups and then a significant attenuation of NF-kB p65 was observed among the Sal B treated group as compared to PBS group after 28 days of the treatment ( Figures .cP < 0.01 is found significant as compared to the PBS control group. The use of Sal B therefore can reduce tissue damage by inhibition of neutrophil infiltration.It was observed that infiltration of leukocytes is increased in SCI in spinal cord tissues but it can be controlled and reduced by the help of Sal B. The evaluation of MPO activity in subgroups of both Sal B and PBS control groups was performed and it was observed that the treatment of Sal B can downregulate the infiltration of spinal cord tissues by neutrophils. A comparison of result of MPO activity in the fourth subgroup of both Sal B and PBS is shown in In the present study it can be concluded that the use of Sal B significantly improved the locomotor function recovery in rats with induced spinal cord injury as compared to the PBS group where Sal B was not administered. A dose of 5\u2009mg/kg/day improved BBB score in rats as evident in the results as compared to the PBS control group and moreover the cavity area was also reduced in this group. The most obvious improvement was observed in the group that received Sal B for 28 days after injury. There can be many reasons attributed to this recovery including the protective effect on neural cells that were injured by SCI and recovery of these neurons. A number of particular genes that are called neuroprotective genes can be induced in a variety of conditions like electrical stimulation, cerebral ischemia, and brain injury ; therefo\u03b2, the main catalytic subunit of IKK, can result in reduced inflammatory cells and neuronal apoptosis after SCI in rats [\u03b2 is the main catalytic subunit of IKK that can activate NF-kB by phosphorylation of inhibitory protein of IkB [\u03b1 level were monitored in both groups where Sal B group showed a significant decrease in the expression of NF-kB p65 and IkB\u03b1. NF-kB p65 is one of the different Rel family proteins like RelB, c-Rel, p50, and p52. The results of Sal B group showed downregulation of infiltration of spinal cord tissues by neutrophils providing additional evidence of therapeutic potential of Sal B. Different compounds have been used from Chinese herbal medicine for this purpose and has shown successful results in the treatment of SCI in rats as, for example, Lua et al. [ Caragana jubata and Rhus verniciflua to exhibit inhibition of NF-kB pathway and help regulate the expression of these genes inducing secondary inflammation in SCI. However, the use of Sal B alone for treating SCI in rats is the first report of this potential use of the compound to the best of our knowledge. Also the parameters studied are physiological as well as cellular expressions, both providing significant evidence of the possible uses of the Sal B; therefore, further studies in this regard will be helpful in exploring this new possible therapeutic agent. The selective inhibition of stimulatory pathways along with the physiological recovery like cavity reduction, improvement in motor functions provides a possible incite for future investigations that should be explored further.It has been reported that secondary inflammation is regulated by IKK/NF-kB pathway  and if t in rats . Studies in rats . NF-kB b in rats , and IKKn of IkB . It is ta et al.  recently"}
+{"text": "In rat mesenteric artery rings, KA-acetoxy induced a concentration-dependent relaxation in vessels precontracted with phenylephrine. In the absence of endothelium, the vasorelaxation was significantly shifted to the right without reduction of the maximum effect. Endothelium-dependent relaxation was significantly attenuated by pretreatment with L-NAME, an inhibitor of the NO-synthase (NOS), indomethacin, an inhibitor of the cyclooxygenase, L-NAME + indomethacin, atropine, a nonselective antagonist of the muscarinic receptors, ODQ, selective inhibitor of the guanylyl cyclase enzyme, or hydroxocobalamin, a nitric oxide scavenger. The relaxation was completely reversed in the presence of L-NAME + 1\u2009mM L-arginine or L-arginine, an NO precursor. Diterpene-induced relaxation was not affected by TEA, a nonselective inhibitor of K+ channels. The KA-acetoxy antagonized CaCl2-induced contractions in a concentration-dependent manner and also inhibited an 80\u2009mM KCl-induced contraction. The KA-acetoxy did not interfere with Ca2+ release from intracellular stores. The vasorelaxant induced by KA-acetoxy seems to involve the inhibition of the Ca2+ influx and also, at least in part, by endothelial muscarinic receptors activation, NO and PGI2 release.The objective of the study was to investigate the mechanism of the relaxant activity of the  The use of alternative therapies, herbs, and supplements occurs at a very high rate among patients with cardiovascular disease including hypertension . HyperteCroton zambesicus is used in traditional medicine in Africa to treat hypertension. The diterpenes isolated from the extract of plant induced vascular relaxation and antihypertensive effect oxadiazoloquinoxalin-1-one (ODQ) . KA-acetoxy was solubilized in a mixture of distilled water cremophor at a concentration of 10\u2009mM and diluted to the desired concentration with distilled water just before use. Indomethacin was dissolved in 0.5%\u2009w/v sodium bicarbonate. ODQ was prepared as stock solution dimethyl sulfoxide (DMSO). EGTA was added in the Ca2+-free Tyrode's solution. The other compounds were freely dissolved in distilled water. The final concentration of cremophor and DMSO in the bath never exceeded 0.01% was without effect when tested in control preparations (data not shown).The following drugs were used: atropine sulfate, acetylcholine hydrochloride (ACh), indomethacin, Nad libitum.Male Wistar rats (200\u2013300\u2009g) were used in all experiments. Experimental protocols and procedures were approved by the Laborat\u00f3rio de Tecnologia Farmac\u00eautica Animal Care and Use Committee. Animals were housed under conditions of controlled temperature ((25 \u00b1 1)\u00b0C) and lighting (light on 6:00\u201318:00\u2009h) and had access to food and tap water 2: 2.0; MgCl2: 1.05; NaH2PO4: 0.42; NaHCO3: 10.0; and glucose: 5.6.), gassed with carbogenic mixture (95% O2 and 5% CO2), and maintained at 37\u00b0C for isometric n tension recordings. The stabilization period was of 1\u2009h under a resting tension of 0.75\u2009g. During this time, the solution was changed each 15\u2009min to prevent the accumulation of metabolites. The isometric tension was recorded by a force transducer  coupled to an amplifier recorder . Endothelium was removed by gently rubbing the intimal surface of the vessels. The presence of functional endothelium was assessed by the ability of acetylcholine (ACh) (10\u2009\u03bcM) to induce more than 80% relaxation of precontracted vessels with Phe (10\u2009\u03bcM). The absence of the relaxation to ACh was taken as evidence that the vessel segments were functionally denuded of endothelium.Rats were killed by stunning and exsanguination. The superior mesenteric artery was removed, cleaned from connective tissue and fat and sectioned in rings (1-2\u2009mm), which were suspended by cotton threads in organ baths containing 10\u2009mL of Tyrode's solution . Under the sustained contraction elicited by Phe the vessels were exposed to cumulative concentrations of KA-acetoxy (10\u22126\u20131\u2009mM).In the first set of experiments, the ability of KA-acetoxy to cause vascular relaxation was evaluated in both endothelium-intact and endothelium-denuded mesenteric artery rings previously contracted by Phe (10\u2009\u22126\u20131\u2009mM) were added cumulatively to organ bath. The extent of relaxation was expressed as the percentage of phenylephrine- or KCl-induced contraction.In the second set of experiments, after the stabilization period, rings without endothelium were precontracted with KCl 80\u2009mM on the tonic phase and different concentrations of KA-acetoxy (10\u03bcM), a nonselective antagonist of the muscarinic receptors, L-NAME (100\u2009\u03bcM), an inhibitor of the NO-synthase (NOS), L-NAME plus L-arginine (1\u2009mM), NOS substrate, L-arginine alone, indomethacin (10\u2009\u03bcM), an inhibitor of the cyclooxygenase (COX), L-NAME + indomethacin, ODQ (10\u2009\u03bcM), selective inhibitor of the guanylyl cyclase enzyme, and hydroxocobalamine (30\u2009\u03bcM), a nitric oxide scaveng, separately.To investigate the possible mechanism(s) responsible for KA-acetoxy induced relaxation, the preparations with endothelium were precontracted with Phe for 30\u2009min after being early incubated with one of the following inhibitors: atropine , and then concentration-response curves to KA-acetoxy were obtained. The TEA was added 30 minutes before the contractions with Phe.Phe-induced sustained contractions were obtained in endothelium-intact and endothelium-denuded mesenteric artery rings incubated with tetraethylammonium, a nonselective inhibitor of K2+ channels, superior mesenteric artery rings were bathed for 15\u2009min in Ca2+-free Tyrode's solution, prepared by omitting only CaCl2 and then exposed for an additional 15\u2009min to a high K+ (60\u2009mM) Ca2+-free solution. Under this new experimental condition, cumulative concentration response curves to CaCl2 (ranging from 1\u2009\u03bcM to 10\u2009mM) were obtained. KA-acetoxy  was added to the preparations for 30\u2009min, and then a new cumulative concentration-response curve for CaCl2 was determined. The maximal contraction obtained with the control concentration-response curve to CaCl2 was taken as 100% and all values were calculated as a percentage of the maximal response. Each preparation was exposed to only one diterpene-concentration. All experiments were done using endothelium-denuded superior mesenteric artery rings.In order to access the effects of KA-acetoxy on voltage-gated Ca\u03bcM Phe or 20\u2009mM caffeine in Ca2+-free solution before and after incubation with KA-acetoxy  for 20\u2009min. The results were expressed as percentages of the response induced by Phe or caffeine alone.The effect of KA-acetoxy on phenylephrine- or caffeine-sensitive calcium intracellular stores was assessed by using a protocol described by Sakata and Karaki . The traEmax\u2061  and pD2 (\u2212log\u2061EC50). Results are expressed as means \u00b1 standard error of the mean (SEM). Student's t-test and one-way analysis of variance (Anova) using Bonferroni's posttest was used to analyse the data, and results were considered significant when P < 0.05.In order to study the effect of KA-acetoxy on inducing relaxation, two pharmacological parameters were analysed: the Emax\u2061 as compared to the control. In rings without endothelium precontracted with K+-depolarizing solution (KCl 80\u2009mM), the concentration-response curves for KA-acetoxy was significantly rightward shifted no change in Emax\u2061 as compared to the phenylephrine-contracted endothelium-denuded vessels (pD2 = 3.6 \u00b1 0.1 and Emax\u2061 = 87.9 \u00b1 4.7%).The magnitude of contraction induced by Phe in rings with and without endothelium functional was 0.42 and 0.44\u2009g, respectively. Similarly, the magnitude of contraction induced by KCl in rings without endothelium was 0.40\u2009g. There were no significant differences between the magnitudes.\u03bcM), indomethacin (10\u2009\u03bcM), or L-NAME (100\u2009\u03bcM) plus indomethacin (10\u2009\u03bcM) significantly shifted to the right the concentration-response curves of KA-acetoxy. In the same manner, after soluble guanylyl cyclase inhibition (ODQ 10\u2009\u03bcM), HDX (30\u2009\u03bcM), a nitric oxide scaveng, or muscarinic blockade (atropine 1\u2009\u03bcM) reduced KA-acetoxy-induced relaxation and produced a rightward displacement of the concentration-response curve for the compound. However, L-arginine (1\u2009mM) or L-arginine plus L-NAME (100\u2009\u03bcM) had not significant effect on KA-acetoxy-induced relaxation (The incubation with L-NAME (100\u2009laxation . The mea\u03bcM), TEA (5\u2009mM), a nonselective inhibitor of K+ channels, was not able to change KA-acetoxy relaxations . These endothelium-derived relaxing factors diffuse to adjacent smooth muscle cells and cause them relaxation , 22. To Km values of the enzyme of L-arginine are 1.5 to 2.3\u2009\u03bcM [L-arginine, a NO precursor, antagonized the effect of L-NAME, but when added alone it did not affect the relaxations induced by KA-acetoxy. The inability of this NO synthase substrate to increase the relaxation induced by KA-acetoxy may be explained in terms of there being sufficient amounts of L-arginine in the vascular endothelium. The o 2.3\u2009\u03bcM . Moreoveo 2.3\u2009\u03bcM , caused 3 subtype) produces an intense dilation, despite the lack of vascular cholinergic innervation [3 receptors [Emax\u2061. These results suggest that diterpene partly could be acting via endothelial muscarinic receptor activation and consequent of participation of cGMP pathway.In most vascular beds, the stimulation of muscarinic receptors  [\u03b11-adrenoceptor agonists, such as phenylephrine, are initiated by Ca2+ release from intracellular stores, which is followed by activation of Ca2+-activated channels causing depolarization of the vascular smooth muscle cell membranes and activation of voltage-gated Ca2+ channels [+-induced contraction in smooth muscle is mediated by cell membrane depolarization and an increase in calcium influx through VOCCs [The maintenance of smooth muscle contraction depends on Ca, resp.) . It is wchannels . Whereasgh VOCCs . In both2+ channels. Based on this assumption, we evaluated the effect of KA-acetoxy on endothelium-denuded rings precontracted with K+-depolarizing solutions (KCl 80\u2009mM). KA-acetoxy was capable to inhibit contractility induced by KCl (80\u2009mM) in endothelium-denuded mesenteric rings. This result suggests that KA-acetoxy could inhibit Ca2+ influx through VOCCs.Thus, it is proposed that the residual vasorelaxant effect of the diterpene is due to a mechanism independent of endothelium, possibly a blocking activity on the Ca2-induced contractions in a depolarizing medium without calcium. This protocol was based on the fact that CaCl2-induced contractions are elicited, almost exclusively, through Ca2+ influx, since the depolarization promoted by high concentrations of extracellular K+ induces the opening of voltage-dependent Ca2+ channels [2 concentration-response curve significantly reducing the maximal response.In order to strengthen the above hypothesis, KA-acetoxy was tested in the presence of CaClchannels . Under t2+ is mainly regulated by IP3 receptor system (IP3Rs) and ryanodine receptor system (RyRs). The former induces Ca2+ release directly when the receptors are bound to IP3. The later may function through a Ca2+ induced Ca2+ release (CICR) mechanism when the receptors are activated by caffeine [2+-free solution, in the absence and presence of diterpene. Thus, KA-acetoxy not inhibited transient contractions induced by phenylephrine and caffeine, suggesting that the compound does not interfere in the calcium mobilization of calcium intracellular stores.The release of intracellular stored Cacaffeine . To inveent-kaur-16-en-19-oic acid). Similarly, KA-acetoxy was able to induce vasorelaxation vascular tissues such as kaurenoic acid. The mechanism involved in the vasorelaxant action has some similarities, such as extracellular Ca2+ influx blocked and activation of NO-cGMP pathway [KA-acetoxy is a closely related derivate with kaurenoic cid  (unpublished data). It is well known that small arteries, as the superior mesenteric artery, play an important role in the determination of the peripheral resistance and in the regulation of blood pressure . We can In addition, only substances that inhibit contractions in KCl test model are worth further examination in a search for naturally occurring calcium-antagonists. In this context, the KA-acetoxy was able to inhibit contractility induced by KCl. In this context, it is possible to suggest that the KA-acetoxy could exert antihypertensive action in vivo as other diterpenes , 14.2 release, as well as probably the participation of EDHF. Endothelium-independent relaxation KA-acetoxy acts through inhibition of the Ca2+ influx.In summary, the present study demonstrated that the KA-acetoxy produced a concentration and endothelium-dependent and -independent vasorelaxation in superior mesenteric artery rings. Endothelium-dependent relaxation appears to be due to endothelial muscarinic receptors activation, NO and PGI"}
+{"text": "Aim. Spindle and kinetochore-associated protein 1 (SKA1) is one subtype of SKA, whose protein can make spindle microtubules attach steadily to the kinetochore in the middle of mitosis. At present, there are fewer researches on the relationship between SKA1 expression and tumor development. Methods. In this study, immunohistochemical analysis was used to determine the expression of SKA1 in papillary thyroid carcinoma (PTC) and adjacent tissues. We used quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis to further verify the results. Results. We found that SKA1 expression was significantly higher in PTC tissues than normal adjacent tissues (P < 0.05). There existed a significant correlation among a higher SKA1 expression, including lymphoid node (P = 0.005), clinical stage (P = 0.015), and extrathyroid invasion (P = 0.004). Survival analysis showed high SKA1 expression in PTC patients more likely to relapse after surgery. Conclusion. High SKA1 expression is predictive of poor prognosis of PTC, implying that SKA1 may be a promising new target for targeted therapies for PTC. Thyroid cancer is the most common malignant tumor of the endocrine system and the most common head and neck tumor. Each year, new cases of thyroid cancer account for 1\u20135% of all cancer cases . ThyroidIt is well known that, during the cell cycle, the proper formation and depolymerization of the spindle play an essential role in maintaining normal mitosis of eukaryotic cells. However, chromosomal division abnormalities can cause genetic instability and ultimately induce PTC development . SpindleThe subjects of this study were 123 patients with PTC who had undergone surgical intervention at the Department of Head and Neck Surgery of the Affiliated Tumor Hospital of Xinjiang Medical University between 2005 and 2009. All patients did not receive any therapy prior to surgery and were diagnosed with primary PTC based on the pathological findings. All 123 patients had complete clinical data, including age, sex, tumor size, lymph node status, clinical stage, and extrathyroidal invasion. Patients were selected for this study only if follow-up examinations and clinical data were available. The TNM stage was defined according to the 7th edition of the TNM classification of the International Union Against Cancer. The study was approved by the ethics committee of the Affiliated Tumor Hospital of Xinjiang Medical University. Written informed consent was obtained from each patient for the use of resected samples for research.\u03bcm slices and embedded with paraffin after fixation with 10% formaldehyde. Immunohistochemistry with the SP method  was used for testing by applying PBS instead of primary antibodies as the negative control, according to the manufacturer's instructions. The results were determined by two independent pathologists using a double-blind method. Five high power fields (200x) were randomly selected, and in each field 200 tumor cells were recorded, totaling to 1000 cells. According to the staining intensity, the cells were assigned the following point values: 0 points for no staining; 1 point for pale yellow staining; 2 points for brown-yellow staining; and 3 points for dark brown staining. The proportion of positive cells was assigned a value between 0% and 100%. The immunohistochemistry expression level was determined according to the product of the staining intensity and the proportion of positive cells. When the product was below 150%, this was considered low expression of SKA1, and when the product was above 150%, this was considered high expression of SKA1.Biopsy tissue samples were sectioned into 4-\u00b5L) was used for 2% agarose gel electrophoresis, and the grayscale analysis was performed with Quantity One software . The relative expression level of SKA1 mRNA was determined by the ratio of the grayscale value of SKA1 mRNA and that of internal reference GAPDH mRNA.The tissue samples were removed from liquid nitrogen, and RNA was extracted using Trizol reagent and a reverse transcription kit . The PCR primers used were SKA1-F: 5\u2032-TGATGTGCCAGGAAGGTGAC-3\u2032, SKA1-R: 5\u2032-CAAAGGATACAGATGAACAACAGC-3\u2032, GAPDH-F: 5\u2032-GTGGACATCCGCAAAGAC-3\u2032, and GAPDH-R: 5\u2032-AAAGGGTGTAACGCAACTA-3\u2032. All primers were synthesized by Shanghai Yingjun Biotechnology Company . The PCR reaction conditions were as follows: initial denaturation for 2\u2009min at 94\u00b0C, 35 cycles of denaturation for 30\u2009s, annealing for 30\u2009s at 94\u00b0C, and extension for 1\u2009min at 72\u00b0C, followed by 10\u2009min at 72\u00b0C. PCR reaction product  at 4\u00b0C overnight. Goat anti-rabbit IgG secondary antibody  marked with horseradish peroxidase and diluted at 1\u2009:\u20091000 was added and incubated at room temperature for 1.5\u2009h. The membrane was washed three times in TBST. The relative level of SKA1 protein was determined by the grayscale value of various protein straps and the ratio of the grayscale value of SKA1 to GAPDH protein.After protein extraction using total protein lysis buffer, the protein concentration was determined by the BCA method. In brief, 80\u2009 SKA1 mRNA expression in PTC was compared with expression in normal adjacent tissues using the Mann-Whitney test (for 2 groups) or the Kruskal-Wallis test (for more than 2 groups). Kaplan-Meier analysis was used for survival analysis, lymph node recurrence-free survival (LNRFS), and distant recurrence-free survival (DRFS) calculations. LNRFS was defined as the time from the date of surgery to the date of lymph node relapse, and DRFS was defined as the time from the date of surgery to the date of distant recurrence [P < 0.05.The chi-squared test was used to analyze the associations between SKA1 expression and the clinicopathological features of PTC.currence , 7. Cox  SKA1 mRNA expression in 45 cases of PTC and normal adjacent tissues using qRT-PCR and Western blot analysis. The mRNA levels between PTC tissues and normal adjacent tissues were calculated with the comparative Ct method (2\u2212\u0394\u0394ct). The results indicated that SKA1 mRNA and SKA1 protein expression was significantly higher in PTC tissues than in normal adjacent tissues  showed high SKA1 expression, with only 37% of the samples from normal adjacent tissues see . To confP = 0.005), clinical stage (P = 0.015), and extrathyroidal invasion (P = 0.004); however, no association was found between SKA1 expression and other clinicopathological features. According to the data, SKA1 expression in tumors might be useful for identifying the degree of PTC.P < 0.001) and DRFS (P < 0.001)   and 5. W< 0.001) .An equal distribution of chromosomes depends on the precise regulation of mitosis during each cell cycle, and the correct formation and depolymerization of the spindle are important for the regulation process . Abnorma SKA1 gene is located on chromosome 18q21.1, contains 255 amino acids, and is approximately 30\u2009kDa in size [The in size . SKA1 is in size . The SKA in size . Its car SKA1 in a transgenic mouse model resulted in spontaneous tumorigenesis [ SKA1 inhibited hepatocellular carcinoma cell proliferation by inducing cell cycle arrest in the G0/G1 phase [Li et al.  found thigenesis . In addiG1 phase . The fewG1 phase , 21, 22.To the best of our knowledge, this is the first report on the relationship between SKA1 and prognosis in patients with PTC. High SKA1 expression is predictive of poor prognosis in PTC, implying that SKA1 might be a promising new target for targeted therapies for PTC. However, to determine the mechanism by which SKA1 upregulated expression promotes PTC generation and development and whether SKA1 also has abnormal expression in other solid tumors, further study is needed."}
+{"text": "The x-axis for"}
+{"text": "To assess feasibility of the harmonic Osteovue blade (HOB) for use in the soft tissue approach for dogs undergoing hemilaminectomy and to compare outcomes between dogs undergoing HOB or traditional approach (TRAD).A prospective randomized clinical trial was performed using 20 client-owned dogs with thoracolumbar intervertebral disk extrusion requiring hemilaminectomy. Dogs were randomly assigned to HOB or TRAD. Neurologic function and pain scores were assessed pre-operatively. Intraoperative blood loss and surgical approach time as well as postoperative pain and wound healing scores were recorded. Additionally, neurologic recovery and owner perceived quality of life were recorded at day 10 and 30 postoperative.There was no significant difference in sex distribution, weight, age, preoperative neurological grade and pain score, and perioperative outcome measures between groups. Intraoperative total blood loss was minimal for HOB and TRAD (median: 0 ml (range 0\u20139) and 2.2 ml (range 0\u20136.8), respectively; p = 0.165) and approach times were similar (median: 7 min (range 5\u201312) and 8 min (range 5\u201313), respectively; p = 0.315). While changes in wound healing scores were similar, changes in postoperative pain scores and neurological function were significantly improved in the HOB compared to the TRAD group. Postoperative complications in the HOB group consisted of automutilation of part of the incision and development of a small soft, non-painful subcutaneous swelling in 1 dog each.The HOB is a safe and effective tool for the soft tissue approach for routine spinal surgery in dogs and is associated with decreased pain and increased neurological function post-surgery. Laminectomies are among the most common procedures performed in canine neurosurgery. Soft tissue dissection during the surgical approach is similar to that used in human spine surgery, traditionally incorporating a mixture of sharp dissection with scalpel blade or scissors, blunt dissection with periosteal elevators, and electrocautery  While blVarious methods including acute normovolemic hemodilution, controlled hypotensive anesthesia, or medical intervention with drugs such as anti-fibrinolytics and vasoconstrictors have been reported to decrease intraoperative bleeding during human spine surgery \u201310. AlteStandard instruments for soft tissue approach to the human spine include electrocautery and periosteal elevators such as the Cobb elevator. To circumvent some of the potential complications associated with these standard instruments, the harmonic scalpel was developed. An earlier model of the harmonic scalpel  was previously evaluated for its potential efficacy in posterior spinal instrumentation in humans . In thisThe purpose of this study was to establish feasibility and safety of the harmonic Osteovue blade (HOB) in a relevant spontaneous large animal model of disease: canine patients with spontaneous thoracolumbar (TL) intervertebral disk extrusion (IVDE) requiring spinal cord decompression via hemilaminectomy. Dogs undergoing surgery for IVDE were randomized to have HOB or traditional sharp dissection and electrocautery (TRAD) used for the surgical approach. Outcome assessments included surgical approach time, degree of intraoperative bleeding, postoperative surgical pain scores and wound healing scores. Results of this trial demonstrated that the HOB was safe and effective for the soft tissue approach for routine spinal surgery in dogs and was associated with decreased pain and increased neurological function post-surgery.The clinical study was conducted as a blinded, randomized clinical trial and was coordinated by the Clinical Trials Office at the OSU CVM following Good Clinical Practice guidelines . This clDogs with naturally occurring IVDE involving the TL spine undergoing decompressive surgery by hemilaminectomy were eligible for inclusion. Dogs had to meet the following criteria: intact deep nociception in pelvic limbs on pre-operative neurological evaluation, evidence of spinal cord compression secondary to a single site IVDE between T10 and L6 based on advanced imaging, body weight between 4\u201330 kg, and lack of significant underlying systemic disorders including cardiac, endocrine, renal or hepatic disease that may impair postoperative recovery and wound healing. Informed owner consent was required prior to inclusion of dogs into the study. All dogs underwent neurological examination and were graded according to the following scale: 0 = normal, 1 = spinal hyperesthesia only, 2 = ambulatory paraparesis, 3 = non-ambulatory paraparesis, 4 = paraplegia, deep nociception present.Following enrollment, dogs were randomly assigned to undergo surgery using the traditional approach with sharp dissection and electrocautery  or an approach using the Harmonic Osteovue Blade . Owners were blinded to the type of approach used on their dog. Diagnostic imaging included computed tomography (CT), CT plus intrathecal contrast or magnetic resonance imaging (MRI). For all dogs, except for 2 that were younger than 3 years of age, complete blood counts and chemistry panels were performed. Pre- and immediate postoperative PCV /TP were recorded for all dogs. The anesthetic regimen was routine for all patients and included the following: acepromazine (0.02\u20130.05mg/kg IM or IV) and hydromorphone (0.05\u20130.1mg/kg IM or IV) for premedication; propofol (4-6mg/kg IV) for induction; and isoflurane gas inhalation for maintenance anesthesia. Intraoperatively, dogs received constant rate infusions of lactated ringer solution (3-5mg/kg/hr IV) and fentanyl (5-10mcg/kg/hr IV)If required for cardiovascular support, dogs received glycopyrrolate (5mcg/kg IV). All dogs received cefazolin perioperatively (22mg/kg IV every 90 minutes).All dogs underwent hemilaminectomy via a standard dorsolateral approach . SurgicaIn the TRAD approach, scalpel blade, tenotomy scissors, periosteal elevators and electrocautery were used for sharp and blunt dissection to elevated paraspinous musculature, and hemostasis was achieved with electrocautery . In the HOB approach, all dissection was performed with the harmonic Osteovue blade, using a blunt periosteal elevator for retraction only. The HOB setup included a hand piece, generator, foot switch and the Osteovue blade itself .Throughout the procedure, the maximum setting for cutting was used where the HOB moves at 55,000 Hz with an amplitude of 70 micro meters. Hemorrhages were controlled with the harmonic blade only. In both groups, final exposure for hemilaminectomy was provided using Gelpi retractors.During the approach, no wound lavage was performed and no sponges were used. Blood was cleared from the surgical field using suction only. At the end of the approach , blood was collected from the suction container and the amount measured to determine blood loss. Observations were made regarding amount of tissue bleeding during the approaches and subjective hemorrhage control by cautery and HOB.Closure was the same for all dogs using either 2.0 or 3.0 polydioxanone  in a continuous pattern for fascia, 3.0 poliglecaprone 25  in a continuous patter for a combined closure of the subcutaneous and intradermal layers, and 316L stainless steel staples for skin closure . A Telfa island dressing was applied to the incision until the next day .After completion of surgery, all dogs received a transdermal fentanyl patch (2-4mcg/kg/hr) and recovered on IV fluids and a constant rate infusion (CRI) of Fentanyl for a minimum of 12 hours. If preoperative anti-inflammatory drugs had been administered, they were continued as necessary. If no preoperative drugs had been administered, dogs received postoperative oral NSAIDs. Additionally, dogs received oral tramadol as needed after the Fentanyl CRI was discontinued. Some dogs also received oral gabapentin for additional pain management.For the duration of hospitalization, each dog underwent daily neurological assessment. Additionally, wound related parameters and level of pain were evaluated daily by two clinicians independently, one of which was unaware of the type of surgical approach. The wound assessment form included questions using numeric rating related to discharge, swelling, bruising and incisional pain . Wound aFollowing discharge from the hospital, dogs were evaluated at the OSU VMC at 2 additional time points: 10 days postoperatively at time of skin staple removal and 30 days postoperatively at time of neurologic recheck. At both time points, a complete neurological examination was performed and the current neurologic grade was recorded. This was used to determine neurologic improvement between the preoperative neurologic grade and the subsequent evaluation time points (change in neurologic grade). Two independent evaluators assessed pain and wound healing as described previously. Improvement of pain was also determined relative to the preoperative values and the subsequent evaluation time points and calculated by the change in pain scores and pain VAS.Owners completed a quality of life (QOL) questionnaire form prior to the surgical procedure and at days 10 and 30 post surgery . This prQuantitative data were assessed for normality by plotting histograms, calculating descriptive statistics, and performing the Anderson-Darling test . Quantitative data were described using the median and range due to the small sample size and the apparent violation of the normality assumption. Comparisons between the Harmonic Osteovue Blade (HOB) and traditional surgery approach (TRAD) groups were performed using Mann-Whitney U tests . Categorical data were described using proportions and 95% mid-P exact confidence intervals. Categorical data were compared between HOB and TRAD surgical approaches using chi-square and Fisher exact tests . Statistical analyses for the subjective scales  were performed on the average value of two independent evaluators and inter-observer repeatability was assessed by calculating the coefficient of variation. Non-parametric Mann-Whitney U tests were used to compare changes in the ordinal scale data between HOB and TRAD groups. Statistical results were interpreted at the 5% level of significance.Dogs enrolled into this study included 9 Dachshunds, 6 mixed breed dogs, 2 Beagles, 1 Wheaton Terrier, 1 Silky Terrier and 1 Shih Tzu. Median weight was 9.4kg (range: 4\u201329.1kg). Median age was 75.8 months (range: 30\u2013144 months). There were 10 spayed females, 7 neutered males, 2 intact females, and 1 intact male dog. There was no significant difference of sex distribution between the 2 groups (p = 0.170). Pre-referral treatments included administration of non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, tramadol, gabapentin, and cephalexin. 4 dogs received no medical treatment prior to referral. Preoperative blood work in 18 dogs did not reveal evidence of underlying systemic disorders. Complete blood work was not performed in 2 dogs less than 3-years of age that had no history or evidence of systemic disease on physical examination. Preoperative factors were not significantly different between surgical groups .Neurologic grades measured preoperatively were: grade 1 (n = 1), grade 2 (n = 7), grade 3 (n = 7), and grade 4 (n = 5). Advanced imaging consisted of CT (n = 15), CT/myelogram (n = 4), and MRI (n = 1) and localized a single site extradural compressive lesion to the following locations: T11-T12 (n = 3), T12-T13 (n = 2), T13-L1 (n = 4), L1-L2 (n = 3), L2-L3 (n = 3), L3-L4 (n = 4), and L4-L5 (n = 1).Perioperative factors were not different between surgical groups .Bleeding from soft tissues was subjectively controlled by HOB and TRAD and blood loss during the approach in both groups was considered minimal. Neither HOB nor electrocautery was used for venous sinus bleeding from within the vertebral canal. Venous sinus bleeding did not occur or was considered minor in 19 of 20 dogs. In 1 dog (TRAD group), venous sinus bleeding was considered moderate and part of an absorbable gelatin sponge was used to aid coagulation. The post-operative PCV/TP was lower in all dogs compared to pre-operative; however, there was no difference between the 2 treatment groups.There was one intraoperative complication in the HOB group. The spinous process of one of the approached vertebrae was found to be fractured at the conclusion of the hemilaminectomy and was subsequently removed. No obvious bone damage in this area had been noted prior to hemilaminectomy. There were no intraoperative complications noted in the TRAD group.Improvement over time in neurological grade and pain was documented as the change in grade/score compared to initial preoperative values. The HOB had a better improvement in neurological grade at time of discharge and at day 10 recheck. At discharge, neurologic function was grade 1 (n = 1), grade 2 (n = 7), grade 3 (n = 7), and grade 4 (n = 4). By day 10, all but one dog had improved in neurologic function and all had motor function present . By day 30, 2 dogs were normal (grade 0) and the remaining 17 were able to walk well with residual ataxia and some weakness (grade 2).The postoperative wound healing scores were not significantly different between the 2 groups at any time point. These scores were solely based on visual scoring of the wound and not on photographic appearance. Postoperative wound complications occurred in 2 HOB dogs. They consisted of formation of a small, non-painful swelling associated with the incision noted at day 30 recheck  and self-mutilation of an apparently healed incision with partial wound opening on day 10 without prior wound related issues. The latter underwent primary closure of the cleaned wound and demonstrated routine healing thereafter. There were no postoperative complications noted in the TRAD group.The HOB had a better improvement in postoperative pain (both questionnaire scores and VAS) over the entire study period. Two dogs experienced spinal pain presumably unrelated to the study/type of approach. One dog in the HOB group was described by the owner as being pain free until sudden onset back pain occurred on day 10 . Pain resolved in this dog within a few days with conservative management; no further diagnostics were pursued. One dog in the TRAD group continued to demonstrate general back pain and pain on palpation of the surgery site after discharge from the hospital. Back pain had deteriorated significantly at the 10 day evaluation and the dog was euthanized on day 14 due to lack of response to medical therapy. This dog had demonstrated normal wound healing without incisional complications up to this point. No further diagnostics were pursued prior to euthanasia. Results of comparisons of postoperative factors are listed in Results from questionnaire data are presented in This is the first report of the use of the harmonic Osteovue blade in canine patients undergoing spinal decompression for thoracolumbar disk extrusions. Client owned dogs with spontaneous diseases such as cancer, osteoarthritis and diabetes have been increasingly incorporated into preclinical testing as models for similar human conditions as these can be more reflective of clinical outcome than data generated using induced models in laboratory animals. This has also been the case for dogs with spontaneous spinal diseases and associated surgical interventions including intervertebral disk degeneration and fibrosis development secondary to laminectomy , 31. DatSubjectively, the Osteovue blade demonstrated ease of use particularly for exposure of the affected vertebrae, requiring only the HOB and a Freer elevator for temporary retraction. There appeared to be excellent control of surface hemorrhage from subcutaneous tissue and muscle, and larger bleeding vessels could be spot coagulated effectively . Due to the risk of iatrogenic damage, bleeding from the venous sinus within the spinal canal was not coagulated using the HOB or electrocautery. While sinus bleeding was subjectively rated as absent or minimal in most patients and bleeding from cancellous bone during the hemilaminectomy appeared similar among dogs, both could have affected postoperative PCV and TP.Removal of soft tissues from and around bone was possible without visibly gauging or burning the bone surface or breaking the HOB. Importantly, the sound caused from blade vibrations was altered during contact with bone, providing a ready indication during the surgical procedure and thus avoiding potential complications such as thermal injury to the bone structure that could lead to thermal necrosis. The only observed intraoperative complication in the HOB group was base fracture of a spinous process of one of the 2 vertebra where the hemilaminectomy was performed. The fracture noted at the end of the surgery prior to wound closure and was believed to be secondary to undercutting of supporting bone during hemilaminectomy. The affected dog was very small with thin spinous process bone, which would not require a tremendous stress riser to induce a fracture if weakened. While retrospective evaluation of video recording of the surgical approach in this patient did not identify prolonged HOB contact with bone, it cannot be entirely ruled out as the cause of the injury.Despite validated pain scales and assessment tools, evaluation of postoperative pain in dogs can be challenging. We chose the Glasgow composite pain scale for assessment of generalized signs of pain and wound pain while dogs were in the hospital . Dogs inWhile a variety of wound assessment scores have been described to evaluate healing of open wounds, such as pressure sores or granulating wounds , 36, theWhile not reaching significance , lower blood loss occurred in the HOB group compared to the TRAD group Click here for additional data file.S2 FileDocument to record various aspects of pain related factors for individual patients.(DOCX)Click here for additional data file.S3 FileDocument with various questions relating to the patient\u2019s owner perceived quality of life.(DOCX)Click here for additional data file.S1 FigExamples of incision photographs of one dog with TRAD and one with HOB approach on day of surgery and 1, 2, and 3 days postoperatively.(JPG)Click here for additional data file.S1 DataDataset of evaluated factors of all patients, including patient and surgery factors, neurologic grading, pain questionnaire and visual analog scores, incision healing scores, owner perceived neuro status and function, and quality of life factors.(XLSX)Click here for additional data file."}
+{"text": "Computational models can provide detailed information about molecular conformations and interactions in solution, which is currently inaccessible by other means in many cases. Here we describe an efficient and precise coarse-grained model for long polysaccharides in aqueous solution at different physico-chemical conditions such as pH and ionic strength. The Model is carefully constructed based on all-atom simulations of small saccharides and metadynamics sampling of the dihedral angles in the glycosidic links, which represent the most flexible degrees of freedom of the polysaccharides. The model is validated against experimental data for Chitosan molecules in solution with various degree of deacetylation, and is shown to closely reproduce the available experimental data. For long polymers, subtle differences of the free energy maps of the glycosidic links are found to significantly affect the measurable polymer properties. Therefore, for titratable monomers the free energy maps of the corresponding links are updated according to the current charge of the monomers. We then characterize the microscopic and mesoscopic structural properties of large chitosan polysaccharides in solution for a wide range of solvent pH and ionic strength, and investigate the effect of polymer length and degree and pattern of deacetylation on the polymer properties. Thus Chitosan is composed of (1-4) linked units of 2-amino-2-deoxy-\u03b2-d-glycopyranose (GlcN) as well as, because deacetylation is never complete, GlcNac monomers. Typically, the polymers are referred to as chitosan if the degree of deacetylation (DD) is larger than 50%. The amino groups of the deacetylated monomers can be protonated in mildly acidic conditions, making the polymers soluble and Chitosan one of the rare cationic polymers [2 or positively charged Chitin is one of the most abundant natural biopolymers and the most abundant amino-polysaccharide on the planet . Its mairegation .As it is readily available and possesses many desirable properties including bio-compatibility, bio-degradability and low nanotoxicity, Chitosan has found many applications in food, cosmetic, biomedical and pharmaceutical industry , 8, 9. P\u03b1-Chitin, however different types of helical structures are also observed for certain experimental conditions [A precise understanding of the relation between structural characteristics of Chitosan in aqueous solution and these factors is of great interest for the efficient engineering of Chitosan based materials. In crystalline Chitosan, the polymers typically have an extended twofold helical structure \u201313 similnditions \u201317. Infonditions \u201320.Molecular simulations can provide valuable insights into the molecular structure under different conditions. However, the large size and complex interactions of polysaccharides present severe limitations for accurate computational modeling. To make matters more complicated, the strong pH dependence of the amino group charge requires careful treatment. Nonetheless, all-atom models have been applied to study the conformational flexibility of different chitosan oligosaccharides  and the \u03b1-Chitin [Coarse grained (CG) simulation models, which group a number of atoms together into one interaction center can overcome some of the above limitations, however, at the cost of the detailed chemical information. Different strategies for the parametrization of bottom-up CG models for different polysaccharides, which preserve as much chemical detail of the atomistic system as possible have been pursued in the past \u201327. In a\u03b1-Chitin  crystal [GlcNH3+  monomers\u03d5 and \u03c8 shown in \u03d5 and \u03c8, which is incorporated in the model via potential of mean force (PMF) maps obtained from all atom sampling methods.One strategy that has been effective for sampling the conformations of large polysaccharides in solution, is based on the conformational preferences of the most flexible degrees of freedom, the glycosidic angles Here, we describe and validate a similar CG model for the precise characterization of Chitosan polysaccharides in solution, which samples the conformations of all glycosidic bonds using pivot move Monte Carlo (MC) simulations. The monomers are mapped into one CG interaction center, on which steric and electrostatic interactions act. PH and ionic strength of the solution enter the model via titration moves on the GlcN monomers, taking into account both the intrinsic pKa value of the monomers and the local electrostatic environment created by nearby charges. We observe that the details of the glycosidic PMFs can significantly affect the structural characteristics of long polymer chains. Furthermore, a change in the charge state of a GlcN Monomer may affect the free energy map of the glycosidic links at both ends, slightly shifting the position of the minima and their relative depths. Therefore, we calculated a complete set of nine different PMF maps depending on the monomer types and charge state at both ends of the glycosidic link. In a protonation move, the corresponding dihedral maps are adjusted according to the new charge state. This procedure is found to greatly improve agreement with experimental observables.In the following sections, first a detailed description of the model, CG force field, and simulation protocol is given. Then the model is validated against equilibrium properties of chitosan polymers and applied to systematically investigate their dependence on degree of polymerization (DP), degree and pattern of deacetylation, ionic strength and pH.i.e. SPC [All-Atom MD simulations were used to define the topology and bonded interactions of the CG model. Simulations were performed in GROMACS 4.6.1 , 41, usii.e. SPC , SPCE [4i.e. SPC , Tip3p, i.e. SPC , Tip4pewi.e. SPC  and Tip5i.e. SPC .The sugar molecules were solvated in a cubic box of 4x4x4 nm. Initial structures were energy minimised by 100000 steps of steepest descent followed by 200 ps equilibration using the stochastic dynamics integrator with a 2 fs timestep at a temperature of 298 K. The pressure was set to 1 bar with the berendsen barostat . Bonds i\u03d5 and \u03c8. For computational efficiency, Metadynamics on a grid was used with 200 bins for each angle. To obtain smooth sampling, a deposition frequency of 25000 steps, i.e. 50 ps was used with a Gaussian width of 0.25 Rad, which corresponds to half of the standard deviation of the fluctuations of the dihedral angles in the unbiased simulations and a Gaussian potential height of 0.5 kJ/mol. The Maps were sampled for 200 ns.Metadynamics simulations \u201355 were \u03c8 and \u03d5 was used [Previous MD simulations found that each glycosidic link is independent of its neighbors , and PMFwas used .\u03c8 and \u03d5. The topology of the CG model retains the atoms defining the two dihedrals, illustrated by the thick bonds in 2 -GlcNH2. The atoms of the carbohydrate rings are mapped into additional CG interaction sites, on which steric and electrostatic forces act. These are centered on the average center of geomerty of the ring atoms for the QM optimized structure of the sugar monomer.The CG polymer model makes use of the restricted conformational space of The interactions that describe the CG forcefield are bonded interactions along the polymer and non-bonded interactions between remote polymer sites. Water molecules are not explicitly represented in the model. The effect of hydration are implicitly included via the glycosidic PMF maps. The details of the different interaction potentials are described in the following:Bonded interactions between successive monomers are represented by the free energy maps of the glycosidic torsion angles \u03c8 and \u03d5. These maps capture the strong interdependence between the two angles, and indirectly incorporate all the factors contributing to the conformations of \u03c8 and \u03d5 at the atomistic scale, such as van der Waals and electrostatic interactions between the bulky rings, hydrogen bond formation, solvation effects and conformational entropy of the link. The strong conformational restriction of the glycosidic links is reflected in the relatively small areas of the minima.Excluded volume effects were included using a repulsive Lennard-Jones (LJ) potential of the formr is the distance between the monosaccharide centers, \u03b5LJ and \u03c3LJ are the LJ energy parameter and radius, respectively and rc = 2(1/6)\u03c3LJ is the cutoff radius, corresponding to the minimum of the LJ potential. \u03c3LJ was chosen such that the surface area of the LJ sphere is equivalent to the molecular surface area of the monomer. As the LJ interactions are purely repulsive, results are very insensitive to the value of \u03b5LJ and thus the parameters for carbon \u03b5LJ = 0.6276 kJ/mol was chosen. Lorentz-Berthelot mixing rules were used.Electrostatic interactions between non-adjacent charged monomers in aqueous solutions of different ionic strengths were modeled with Debye-Huckel interactions.z, equal to 0 or 1, is the valence of the chitosan monomers, \u03bbB is the Bjerrum length equal to 7.14 \u00c5in water at 298 K and \u03ba\u22121 is the Debye length. The ionic strength cs of the solution enters the interaction through the relation cs. A semi-grand canonical ensemble is employed for modeling the titration of the weak base monomers, similar as in previous studies [zi = 0 or zi = 1 according to their protonation state. The free energy contribution from the titration state of the polymer is written as the sum over all Nt titratable monomers in the polysaccharide\u03bci of protonating the individual monomers and the second term describes the contribution of the screened electrostatic interactions with the other charged sites in the polymer, including also nearest neighbors.Each deacetylated Chitosan monomer represents a weak base, which can be neutral or protonated to a positively charged site. The protonation state of these monomers will depend on the pH of the solution, but also on the local electrostatic environment defined by the charges of the surrounding monomers and  studies \u201361. The i, by i lies between 6.0 and 7.1. Therefore, here we chose a suitable value for pKi from fitting experimental curves for the degree of dissociation \u03b1 as a function of pH, as described below.The chemical potential for protonation is related to the pH and the intrinsic dissociation constant of a single site, pKThe polysaccharide\u2019s conformational space is sampled with Metropolis MC simulations. Trial conformations were generated using simple Pivot Moves (PM) , 63 aroukBT.Each PM MC step represents a move of the torsion angles of one randomly chosen glycosidic bond, to a random position on the corresponding PMF map. To maximize sampling of the entire map, no limit on the step size is used. Instead, only steps to regions of the map with energies below a chosen cut-off are attempted, to achieve reasonable acceptance rates. For most cases, this cutoff was chosen to be 7 Nt protonation moves are performed on randomly chosen titratable sites [In addition to the conformation altering moves, for each PM MC move le sites , 61. ThiSimulations are stopped and the systems considered to be in equilibrium when the glycosidic angles have sampled all accessible regions of the PMF maps and the physico-chemical properties of the polymer show equilibrium distributions. The number of steps required to achieve this depends on the system conditions, as those determine the acceptance rate.Simulations were performed with a code based on the cross platform. Net framework Mono  and OpenLP is defined by the decay of the bond vector correlation Ck = \u2329bi \u22c5 bi+n/|b|2\u232a, as Ck = exp(\u2212nb/LP). For polyelectrolytes, Ck is determined not only by the stiffness of the bonded structure, but also by the repulsive electrostatic interactions of charged monomers. Therefore, in the context of polyelectrolytes, the concept of an electrostatic persistence length has been introduced [LP are often decomposed into an intrinsic part LP,0, corresponding to the polymer stiffness at infinite cs, and an electrostatic contribution LP,e.The persistence length troduced , 67, to Ck, as seen in the example shown in cs = 0.001. The different scales make the identification of LP from fitting ln(Ck) ambiguous. To determine the influence of the separate contributions to LP, we obtain LP,0 from a set of separate simulations without the electrostatic interactions, as illustrated in LP,0 and LP are obtained by fitting ln(Ck) from both simulations. For LP the fit is limited to large n, where a linear decay is approached.As described previously , the difAlthough the Amber-Glycam force field has been parametrized in combination with the TIP3P water model, it is frequently also used with the TIP5P water model. Recent MD simulations of saccharide solutions have observed a large effect of the water model on the solution properties , and sug2 monomers are weak bases, so that the Chitosan polymers are made up from the three monomeric building blocks GlcNH2, Chitosan is rarely 100% deacetylated and the deacetylated GlcNHt low pH  suggestet low pH .kBT all present a similar overall topology, with the main minima present at the same angles. However, both the size of the main minimum and the energy difference to the second minimum, \u0394G2, and thus the population of this second minimum, depends on the link type. Some of the main features contributing to the conformational flexibility of the links are summarized in Here we calculated a complete set of nine PMF maps, to include all combinations of charge state and acetylation. The maps, shown in G2 = 1.35kBT. The link type with the highest \u0394G2 is the neutral GlcNH2 -GlcNH2 dimer.The general features of the maps shown here are in good agreement with the findings obtained in . The mapG2, whereas the strong electrostatic interactions of two adjacent charged units  clearly shows the effect of the map features on the mechanical polymer properties. The data shows that the flexibility is mainly, but not completely, determined by the depth of the second minimum. The most flexible polymers with LP,0 = 2.38 nm are produced by the 2 map with LP,0 = 4.14 nm. These two maps also posses the lowest \u0394G2  model. A comparison of several sets of light scattering data obtained with similar conditions showed, that most values of RG follow the same dependence on the DP [cs close to 0.3 follow a similar exponent, but as expected, lower values of RG [LP. Similarly, as our model reproduces the reported values of RG well, good agreement for LP could be expected. However, here the relevant question is, how the value obtained in analogy to experiment from the WLC model agrees with the microscopic value found from the decay of Ck.Experimentally, n the DP , 89, 90.es of RG , 88, 95.LP,0 from the radius of gyration of the polymer in solution, i.e. in a perturbed state, can be obtained following the model of Odijk and Houwart [LP is calculated using an iterative procedure from the Benoit Doty [L is the polymer\u2019s contour length. This estimate of LP is used to evaluate the electrostatic excluded volume parameter RG,0, is obtained from RG = \u03b1elRG,0. This process is iterated until the results converge.An estimate  Houwart , which poit Doty  relationLP,e isLP,0 = LP\u2212LP,e. However, the scaling of LP,e with \u03ba\u22122 is somewhat controversial in the literature. It is predicted by several stiff polymer models [LP \u221d \u03ba\u22121 instead [\u03ba\u22121 was observed at large \u03ba\u22121 [LP depends linearly on \u03ba\u22121 for all values of \u03ba\u22121 used, in agreement with experimental data from [LP,0 predicted from the Odijk model. In addition, In some cases, a polydispersity correction to the z-averaged Radius of Gyration, can lower the estimated value of the persistence length significantly [The expression proposed for r models , 67, whe instead . Detailearge \u03ba\u22121 . Howeverata from . This dificantly , 89. HowLP for different cs at pH = 4, we find that i) approximating cs \u2265 0.1, for polymers with DP \u2248 1000; ii) the values for the total persistence length using the approximation LP,0 obtained from the WLC model and cs, where LP,e becomes negligible, and for very low cs, where presumably the proposed \u03ba\u22122 relation is approached. For intermediate values however, LP,e which are small compared to the total persistence length, and therefore much larger values of LP,0. Using the linear relation LP = 7.06 \u2217 \u03ba\u22121 + 8.6 obtained from fitting the data instead, values of LP,0 between 7.7 and 10.1 are found for all cs, which agrees well with the values between 8.5 and 11.5 obtained from the microscopic definition. The estimates of LP,0 between 6 and 12 nm found by several experimental studies performed at pH 4.5 following this procedure [Comparing the WLC values to the microscopic rocedure , 90, 93 i were reported to depend on DD [Previous work has shown, that solution properties and self-assembly of Chitosan depend strongly on the DD. Furthermore, also the titration behavior and estimates of the pKnd on DD . Three rnd on DD  to aggrend on DD , 100 andnd on DD , 80. Forapp decreases more rapidly with \u03b1 at low ionic strength. However, for low values of DD, both the cs dependence and the dependence of pKapp on \u03b1 become less significant, as the spacing between charges along the backbone is greater, so that screening sets in at much lower cs.\u03b1 < 0.6, for all ionic strengths. For higher values of \u03b1, the simulated curves continue to increase linearly and meet at pKapp = pKi for the neutral polymers at \u03b1 = 1. The experimental values for pKapp on the other hand begin to deviate from the linear dependence, towards lower values, and different extrapolations for pKi are found for different ionic strengths.The data for the highly deacetylated polymers Click here for additional data file."}
+{"text": "The Four Free and One Care Policy (HIV/AIDS-related free services) has been in place in China since 2004. However, linkage to human immunodeficiency virus (HIV) care is not yet achieved very well among people living with HIV. We conducted a qualitative study to explore individual and contextual factors that may influence a linkage to HIV care from the perspective of young HIV-infected men who have sex with men (MSM) in a highly centralized HIV care context of China.Purposive sampling was used to recruit 21 HIV-infected MSM in Shandong Province, with in-depth interviews conducted between March and July 2015. Thematic content analysis was subsequently used for data analysis.Key barriers and facilitators related to a linkage to HIV care emerged from participants\u2019 narratives. The barriers included perceived healthy status, low health literacy, and stigma associated with receiving HIV care. The facilitators included an awareness of responsibility, knowledge associated with health literacy, social support, and trusting and relying on services provided by the Center for Disease Control and Prevention (CDC) and the government. These were related to the quality of current HIV counselling and testing, service promotion, and the cost and placement of these HIV services.In order to improve the MSM linkage to HIV care in China, it is imperative to improve the quality of the current on-going counselling and testing. Further critical linkage support includes increasing supportive services among local CDC systems, designated hospitals and community-based organizations (CBOs), and more financial support for HIV/AIDS related testing, medical checkups and treatments. Linkage to care is a critical step in the HIV continuum of care . The WorIn China, the HIV epidemic has been expanding rapidly among men who have sex with men (MSM), accounting for 17.4% of people living with HIV (PLWH) . In ordeA nationwide study of HIV-infected persons in the United States found that significant disparities existed in the continuum of HIV care among different subgroups . There cIn China HIV care is highly centralized with the Centers for Disease Control and Prevention (CDC) in charge of HIV/AIDS related counselling and testing. This is done through cooperation with designated hospitals to provide medical checkups and antiretroviral drugs (ARV) for PLWH . An indiThis qualitative study was conducted in Jinan and Qingdao, two major cities in Shandong Province, China, from March to July 2015. Ethical approval was granted by Institutional Review Board of the University of North Carolina, Chapel Hill; University of California, San Francisco; Shandong University, and Shandong CDC. We defined the linkage to HIV care as completing a confirmatory testing  or undertaking CD4 testing after the diagnosis. Based on the literature and the findings of our previous studies in China, we refined our semi-structured in-depth interview guide to add questions exploring participants\u2019 local life experience as a tongzhi/gay and being HIV-infected in order to understand their relationships and interaction with peers, friends and healthcare staff. The interview guide featured perception, cognition, and experiences of linkage to HIV care, as well as its related psychosocial, economical, and environmental factors. Main questions included \u201cAfter you were diagnosed with HIV infection, how did you feel and what did you do?\u201d \u201cIn your point of view, what are the advantages and disadvantages of the HIV-related healthcare services, such as confirmatory testing and CD4 testing after the diagnosis?\u201d \u201cDid you receive these HIV-related healthcare services? Why did you receive these services/why not?\u201d \u201cWhat factors facilitate or impede your access to these HIV-related healthcare services?\u201dPurposive sampling scheme was adopted, which planned to recruit approximately 20 HIV-infected MSM, including about half of these MSM being linked to HIV care and another half being not linked or facing delays in their linkage to HIV care. The inclusion criteria included: (1) HIV-infected , (2) men who reported to having had sex with men in the past 12\u00a0months, and (3) aged between 18 and 35 years at the time of HIV diagnosis. According to the above scheme, the local CDC worked with community-based organizations (CBOs) to source eligible participants by way of convenience through their contacts and networks. They contacted 29 potential participants who were eligible, 21 of them agreed to participate, and the other 8 potential participants refused. Potential participants were informed about the current study aiming to understand how they perceived and/or experienced HIV-related healthcare services, in particular, the uptake of confirmatory testing  and CD4 testing after the diagnosis of HIV infection. After potential participants agreed to participate, appointments were set up and the interviews were arranged in a private room in the local CDC or CBO office. Participants were informed that refusal would not affect their right to use healthcare services, that the interview content would be digitally recorded and transcribed verbatim, and that privacy protection was assured. Written informed consent was then obtained. The first author conducted all of the interviews using mandarin Chinese and participants were informed not to provide their real names, working and home addresses in the interview for the purpose of confidentiality protection. Each interview lasted approximately 90\u2013120 min. In order to compensate for participants\u2019 time spent and transportation costs, we provided each participant 100 RMB (about $16 USD) in cash after completion of the interview. The digital recording was transcribed verbatim in Chinese by student assistants.Socio-demographic data were summarized for sample description. We then conducted thematic content analysis on the transcripts.  Each intWe recruited a total of 21 eligible participants (Table\u00a0Three major barriers to linkage to HIV care were identified: (1) perceived healthy status; (2) low health literacy; and (3) stigma associated with receiving HIV care.Some participants reported feeling in a good physical condition with no health concerns as a justification for not linking to HIV care. For example, one participant (JN03) said: \u201cWhen I was diagnosed as HIV-infected, I didn\u2019t want to come out of this place, taking CD4 testing and so on. I felt good physically. Nothing happened. It should be no problem. \u2026I felt very young, very healthy, and did not want to accept [the services in] CDC and did not want to come here.\u201d Another participant (QD08) also described a similar view: \u201cIn terms of physical condition, there\u2019s no big problem in my body, just the same as before, very good, no difference. I think it was not necessary [to visit CDC].\u201d In this regard, some of these HIV-infected MSM interviewed, in particular younger informants, usually perceived themselves to be in a good health and therefore did not take their infection of HIV seriously. This misconception may have been related to low health literacy levels.Many participants lacked a satisfactory knowledge that enabled them to get access to HIV care, such as what services, where to get access, benefits and potential risks. This resulted in ignorance of dealing with their illness, or made them confused and scared. For example, one participant (JN05) described: \u201cI was confused. What\u2019s CD4? What\u2019s viral load? What medicine should I take? Why to take medicine? What benefit? I had no idea at all. \u2026 Then, I ran away. Anyway, I felt very scared at that moment.\u201d Some HIV-infected MSM appeared to have a low health literacy about HIV/AIDS and its related services and therefore rejected free HIV care provide by the CDC.Another participant (JN02) described: \u201cShe [a CDC staff] didn\u2019t say anything at all on the phone, just asking me to take my ID card to make a diagnosis. Why should I have a diagnosis? What\u2019s the difference between a diagnosis and non-diagnosis? Because I had no idea of CD4 testing. I thought, when I really get sick, it should not be too late to take medicines. I therefore just stayed at home, and didn\u2019t think about it at all.\u201d In this regard, due to a low health literacy, some HIV-infected MSM may regard HIV/AIDS as a general disease, which should only be treated when feeling sick.The HIV care provided in the CDC were perceived as negative by some participants, because it was a setting associated with HIV/AIDS, a highly publically stigmatized disease. This perception has also been labeled as \u201ccourtesy stigma\u201d, a type of stigma by association . For thiThese MSMs\u2019 negative perceptions of the CDC health care settings could well escalate further anxiety associated with attendance and concordance with care. For example, one participant (JN03) described: \u201cI didn\u2019t know much about this disease. I didn\u2019t want to visit this kind of place [CDC] and test for CD4. It\u2019s jumbled. I was afraid of encountering acquaintances, feeling awkward. Furthermore, this disease is so\u2026[stigmatized].\u201d In this regard, some HIV-infected MSM held negative perceptions of the services provided in the CDC setting and thought they may encounter embarrassing situations.Several facilitators to HIV care were identified, including: (1) an awareness of responsibilities; (2) knowledge associated with health literacy; (3) social support; and (4) trusting and relying on CDC services and the government.Many participants who were linked to HIV care expressed their sense of self responsibility. For example, one participant (JN01) said: \u201cIt\u2019s about how you look at yourself. \u2026 You went out to have sex and got infected. It\u2019s your own behavior. You should take your own responsibility, right!\u201d Some HIV-infected MSM also realized their responsibilities to their families and friends. Another participant (QD09) also said: \u201cIt\u2019s mainly responsible to my own health, both physically and psychologically, and it\u2019s also responsible to my friends and families.\u201d He elaborated further: \u201cIt\u2019s hard to say taking responsibility. It\u2019s about not doing too much harm to them. After all, closer cooperation to services results in a better life. Families and friends would worry less. Poor cooperation would easily result in transmission to others.\u201d In this regard, some HIV-infected MSM realized that linkage to a continuum of HIV care would diminish harm and improve their lives.Some participants reported when they had knowledge about HIV/AIDS and associated self-care, they actively took part in HIV care . For example, a participant (QD10) said: \u201cI realize knowledge is very important. \u2026 I insist in taking a CD4 test every three months. \u2026Because I think, if there is a change of the value, I can see it in every three months.\u201d Another participant (JN07) also expressed his view that \u201cIt\u2019s for my own health. Anyway, I feel that I have to do the test twice per year, and it\u2019s for my own health. It\u2019s good if nothing happens. But if there\u2019s something going on, I will be informed timely.\u201d These HIV-infected MSM with a sound knowledge about HIV/AIDS and health literacy of self-care were able to monitor their own health through actively engaging in the HIV care.Some participants reported obtaining social support from friends and work force peers. For example, friends encouraged and reminded them to receive HIV care, thereby providing psychological and spiritual comfort. A participant (JN04) described: \u201cMy friend helped me a lot. \u2026 He reminded me some things that I didn\u2019t know. \u2026 He told me some things that were helpful for me. It\u2019s a kind of comfort, improving me. \u2026 At least, I obtained comfort spiritually. Sometimes if only me, I really didn\u2019t want to come [to CDC]\u201d. Friendships provided multi-functional support, not only reminding him but also supporting him both spiritually and psychologically. Another participant (JN05) shared similar experiences describing: \u201cHe [a friend] enlightened me. We chatted, had food and played together. \u2026 Gradually I calmed down. \u2026 He has a lot of knowledge and explained to me. \u2026Gradually I got to know that it\u2019s not so terrible, and then I changed slowly. \u2026 I trusted him and thought it should be nothing more than a diagnosis. \u2026At that time I have accepted it and tried to have a change.\u201d Friends and peers were able to not only share HIV related knowledge and information about services, but also provide companionship, which is crucial for HIV-infected MSM to link to HIV care.Of critical importance for these MSM was the obtaining of personal care and support from healthcare professionals even out of office hours. A participant (JN02) said: \u201cThe doctors here get along very well with patients like us. The doctor spent time talking and chatting with us. He taught me to make tea using honey and lemon together, which does a lot of good in our body. He told me a lot of relevant knowledge.\u201d This positive interaction between healthcare professionals and HIV-infected MSM establishes a patient/doctor rapport. Moreover, QQ (a widely used online media platform in China) has been used by local CDC to set up support groups for HIV-infected individuals. Another participant (QD05) who was a member of a QQ support group described: \u201cFor HIV-infected individuals like us, the services provided by CDC are currently imperative. They really help us a lot. Without their help, we perhaps are completely filled with questions and do not know where to start. We do not know how to take treatments. With their help, we can at least know when to take medicine, when to do testing, such as CD4 and viral load testing. In this regard, their care is very helpful for us.\u201dOne strength of CBO is being close to the MSM communities and able to provide personalized services to specific HIV-infected MSM. A participant (QD03) recalled: \u201cXX (a CBO member) stated \u2018Don\u2019t worry too much, CDC has been doing this work for a long time, they perform well in protecting confidentiality. He then talked to me for a while. I then agreed [to conduct a diagnostic test in CDC]\u2019.\u201d The CBO provided psychological counselling to these men, and accompanied them to go through the linkage process, which made these men feel they were supported and encouraged. Another participant (QD05) described: \u201cBefore taking a test, CBO will provide you with psychological counselling. When people feels relaxed and no longer afraid of AIDS, the CBO will then provide you with a HIV test. \u2026 If you let me do it by myself, I'm definitely not going to do so. If I go to do it by myself, I feel afraid. To come here (CBO), no matter to do a testing or not, at least someone will accompany me, giving me spiritual support, cheering me up and giving me encouragement.\u201dMany participants trusted and relied on the CDC and the government. For instance, a participant (JN06) described: \u201cNow there is a CDC staff taking care of us. At least there is a leader in our circle. If we have any questions, we can ask the doctor and he will give us answers. At least we are not alone. \u2026 If nobody manages us, we will be very blind and alone. If things turn upside down and disorderly, our illness will become more and more serious. If there are any complications, the doctor can help us to control it in advance. I feel hope anyway.\u201d These men accepted the HIV/AIDS-related services from CDC. Some participants also expressed an expectation of greater financial support for medical checkups and treatment, more service sites, as well as better training and support for CBOs. Another participant (JN01) said: \u201cThe government has provided us with free medication and all of the services provided by the government are free. \u2026 There is no heavy economic pressure. \u2026 This only aims to make us live healthy. There is no reason to refuse cooperation.\u201dFew qualitative studies have investigated issues related to the linkage to HIV care from the perspective of PLWH in China, in particular delayed diagnosis, late entry care after a HIV diagnosis, and factors influencing the decision to seek care , 18. In Our findings contend that the quality of current HIV counselling and testing needs to be improved. Our participants testified to having a lack knowledge of HIV care and their own health status, were worried about stigmatizing settings in the CDC and perceived multiple barriers to accessing HIV care . As a result, some HIV-infected MSM reported not linking into the formalized HIV care available . This was seen by our informants to be related to the limitations of current HIV counselling and testing, in particular, the pre-test and post-test counselling services. Researchers have argued that HIV diagnosis alone is not enough to ensure that PLWH will get adequate and timely health care, and therefore ask for greater emphasis on the core value of linkage to HIV care in counselling and testing recommendations . CounselIt is also crucial to provide more training for HIV counselors, which contribute to high rates of linkage to HIV care . In suchBeside the suggested improvements to local HIV counselling and testing, it is also important to provide support services to promote linkage to HIV care. Our analysis indicates that social support from peers, families and friends, healthcare professionals, and CBO services promotes the linkage to care for these young adult HIV-infected MSM. This is consistent with studies arguing that support services, such as case management, outreach, health navigator, mental health services, motivational interviewing, and support groups or educational activities, are crucial in improving linkage to HIV care , 24. GivThe cost and location of HIV/AIDS-related services are also an important concern. The analysis shows that many HIV-infected MSM accepted the health management of HIV/AIDS in China\u2019s CDC system and showed a willingness in trusting and relying on the government. This was expressed as expecting more service sites in the CDC system and designated hospitals, more financial support in HIV/AIDS related medical checkups, and more training and financial support for effective CBOs. Government support is crucial in linking these men to HIV care, because having public medical insurance and being self-funding were reported to be negatively associated with linkage to HIV care . It has The current study is subject to some limitations. Firstly, the themes analyzed were limited by the extent to which individuals disclosed the issues that were relevant and important to them in a comparatively short time. Secondly, the sample was small and the sampling of participants was limited to social networks of local CDC and CBO. Thirdly, it was difficult to recruit HIV-infected MSM who had refused to contact the CDC or the CBO. As an alternative strategy, we recruited HIV-infected MSM who had refused to undertake WB, or lost to follow-up for CD4 testing. Finally, since this was a context specific qualitative study, there are associated transferability concerns.The results have advanced knowledge regarding the barriers and facilitators related to linkage to HIV care among HIV-infected young adult MSM in contemporary China. In order to improve the MSM linkage to HIV care in China, it is imperative to improve the quality of the current on-going counselling and testing. Further critical linkage support includes increasing supportive services among local CDC systems, designated hospitals and community-based organizations (CBOs), and more financial support for HIV/AIDS related testing, medical checkups and treatments."}
+{"text": "ST, MDS plot, AMOVA and SAMOVA. Haplogroups were classified and their geographical distribution was visualized using surface interpolation maps. Results of the present study showed that Poles are characterized by the main West Eurasian mtDNA haplogroups. Furthermore, the level of differentiation within the Polish population was quite low but the existing genetic differences could be explained well with geographic distances. This may lead to a conclusion that Poles can be considered as genetically homogenous but with slight differences, highlighted at the regional level. Some patterns of variability were observed and could be explained by the history of demographic processes in Poland such as resettlements and migrations of women or relatively weaker urbanisation and higher rural population retention of some regions.The aim of the present study was to define the mtDNA variability of Polish population and to visualize the genetic relations between Poles. For the first time, the study of Polish population was conducted on such a large number of individuals (5852) representing administrative units of both levels of local administration in Poland (voivodeships and counties). Additionally, clustering was used as a method of population subdivision. Performed genetic analysis, included F It is used especially in population genetics and molecular evolution studies and allows to understand the question of human migration and settlement from different regions of a country or the whole world , 2. MatemtDNA variability in Polish population was studied in comparison to Russians , 2 or asNational population biobanks and sample repositories store human biological material for the use mostly in genetic research to connect the lifestyle and medical history with genetic traits. Genetic and molecular information associated with the data about the sample donor can also be used in population studies . Furthern\u2009=\u200916) and counties (n\u2009=\u2009349) with the more natural division into clusters (n\u2009=\u200980) which may largely correspond to geographic regions.The aim of the present study was to determine mtDNA variability of the Polish population, including geographical and historical context. For this purpose, obtained haplotypes of 5852 individuals were classified into major haplogroups and subhaplogroups, and their distribution for units of the first (voivodeship) and second (county) level of local government and administration in Poland was analysed. For the first time, the study of the Polish population was conducted on such a large number of individuals. The gathered data set was then clustered on the basis of genetic information as well as the information about the place of origin, letting us to compare the quite artificial division into voivodeships .The studied population consisted of individuals recruited between 2010 and 2012 within the TESTOPLEK research project. All samples belonged to the POPULOUS collection which is registered since 2013 in the BBMRI catalog , 20. TheK-means clustering method applied to spatial coordinates was used to merge individual counties in larger geographic groups (clusters) on the basis of the nearest mean. Each cluster Fig.\u00a0 is repreThe list of clusters containing the information about the cluster number to which each county was assigned as well as the name of the corresponding geographic region, is gathered in Supplementary Table\u00a0Infinium HTS Human Core Exome PLUS microarrays were used to genotype DNA samples for 551,945 SNPs according to the manufacturer\u2019s protocol . Qualitative analysis was performed to identify outliers and artefacts on the microarray. Samples were excluded if call rate was below 0.94 and if the 10% GenCall parameter was below 0.4. Visual inspection was conducted to investigate the heteroplasmy, which was detected only in a few cases.Applied microarrays allowed the identification of 323 SNPs (single nucleotide polymorphisms) in mtDNA (Tab. Shttp://phylotree.org/tree/index.htm) [ST values [ST with cmd scale function in R ver. 3.4.2 [Haplogrep software was then used to classify haplotypes into haplogroups and subhaplogroups  . HaplogrT values , both foT values . To visur. 3.4.2 . Furtherr. 3.4.2  was doneFinally, the geographical distribution of lineages H, U, J, T, HV, K, W, I in Polish population was represented by a surface interpolation map built with QGIS version 2.18.16 .21mtDNA haplotypes belonging to haplogroups Table\u00a0. The mosn\u2009=\u200919) was observed in Silesian voivodeship while the lowest (n\u2009=\u200910) was observed in Holy Cross voivodeship. However, they were represented by as the largest and the smallest sample number; 963 and 72, respectively. Among the voivodeships with the sample number between 200 and 500, 17 haplogroups were observed in Lesser Poland while only 12 in \u0141\u00f3d\u017a voivodeship. Analysis based on Pearson\u2019s Chi-square test was performed to assess the differences between voivodeships infrequencies of 10 main haplogroups . The highest number of haplogroups  as well as for Holy Cross and Pomeranian . Furthermore, Holy Cross was the only voivodeship with FST values higher than 0.01; the differences concerned also Lesser Poland ; Subcarpathian ; Lublin ; Opole  and Kuyavian-Pomeranian   Fig.\u00a0.ST values) were observed for the same voivodeships as in Holy Cross comparison. However, FST values were lower in each case: Lesser Poland ; Subcarpathian ; Lublin ; Opole  and Kuyavian-Pomeranian . As in the case of Holy Cross, the highest FST values for this region were observed with West Pomeranian  and Pomeranian   Fig.\u00a0.ST values for numerous voivodeships: apart from the above-described Holy Cross and \u0141\u00f3d\u017a, also Warmian-Mazurian ; Podlaskie ; Lower Silesian ; Lesser Poland  and Silesian   Fig.\u00a0.ST analysis was also performed for clusters. In this case, FST estimates were positive, quite low and ranged between 0 and 0.07907 (Table\u00a0ST estimates were identified between clusters: 71 (Choszczno and Drezdenko counties) and 41   and also between clusters: 64  and 23 (Krak\u00f3w county)  , 41, 59  and 64 among many others  Fig.\u00a0. Furtherers Fig.\u00a0.ST values calculated for all voivodeships and clusters are gathered in Tables\u00a0Detailed information about FST values, was constructed to visualize the relationships between voivodeships , 18 (P\u0142ock and Sierpc Lands), 23 (Krak\u00f3w county), 31 , 34 , 41 , 47 (Western Kuyavia), 49 (E\u0142k and Grajewo regions), 64 (northern Mazovia) and 71 (Choszczno and Drezdenko counties) were outside of this group.A MDS plot, on the basis of the pairwise Fips Fig.\u00a0. A groupips Fig.\u00a0. In thisp\u2009=\u20090.01075). Only a small proportion of total variance was attributed to variation among groups also in the case of voivodeships  (Table\u00a0p\u2009=\u20090.00109). Only a small proportion of total variance was attributed to variation among clusters  . With the ncreasing number of groups we could observe downward sloping trend with fluctuations, so we could identify local maxima . When dividing into groups corresponding to local maxima, the following clusters were separate: no. 64 (7 times), no. 34 (6 times) and no. 71 (5 times) while cluster 37 was grouped with 59 (5 times).When dividing into the maximum number of 33 groups, the variance among groups was equal to 0.32% (p\u2009<\u20090.00001).Analysis of the molecular variance conducted in SAMOVA ver. 2.0 software, based on the mtDNA SNPs and aimed at determining the most probable number of genetically different population groups, showed that the maximal number of significantly divergent groups is 33. The highest variance among groups was observed when the population was divided into 2 groups  were observed for Suwa\u0142ki region and Krak\u00f3w Land (FST\u2009=\u20090.026) and Kuyavia (0.029). However, MDS analysis did not show this region as significantly divergent. When Suwa\u0142ki region was treated as a part of Podlaskie voivodeship, the only difference, based on FST values, was observed with Western Pomerania. Nevertheless, MDS analysis did not show Podlaskie as a divergent voivodeship. In our study, raw data based on SNP was used to compute FST values, while Grzybowski et al. [ST values between this region and Holy Cross and \u0141\u00f3d\u017a voivodeships. However, FST values were still very low and cannot prove genetic separation, which is confirmed by the MDS plot, where Kuyavian-Pomeranian was grouped together with the rest of voivodeships. At the level of clusters, the situation is different. Cluster no. 47 corresponding to Western Kuyavia  was observed as different to many other regions of Poland. FST values ranged between 0.0140 and 0.0660; all were statistically significant. The highest FST value was noted between Western Kuyavia and Northern Mazovia . MDS plot clearly illustrated the separation of this region from the rest of Poland. Interestingly, Eastern Kuyavia  did not show many differences to other clusters, based on FST values. The only two statistically significant differences were observed for cluster no. 34  and 59  compared to Eastern Kuyavia, but FST values were rather low. MDS plot confirms that Eastern Kuyavia is not genetically separate within the Polish population.As mentioned above, the Polish population was the subject of the genetic research, but only in comparison to broader groups of Slavs or Europeans. Grzybowski et al.  made a gavia 0.02. HoweverThe history of Poland, especially in the last century, was marked by extensive human resettlements that took place during and shortly after the Second World War (WWII). The reasons of massive migration of Poles are following: the exile and internal exile of Poles during the September campaign at the beginning WWII; displacement of Poles from areas annexed to USSR ; deportation for forced labour under German rule during World War II; Polish population transfers after WWII connected with the change of borders; economical migrations . All of For the first time, clustering, a method of population subdivision, was used to define the genetic relationships within the Polish population. Additionally, the administrative division of Poland was overlaid on genetic separation in order to present the most complete view of Polish society. Furthermore, for the first time, the study of the Polish population was conducted on such a large number of individuals (5852). All of this makes it difficult to find similar studies to directly relate our findings to results of others. There were important differences in analysis based purely on administrative division and on geographical clustering, which is expected, showing that a large dataset makes it possible to perform a deeper and more relevant analysis.Our comprehensive analysis of mtDNA variability, based on the data from 5852 individuals, allowed us to describe the mtDNA variability of Polish population and genetic relations between Poles. It gives a better insight into mtDNA variability in Poland, with detailed administrative divisions and geographical regionalization. A complete genetic analysis including all voivodeships and most counties of Poland has been performed for the first time. Poles are characterized by the main West Eurasian mtDNA haplogroups, but relatively minor genetic differences observed on the level of voivodeships and clusters may indicate historical and cultural influences. Although the level of differentiation within the Polish population was found to be low, the existing genetic differences can be explained well with geographic distances. Using a large set of data, it was shown that Poles can be considered as genetically homogenous but with slight differences, highlighted at the regional level. The structure of our study allowed us to confirm that intrastate administrative divisions are artificial formations and do not reflect the genetic diversity of specific populations. Spatial information-based clusters are more adequate and in similar studies, researchers should consider grouping available samples based on geographic location, enhancing the quality of analysis in comparison to division into voivodeships and counties. The following study was based only on mitochondrial markers, which can illustrate gene flow in the maternal line. Therefore, conclusions can be drawn exclusively about migrations and settlements of women. Certainly, the present survey could be the basis for further research relating to the historical context of human migration or resettlements, when expanded with an analysis of chromosome Y.Tab. S1Tab. S2_haplotypesTab. S2_markersTab. S3Tab. S4Tab. S5Tab. S6Tab. S7Tab. S8Tab. S9Tab. S10Tab. S11Tab. S12Tab. S13Tab. S14Tab. S15Fig. S1Fig. S2Fig. S3Fig. S4Fig. S5"}
+{"text": "In silico, a structure of the Lacc 6 enzyme of Pleurotus ostreatus was constructed using a laccase of Trametes versicolor as a template. From this structure, 16 mutants with possible resistance at high temperature due to ionic interactions, salt bridges and disulfide bonds were also obtained in silico. It was determined that 12 mutants called 4-DB, 3-DB, D233C-T310C, F468P, 3-SB, L132T, N79D, N372D, P203C, P203V, T147E, and W85F, presented the lowest thermodynamic energy. Based on the previous criterion and determining the least flexibility in the protein structures, three mutants  were selected, which may present high stability at high temperatures without affecting their active site. The obtained results allow the understanding of the molecular base that increase the structural stability of the enzyme Lacc 6 of Pleurotus ostreatus, achieving the in silico generation of mutants, which could have activity at high temperatures.Fungal laccase enzymes have a great biotechnological potential for bioremediation processes due to their ability to degrade compounds such as \u03c1-diphenol, aminophenols, polyphenols, polyamines, and aryldiamines. These enzymes have activity at different pH and temperature values, however, high temperatures can cause partial or total loss of enzymatic activity, so it is appropriate to do research to modify their secondary and/or tertiary structure to make them more resistant to extreme temperature conditions.  One of the greatest qualities studied and demanded by protein engineering in the environmental and biotechnological fields is the search for conformational and structural stability of proteins when subjected to high temperatures, due to the fact that a high percentage of industrial processes are carried out in such conditions.The native structure of a protein is stabilized by physicochemical interactions ; neverthIn studies on protein flexibility, it has been found that mutations through which conformational degrees of freedom are reduced confer structural resistance to proteins . A type On the other hand, the laccase enzymes (EC.1.10.3.2) that belong to the family of multicopper-oxidases, can oxidize numerous compounds with an oxide-reduction potential that varies between 500 and 800 mV, such as the \u03c1- diphenol, aminophenols, polyphenols, polyamines and aryl diamines . They arPleurotus ostreatus has shown a high level of expression under different development conditions and has been partially characterized, determining its molecular weight, pI, optimum pH of activity, Km on different substrates, etc.  in BLAST server  through PDB3  finding the most favorable and thermodynamic residue shift in Lacc 6 by PopMusic server6  making an exhaustive literature search to find all laccases with t1/2 over 55\u00b0C  to struc server6 , each ofhborhood .Once the mutants were built, they were minimized by the steepest descent protocol up to 0.01 kcal/mol, with the NAMD 2.12 package  , using thttp://www.sciences.univ-nantes.fr/elnemo/  , to late/elnemo/ . The the/elnemo/ .Pleurotus ostreatus shown below (UniProtKB: A0A067NQH1_PLEOS), which is constituted by 533 amino acids, where the first 21 residues correspond to the signal peptide.The Lacc 6 sequence of 8 , it is worth mentioning that the alignment was consistent with the template structure and the active sites were perfectly aligned between them. Consequently, 1GYC structure was used as a template to make the model of Lacc 6, the homology between its sequence and 1GYC was close to 62%  with the ability to confer structural stability to Lacc 6 at high temperatures can be see in Table 10, capture the conservation propensity of each mutate residue set in column 3, with this approach we can see that the best single mutant is P203V showing a \u0394\u0394G = \u22121.91 kcal/mol below the Lacc 6.Table Based on the Tables 2) of each alpha carbon (C\u03b1) is plotted as a function of the number of residues, where for all residues, significantly mobile regions are seen above and still below the line in the graph of Figure Once the energy differences were quantified and analyzed, was continued with a Normal Modes study of the thermostable mutants . The intention of Normal Modes Study is know the collective and correlated movements of the lower energy mutant modes . The modR2 below the line at the origin. The higher amplitude values are due to the zones near to the cavity and to the terminal helix, the latter usually being a meaningless section since it is usually an artifact of the software itself, it is usually eliminated before calculations  giving rise to a more active enzyme but less stable with respect to the native enzyme (POXA1b or Lacc 6), same enzyme used in our research.In recent years, computational design has been successfully applied for the thermostabilization of enzymes with potential use in biotechnological processes, mimicking the evolution in the laboratory to develop more stable enzyme variants and, more recently, using rational strategies of computer-assisted enzymatic engineering. Tramentes versicolor by molecular dynamics and other computational techniques in which they predict and rationalize a laccase to provide stability to mutations of multiple sites in its structure, showing that it is possible to generate stable mutants through computational studies, although to carry it out it turned out to be expensive. On the other hand, Pleurotus ostreatus and its mutants. This suggests that it will have greater stability in its structure that will allow it to have oxidase activity at temperatures above the optimum.Normal modes indicate that the nucleus of the mutants is found without significant motions, however, the exposed sections and in particular the domain of the distant copper undergo movements counter-rotation and global compression. Likewise, it is predicted that lower energy mutants such as 3-DB, 3-SB, P203C minimize fluctuations by making the skeleton less flexible in the vicinity of them, decreasing the range of motions. In the same way it is found that the mutants L132T and D468P generate a conformational change in the vicinity of the active site. This work predicts the global movements of the enzyme laccase, while providing a new perspective of these enzymes. This allows us to discuss the character of the movement in multiple structures on the same basis and as expected the normal low frequency modes from the modeling of the elastic network provide a good description of the global movements of this enzyme, which allows us to understand the molecular basis of the structural stability of the Lacc 6 enzyme of RD performed the experimental design, directed the over-all study, and also drafted the manuscript. YM-F, GD-G and MA-R helped perform the analysis of results and revised the manuscript. LH-Z performed the protein modeling and revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Although cadmium (Cd)\u2010induced hepatoxicity is well established, pronounced knowledge gaps remain existed regarding the inherent cellular signaling that dictates Cd toxicity. Specifically, the molecular basis for determining the equilibrium between prosurvival and proapoptotic signaling remains poorly understood. Thus, it is recently revealed that long non\u2010coding RNA (lncRNA) MT1DP, a pseudogene in the metallothionein (MT) family, promoted Cd\u2010induced cell death through activating the RhoC\u2010CCN1/2\u2010AKT pathway and modulating MT1H induction. Here, first the dependency of MT1DP induction on MTF1, an important transcriptional factor in driving the mRNA expression of MT1 members is defined. Additionally, a bridge molecule between MT1DP and nuclear factor erythroid 2\u2010related factor 2 (Nrf2) is established: miR\u2010365. Mechanistically, MT1DP induction under Cd stress decreases the nuclear factor erythroid 2\u2010related factor 2 (Nrf2) level to evoke oxidative stress through the elevation of miR\u2010365, which acted to repress the Nrf2 level via direct binding to its 3'UTR. In contrast to the competing endogenous RNA (ceRNA) mechanism, a new mechanism is proposed: MT1DP elevated the miR\u2010365 level though stabilizing its RNA via direct binding. Collectively, the combined data demonstrate a crucial role of MT1DP in reducing the Nrf2\u2010mediated protection of cells, and this is dependent on the interplay with miR\u2010365. Hence, the study further expands the knowledge of inducible endogenous lncRNA in modulating oxidative stress. However, regardless of the activation of these antioxidant systems, a prolonged oxidative stress will eventually disrupt the equilibrium between the proapoptotic and prosurvival signaling, resulting in inevitable cell death.Long noncoding RNAs (lncRNAs) are a large cluster of noncoding transcripts containing more than 200 nucleotides. Although lncRNAs are weakly evolutionally conserved across different species and are still poorly characterized, increasing evidence has shown that lncRNAs are implicated in regulating or coordinating various vital biological processes, including cell differentiation, cellular homeostasis, genomic imprinting, and organogenesis and are even involved with diverse pathologies .We have recently discovered that a particular lncRNA, MT1DP, functions to shunt the cellular defense to cytotoxicity through crosstalk with MT1H and RhoC under cadmium stress in hepatocytes.22.1Figurep < 0.05). Likewise, the expression levels of MT1DP were increased by more than 20\u2010fold under Cd treatment at 10 and 20 \u00b5m for 6 h relative to untreated cells (p < 0.001), which is suggestive of a close correlation between Cd\u2010triggered oxidative stress and MT1DP induction. When Cd\u2010induced ROS production was bleached by pretreatment with the NADPH\u2010oxidase inhibitor, apocynin , the induction of MT1DP expression was analogously compromised by 70% compared with the control cells in response to 20 \u00b5m Cd . These data indicated that MT1DP induction by Cd treatment is, at least in part, subject to oxidative stress.We recently showed that the lncRNA MT1DP was a crucial regulator in promoting Cd\u2010induced cell death.2.2Figurep < 0.001), the induction of MT1DP levels in MTF1\u2010knockdown cells was much weaker (an \u224870% reduction) than that in the scrambled control cells responding to Cd stress . These findings, therefore, suggest that MT1DP is a downstream target of MTF1.Given that the MT1 subfamily members with metal response elements (MREs) can be recognized and activated by metal\u2010responsive transcription factor 1 (MTF\u20101),p < 0.001), and a reference region (\u221299 to \u22121 in the promoter) without the putative MREs was used as the negative control. These results demonstrate that MT1DP is directly regulated by MTF1 through the MREs at the transcriptional level upon Cd treatment.To test this assumption, subsequent bioinformatic analysis revealed a region with two putative MREs within the promoter region of MT1DP  and then compared the cellular oxidative status between the control cells and the MT1DP\u2010knockdown cells upon Cd treatment. As shown in Figure p < 0.05), whereas no significant change of MDA content was observed in the MT1DP\u2010knockdown cells. To confirm the role of MT1DP in regulating the cellular redox status under Cd treatment, the antioxidant indicators SOD, catalase (CAT), and the glutathione/glutathione disulfide (GSH/GSSG) ratio were also determined. As presented in Figure p < 0.05), signifying that MT1DP is a novel regulator of Cd\u2010induced oxidative stress. Moreover, dichlorofluorescein diacetate (DCF\u2010DA) dye was also used to investigate the effect of MT1DP on Cd\u2010induced ROS production. As shown in Figure p < 0.05), indicating the important role of MT1DP in evoking oxidative stress in response to Cd. To substantiate this finding, the MT1DP content was augmented by the transfection of an MT1DP overexpression construct . These data revealed that MT1DP aggravates Cd\u2010induced oxidative stress.To understand the physiological function of MT1DP induction upon Cd exposure, we used a shRNA strategy to knock down the endogenous MT1DP by \u224880% in the HepG2 cells , which highlights the important role of Nrf2 in combating Cd\u2010induced oxidative stress.Next, we attempted to delineate the molecular basis underlying MT1DP\u2010dependent ROS promotion. As a master regulator, Nrf2 functions to combat oxidative stress\u2010associated cellular injury through diverse mechanisms, including removal of free radicals via the induction of antioxidant components, such as HO\u20101 and SOD1.To identify the link between Nrf2 and MT1DP, we next investigated the alterations of MT1DP and Nrf2 upon their mutual knockdown. As shown in Figure 2.5Figurep < 0.05). However, when the binding site of miR\u2010365 within the 3'UTR of Nrf2 was mutated, the inhibitory effect of miR\u2010365 on the activity of Nrf2\u20103'UTR was also significantly compromised . Similarly, Cd\u2010induced Nrf2 protein accumulation was also effectively inhibited by miR\u2010365 mimics . These results show that miR\u2010365 serves as an intermediate between MT1DP and Nrf2, playing an important role in promoting Cd\u2010induced oxidative stress by suppressing Nrf2 concentrations at the translational level.To interpret the mechanism of how MT1DP regulates the Nrf2 level, we searched for partner molecules that could bind both MT1DP and Nrf2. As shown in 2.6Figurep < 0.05). In contrast, the miR\u2010365 level was increased by 150% in the MT1DP overexpression cells relative to that in the vehicle control cells (p < 0.05). Notably, miR\u2010365 mimics had no significant effect on the level of MT1DP , pointing to a direct interplay interaction. In addition, MT1DP deficiency elicited no effect on the expression of pri\u2010miR\u2010365 ; however, the decline of miR\u2010365 was markedly blocked by the overexpression of MT1DP in the ActD\u2010treated cells (p < 0.05). In contrast, the decline rate of miR\u2010365 was further accelerated in the ActD\u2010treated cells upon MT1DP knockdown . Together, our data demonstrate that MT1DP functions to elevate the miR\u2010365 concentration by enhancing miR\u2010365 RNA stability.To understand the mechanism underlying the regulatory activity of MT1DP on the miR\u2010365 level, an RNA\u2010pull down assay was performed by using MS2 hairpin\u2010tagged MT1DP to elucidate whether MT1DP could directly bind to miR\u2010365. As shown in Figure 3MT1DP is a pseudogene that belongs to the MT1 subfamily and is specifically present in human genome.It has been reported that several miRNAs, such as miR\u2010144, miR\u201028, and miR\u2010153, can negatively regulate Nrf2 levels by directly targeting the 3'UTR of Nrf2 mRNA.In summary, the current study has demonstrated an important role of MT1DP in aggravating oxidative stress in hepatocytes upon Cd treatment through its interplay with miR\u2010365, which functioned as the intermediate to repress Nrf2\u2010mediated antioxidant signaling. Specifically, MTF1 was found to be induced by Cd in order to activate MT1DP expression in the hepatocytes; MT1DP, in turn, elevated the miR\u2010365 level through a protection mechanism, dependent on the direct binding between them. Consequently, miR\u2010365 decreased Nrf2 levels, provoking Cd\u2010induced oxidative stress. Our results reveal a new mechanism underlying the MT1DP\u2010conducted signaling that promotes Cd toxicity in the hepatocytes.4Cell Culture: The human hepatocellular carcinoma cell line HepG2 was obtained from the Cell Resource Center of the Institute of Basic Medical Sciences . Cells were grown in Dulbecco's modified Eagle medium (DMEM)  with 10% fetal bovine serum (HyClone) and penicillin/streptomycin (HyClone) at 37 \u00b0C with 5% CO2.Cell Transfection: miRNA mimics were purchased from Genepharma Biotechnology . The pcDNA3.1\u2010MT1DP, MT1DP shRNA, and MS2\u2010MT1DP were constructed as previously described.Western Blot Analysis: HepG2 cells were lysed with radio immunoprecipitation assay (RIPA) buffer  with protease inhibitor cocktail  using a standard method. After mass quantification using the Bradford protein concentration assay , the cell extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto the nitrocellulose membranes , as described previously.Luciferase Reporter Assay: Cells were harvested 48 h after transfection and luciferase activity was measured in the form of chemiluminescence using the dual\u2010luciferase reporter assay system  following the manufacturer's instructions. The relative luciferase activity was normalized to the according Renilla luciferase activity.Real\u2010Time PCR (qRT\u2010PCR): Total RNA was extracted by Trizol reagent (Invitrogen), and first strand cDNA was synthesized using the Thermo script RT\u2010polymerase chain reaction (PCR) system  according to the manufacturer's instructions. qRT\u2010PCR was performed on an iQ5 PCR instrument , as described previously.TableAntioxidant Components Determination: The cells were treated with or without 20 \u00b5m Cd for 6 h and then were lysed with RIPA buffer at 4 \u00b0C. Next, the cell lysates were centrifuged to obtain the supernatants for further analysis. The MDA content, the activities of SOD and CAT, and the GSH/GSSG ratio were determined using the corresponding assay kits  according to the instructions provided by the manufacturer.ROS Determination: Cells grown in 96\u2010well plates were exposed to various doses of Cd for 6 h. DCF\u2010DA  probe (10 \u00d7 10\u22129m) was then added to the cells and incubated for an additional half hour. Finally, the generation of intracellular ROS was assessed by fluorescence analysis with an emission wavelength of 525 nm and an excitation wavelength of 488 nm on a microplate reader (Thermo Scientific).Statistical Analysis: All data were analyzed by the Student's t\u2010test or one\u2010way analysis of variance test. Each experiment was performed at least three times, and the results were expressed as the mean +/\u2212 standard deviation (SD). p\u2010value less than 0.05 (*p < 0.05) or 0.001 (#p < 0.001) indicated a statistically significant difference.The authors declare no conflict of interest."}
+{"text": "Dual-specificity phosphatases (DUSPs) are a subset of protein tyrosine phosphatases (PTPs), many of which dephosphorylate the residues of phosphor-serine/threonine and phosphor-tyrosine on mitogen-activated protein kinases (MAPKs), and hence are also referred to as MAPK phosphatases (MKPs). Homologue of Vaccinia virus H1 phosphatase gene clone 5 (HVH-5), also known as DUSP8, is a unique member of the DUSPs family of phosphatases. Accumulating evidence has shown that DUSP8 plays an important role in phosphorylation-mediated signal transduction of MAPK signaling ranging from cell oxidative stress response, cell apoptosis and various human diseases. It is generally believed that DUSP8 exhibits significant dephosphorylation activity against JNK, however, with the deepening of research, plenty of new literature reports that DUSP8 also has effective dephosphorylation activity on p38 MAPK and ERKs, successfully affects the transduction of MAPKs pathway, indicating that DUSP8 presents a unknown diversity of DUSPs family on distinct corresponding dephosphorylated substrates in different biological events. Therefore, the in-depth study of DUSP8 not only throws a new light on the multi-biological function of DUSPs, but also is much valuable for the reveal of complex pathobiology of clinical diseases. In this review, we provide a detail overview of DUSP8 phosphatase structure, biological function and expression regulation, as well as its role in related clinical human diseases, which might be help for the understanding of biological function of DUSP8 and the development of prevention, diagnosis and therapeutics in related human diseases. Protein phosphorylation is a key event that controls cellular responses to external cues \u20133. EukarMAPK are switched off by both generic phosphatases and dual-specificity phosphatases (DUSPs) and are further regulated by scaffold proteins, which are usually specific for each of the three major mammalian MAPK pathways , 25\u201327. The DUSP8 gene is a protein-coding gene. Human DUSP8 gene, also known as hVH5, is located in human chromosome 11 p15.5 , 43. The\u03c8hVH-5). \u03c8hVH-5 transcripts were detected in human peripheral tissues as well as in 11 of 14 breast cancer cell lines. In respect to the normal hVH-5 gene, the pseudogene contains several point mutations and a frame shift due to the deletion of 2 bases that would lead to the truncation of the putative \u03c8hVH-5 product. The intronless pseudogene \u03c8DUSP8/\u03c8hVH-5 contains three different gene sequences; DUSP8P1/DUP8P/DUSP8P3 and DUSP8P2/DUSP8P4 were located on chromosome 10q11.22, and DUSP8P5 are positioned on 11q22.2 rather than hVH-5 presents on chromosome 11p15.5 [Surprisingly, analyzing DUSP8/hVH-5 transcripts in mammary carcinoma cell lines discovered a sequence with 88% similarity to hVH-5 transcripts. Because this variant of hVH-5 lacked intronic sequences in its genomic structure, so it might be a processed pseudogene of hVH-5 . For DUSP8, cysteine mutations are necessary to obtain diffraction-quality crystals during structure determination of human DUSP8, possibly because the cysteine residues are susceptible to oxidation. Therefore, the 3D structure and catalytic site containing Ser-246 but not Cys-246 for DUSP8 based on calculation with Pymol is shown in Fig.\u00a0The secondary structure determination of human DUSP8 is identified by protein expression and crystallization using molecular-replacement method with human DUSP10 (PDB: 1ZZW). The final model of DUSP8 at 1.9\u00a0\u212b resolution consists of residues 160\u2013310 of n B Fig.\u00a0a, two sun B Fig.\u00a0b and 157Unlike DUSP5 has distinct and specific dephosphorylated substrate\u2014extracellular signal-related kinase (ERK) , DUSP8 pO-tetradecanoylphorbol-13-acetate (TPA/PMA) and anisomycin [2+ was defined as excitation-transcription coupling, which affected the transcription of DUSP8 in a cell type specific manner [http://alggen.lsi.upc.es), indicating these TFs might be involved in the transcriptional regulation on DUSP8 expression.The expression and activity of DUSP8 can be regulated in a number of ways including gene transcription, protein stability, and phosphatase activity. This multi-level regulation allows for tight control of MAPK activity. Despite DUSP8 is still controversial as an immediate early gene, the expression of DUSP8 mRNA or transcript can be rapidly induced after oxidative stress, heat shock, growth factors, and some small molecular activator such as isomycin \u201359. In Kisomycin . Importaisomycin . In addic manner . FinallyDue to the long half-life of DUSP8 mRNA, post-transcriptional regulation is also an important part of DUSP8 expression. However, the molecules and related mechanisms involved in the post-transcriptional regulation of DUSP8 remain poorly understood, and more research and studies should be needed for further investigation. Thus, here are listed conserved miRNAs, a class of important gene post-transcriptional factors, that may be involved in post-transcriptional regulation of DUSP8 from TargetScan Database , 63 and Ser 515, Thr 518 and Ser 520 in M3/6, which were JNK-induced phosphorylation sites. And after arsenite stimulation, M3/6 mutated at these sites showed reduced phosphorylation compared to wild-type protein. Interestingly, the expression of the M3/6 phosphorylation mutation delayed the time course of JNK phosphorylation and activation via arsenite stimulation, suggesting that JNK phosphorylation of stress-stimulated M3/6 phosphatase led to a decrease in phosphatase activity and an acceleration of JNK activation [\u00ae PTM resources from Cell Signaling Technology, Inc. [http://www.phosphosite.org).As a mitogen-activated protein kinases phosphatase, whether DUSP8 has post-translational modification sites has been widely concerned. Up until now, however, only murine DUSP8, as also known M3/6, has been identified to have phosphorylation sites. It has been shown that M3/6 itself can be phosphorylated by JNK when stimulated with arsenite, but the effect and mechanism of DUSP8 self-phosphorylation has not been studied. Mass spectrometry assay has shown that there are phosphorylation residues tivation , 59. Morgy, Inc.  and makeComplex signaling networks including JNK, ERK, p38 MAPK and ERK5 can be activated when the brain exhibits an initial restriction on blood supply and subsequently resumes blood flow. A study has shown that cerebral ischemia and reperfusion in rat hippocampi could induce JNK undergoing complete inactivation following the intense activation, in which after 4\u00a0h of reperfusion in rat hippocampi, the activity of DUSP8 was significantly upregulated, accompanied by the dephosphorylation of JNK. The inhibitor of DUSP8, anisomycin, might elevate JNK activity following postischemic reperfusion, indicating that DUSP8 was closely correlated with inactivation of JNK following cerebral ischemia. Innovatively, on one hand, the expression level of heat shock protein (HSP70) increased obviously and was participated in the upregulation of soluble cytoplasmic DUSP8 levels, which induced by cerebral ischemia; on the other hand, inhibition of the activity of HSP70 by using quercetin led to restore the activation of JNK via downregulating the cytoplasmic solubility of DUSP8, suggesting DUSP8 involved in the process of inactivating JNK activation in response to cerebral ischemia required the molecular chaperon HSP70 to impetus the calibration of folding defects .Similarly, Rosiglitazone is a synthetic peroxisome proliferator-activated receptor-\u03b3 (PPAR-\u03b3) agonist that prevents cell death following cerebral ischemia in animal models. The study investigated how Rosiglitazone protected neurons from ischemia were conducted in which mice pre-treated with Rosiglitazone were subjected to ischemia for 60\u00a0min and then reperfusion. The infarct volume was reduced by Rosiglitazone after ischemia and reperfusion. And PPAR-\u03b3 antagonists could reverse the neuro-protective effects. Importantly, Rosiglitazone inhibited a significant increase in the expression of phosphorylated JNK and p38 MAPK in ischemic brain tissue. In addition, Rosiglitazone simultaneously increased the expression of DUSP8 at mRNA and protein level in ischemic brain tissue. Furthermore, knockdown of DUSP8 in primary cultured cortical neurons undergoing oxygen\u2013glucose deprivation diminished the effect of Rosiglitazone on the downregulation of JNK phosphorylation. Therefore, the neuro-protective effect of Rosiglitazone after ischemia was dominantly mediated by upregulation of DUSP8 to block ischemia-induced JNK phosphorylation .For diagnosing biomarker, due to Parkinson\u2019s disease (PD) shares pathological and clinical features with patients diagnosed with progressive supranuclear palsy (PSP) and the differences in disease progression, treatment, and clinical management, it is very urgent need to identify other biomarkers that can distinguish among these diseases. By testing DUSP8 and PTPRC for their diagnostic potential through quantitative PCR assay from 138 blood samples with PD, the results have shown that compared to PD patients and healthy controls, the relative abundance of PTRPC mRNA significantly decreased in PSP patients, whereas there was no obvious difference in the expression level of DUSP8, suggesting DUSP8 might not be suitable to become a biomarker for assisting to diagnose PD or PSP .As a novel member of immediate early genes that encode enzymes of MKP family, DUSP8 also plays a unique role in drug addiction. In the nucleus accumbens \u017dNAc., caudate putamen, frontal cortex and hippocampus of rat brain, i.p. injection of cocaine, amphetamine and caffeine induced DUSP8 mRNA expression within 40\u00a0min, with a maximal affect in the NAc. Compared with c-Fos and EGR-1 immediate early genes, the expression of DUSP8 mRNA in the NAc and hippocampus was obvious upregulation and continuous after cocaine injection for 10\u00a0days as a single injection later. This study demonstrated from another perspective that DUSP8 acted as a negative regulator of MAPK and was involved in MAPK-mediated central phschostimulants addiction .High glucose could promote the proliferation and collagen synthesis of rat cardiac fibroblasts, accompanied by the upregulated expression of micoRNA-21, a well-known molecule of extensive studies on multiple diseases, suggested the critical role in diabetic cardiomyopathy. DUSP8, a direct target of miR-21 binding 3\u2032-UTR of DUSP8 inhibited the expression at post-transcriptional level, was involved in regulating cell proliferation and collagen synthesis in cardiac fibroblast via p38 MAPK and JNK/SAPK signaling pathway .Furthermore, a study has documented that DUSP8 gene deletion (knockout) mice exhibited baseline concentric heart remodeling, which was enhanced during stress stimulation. This concentric ventricular remodeling was associated with increased myocardial contractility at baseline and prevention from dilation and heart failure in two different induced pathology models. In addition, loss of DUSP8 resulted in increasing ERK1/2 activation at baseline and after acute pathological stimulation, whereas p38 and JNK kinase were unaffected. In contrast, overexpression of DUSP8 led to decreased phosphorylation of all MAPKs studied, ventricular dilatation and greater propensity for heart failure. This study suggested that DUSP8 regulated the dynamics of cardiac MAPKs signaling pathway, which directly affected ventricular remodeling and heart failure propensity .Renal branching morphogenesis is a necessary process to mammalian kidney development, which is defined as growth and branching of the ureteric bud (UB) and its derivatives. And the formation of the collecting ducts and pelvis of mature kidney also needs UB to grow, branch, and differentiate. Defects with renal branching morphogenesis result in congenital renal dysontogenesis, characterized by aberrant collecting system morphology and low nephron number. Integrin-linked kinase (ILK) is definitely an intracellular scaffolding protein with essential cell specific functions in the embryonic and mature mammalian kidney. Based on whole genome expression analysis of whole kidney mRNA in transgenetic mice with ILK loss in the ureteric cell lineage, the expression of six genes relevant kidney maturity including Wnt11 were downregulated, whereas the expression of DUSP8 increased in ureteric tip cells, accompanying with the downregulation of phosphorylated p38 MAPK in kidney tissues with ILK deficiency. Moreover, overexpression of DUSP8 in murine inner medullary collecting duct 3 (mIMCD3) cells significantly suppressed the activation of p38 MAPK. Furthermore, overexpression of DUSP8 mediated by adenovirus also inhibited ureteric branching and the activity of p38 MAPK. This study vests DUSP8 a brand new function of kidney differentiation and maturation .The proliferation, differentiation, secretion of cytokines, and execution of immune responses of various types of immune cells are inseparable from the strict and orderly regulation of activation and deactivation of MAPK pathway. BAF3 cells were precursor B cells that undergone apoptosis following IL-3 withdrawal or ceramide treatment. JNK/SAPK in BAF3 cells was stimulated by ceramide and responded to IL-3 stimulation during cell proliferation. Expression of DUSP8 in BAF3 cells prevented ceramide from stimulating JNK/SAPK activity. The proliferation of BAF3 cells expressing DUSP8 with IL-3-stimulated was inhibited . In addiFor human colorectal carcinoma (CRC), our previous work observed the effect of antisense oligonucleotides (ASOs) against miR-21 on the growth and metastasis of CRC in vivo using a xenograft model of human CRC. We found that ASOs could effectively inhibit the growth and metastasis of CRC in vivo, accompanied by downregulated expression of miR-21 and reduced transduction of the AKT and ERK pathway. Mechanically, global gene expression analysis showed that the expression of DUSP8, a target of miR-21, was upregulated in tumor mass. Furthermore, overexpression of DUSP8 could remarkably suppress the proliferation and migration of CRC cells in vitro. Finally, downregulation of DUSP8 could abrogate the effects of ASOs against miR-21 on the proliferation and migration of CRC cells, as well as altered transduction of the AKT and ERK signaling pathway. This is the first time we demonstrated that the DUSP8 mediated pathway plays a pivotal anti-cancer role on ASOs against miR-21 inhibiting the growth and metastasis of CRC .In contrast, other study in prostate cancer reported that Phenyl isocyanate (PEITC) was capable of inducing activation of JNK and apoptosis in prostate cancer cell lines with different p53 status. The JNK dephosphorylation and deactivation rate of cells exposed to PETIC was reduced by PEITC directly downregulating the expression level of DUSP8 via proteasome-dependent mechanism, suggesting that PEITC targeting DUSP8, similar as a oncogene in prostate cancer, could restore activity of JNK through inhibiting the expression and activation of DUSP8 . TherefoIn type II diabetes, based on the pyrosequencing of DNA methylation, one study investigating 40 single nucleotide polymorphisms (SNPs) previously associated with type II diabetes a CpG site (introduce or remove), and examining whether these CpG-SNPs were correlated with differential DNA promoter methylation in pancreatic islets of 84 human donors, has revealed that 19 of 40 (48%) type II diabetes-related SNPs with introducing or removing a CpG site. Then, DNA methylation data were successfully generated 16 genes including DUSP8 (SNP ID: rs2334499) from 19 CpG-SNP loci, which had 4 linkage-disequilibrium (LD) SNPs. Combining these 19 type II diabetes-related CpG-SNPs presented strong LD with a total of 295 SNPs including 91 CpG-SNPs .Moreover, another study has shown that evidence of genetic linkage of alcohol dependence was found on chromosome 11p15.5 in autosomal scans of aboriginal populations in Southwestern America. And DUSP8 gene not only met the candidate criteria, but also played a crucial role in various pathways known to constitute pathophysiological mechanisms leading to the development of alcohol dependence, which was sequenced to identify polymorphisms that might be associated with disease. The results revealed that 4 patent polymorphisms were characterized in DUSP8, one of which resulted in an amino acid substitution (DUSP8 C712T). However, there was not significant association between DUSP8 C712T polymorphism and alcohol dependence by using genotyping of analyzing functional of DUSP8 in 463 Southwestern Native Americans .\u03c8hVH-5, in the development of various human diseases, is still unknown. DUSP8 is widely involved in the development of multiple clinical diseases (Fig.\u00a0Accumulating evidence has shown that DUSP8, a unique member of DUSPs family, is emerging as a critical negative regulator for MAPKs pathway and involved in cell oxidative stress response and cell apoptosis, as well as the development of various human diseases. In the future, there still are some core issues, mainly from three aspects, should been further addressed. The first is the regulation mechanism of DUSP8 expression in multiple types of cells and tissues. Even there have been some mechanisms of DUSP8 expression have been investigated as above description. However, there are still some problems need to be illustrated, such as what is the core sequence of promoter of DUSP8 and its related transcription factors or elements in various cells and tissues; what is the mechanism on the protein stability of DUSP8, specially the degradation of endogenous DUSP8 protein, and so on\u2026 The second is the biological structure of DUSP8. Due to the unknown crystal structure of the DUSP8 KIM motif in Rhodanese domain, the analysis and clarification of the recognition mechanism of DUSP8 on different MAPKs substrates would be further conducted. Especially, as a result of DUSP8 itself has multiple potential phosphorylation sites, it is unclear yet whether DUSP8 exerts dephosphorylation function depends on the phosphorylation of its specific sites or whether phosphorylation of different sites is related to the substrate specificity of dephosphorylation. Meanwhile, the origin of nuclear translocation signal on DUSP8 protein sequence and the subcellular co-location of both DUSP8 and its substrates including classical MAPKs such as ERK, JNK and p38 MAPK or other factors also need to be identified. Third, the exact role of DUSP8, including In all, successive research works on the exploration on more exact regulation expression mechanism and biological structure of DUSP8, as well as the connection between DUSP8 and clinical diseases, will undoubtedly provide a better prospect of acknowledgement on DUSPs family member including DUSP8 and the clinical application for DSUPs-based prevention, diagnosis and therapeutic strategies in clinical diseases."}
+{"text": "Trends and hotspots analyses were performed on LDAP results. We found that the focus points of AD research for the past 10 years include 15 diseases, 15 amino acids, peptides, and proteins, 9 enzymes and coenzymes, 7 hormones, 7 carbohydrates, 5 lipids, 2 organophosphonates, 18 chemicals, 11 compounds, 13 symptoms, and 20 phenomena. Our LDAP framework allowed us to trace the evolution of research trends and the most popular areas of interest (hotspots) on disease, protein, symptom, and phenomena. Meanwhile, 556 AD related-genes were identified, which are enriched in 12 KEGG pathways including the AD pathway and nitrogen metabolism pathway. Our results are freely available at https://www.keaml.cn/Alzheimer.About 29.8 million people worldwide had been diagnosed with Alzheimer's disease (AD) in 2015, and the number is projected to triple by 2050. In 2018, AD was the fifth leading cause of death in Americans with 65 years of age or older, but the progress of AD drug research is very limited. It is helpful to identify the key factors and research trends of AD for guiding further more effective studies. We proposed a framework named as LDAP, which combined the  Between 2010 and 2015, the number of Alzheimer's disease (AD) cases has more than tripled, and the prevalence of AD is still increasing AD is a type of brain disease that begins primarily at the age of 65 or older, although 4% to 5% of AD cases are early-onset Many journals, conferences, patents, and associations are dedicated to the study of AD. For example, Alzheimer's Association releases an annual report, which describes the public health impact of AD, including incidence and prevalence, mortality and morbidity, use and costs of care, and the overall impact on caregivers and society For the curation research on AD, the whole world is struggling to find more effective ways to treat the disease, delay its onset and prevent its development. However, this progress is not satisfactory. For example, it is reported that researchers had spent more than 100 billion dollars to find effective drugs to cure AD in clinical treatments in past years. But the results were disappointed as in the failure of the antibody, solanezumab, which held the promise of improving cognition but failed trial tests For clinicians or biological researchers, rapid and effective acquisition of cutting-edge information on research advances from tens of thousands publications is a huge challenge et al. in 2003 D={md}, m\u2208{1, \u2026, M}. The distribution of topic k over vocabulary is denoted as \u03a6={k\u03d5}, k\u2208{1, \u2026, K}, and the distribution of the m-th document over all K topics is denoted as \u0398={m\u03b8}, m\u2208{1, \u2026, M}. A topic is a distribution of terms over a vocabulary. It allows each document to be described as a distribution over topics, which can be expressed as:Latent Dirichlet allocation (LDA) is a topic model algorithm based on probability proposed by Blei w represents a word, d represents a document, and t is the topic. It can be expanded as follows:where m, the distribution of document over topics m\u03b8 and the distribution of topics over vocabulary \u03a6 are sampled from priors \u03b1 and \u03b2, respectively. Then, the topic assignment z for each word is generated from m\u03b8, and the accurate words w are generated according to their respective topic assignment z and the distribution of topics over the vocabulary \u03a6.where, for document To get the topic words of each year, we used Gibbs sampling to estimate the LDA model. Gibbs sampling is a Markov chain Monte Carlo (MCMC) algorithm for obtaining a sequence of observations which are approximated from a specified multivariate probability distribution, when direct sampling is difficult.X = {x1, x2n, ..., x}, and there is no hypothesis of inherent structure between data. Let S be a matrix that depicts the similarity between points, s > s if and only if the similarity between ix and jx is greater than that of ix andk x.The similarity is represented by the reciprocal of cosine similarity corresponding to (3):Affinity propagation (AP) is a clustering algorithm based on the concept of \u201cmessage passing\u201d between data points. AP finds \"exemplars\", which are members of the input set representing clusters The AP algorithm alternates between two messages passing steps to update two matrices:R: r describes the extent to which sample k is suitable for the clustering center of sample i, and it represents the messages from i to k.(a) The responsibility matrix A: a describes the selection sample i choosing sample k as the degree of suitability for the cluster center, and it represents the messages from k to i.(b) The availability matrix R will be constantly updated according to (4).The matrix A will be continually updated according to (5) and (6).The matrix \u03bb\u2208 is introduced when updating the two matrices, as described in (7) and (8):After copious iterations, the final clustering result can be obtained when the two matrices converge. To avoid numerical oscillations, a damping factor ad, disease, alzheimer, and dementia to get high scores in each topic. Therefore, we generated topics using LDA and then clustered them with AP. With the introduction of MeSH, David, and KEGG, the results can be represented from different aspects, such as proteins, diseases, and pathways.This paper presents an LDAP framework, which combines LDA model and AP algorithm to extract research topics. Our proposed framework is depicted in Figure Because an abstract can provide a concise and accurate description of the important content of a paper, necessary information can be obtained from abstracts without reading the full text. We took \u201cAlzheimer's disease\u201d as the entry term to search papers in PubMed and obtained 95,876 papers from 2007 to 2016. Each file is a semi-structured XML document and contains various tags, such as <title>, <abstract>, <pmid>, etc. We extracted the content in <abstract> and <pmid> fields from the raw XML files. PMID is the unique ID for a paper in PubMed. After extraction, each abstract was stored in a corresponding file.We obtained statistics in which the number of papers on AD increased over time. Figure Because the original data contains irrelevant and noisy information that affected the correctness of the final experimental results, a pre-processing operation was necessary to assure the accuracy of the dataset. In our work, the natural language processing techniques such as word segmentation, lemmatization, and stop words removal were applied to the raw data.t=200, hyper-parameters \u03b1=0.25, \u03b2=0.01, and the iteration i=400 by perplexity evaluation. In the AP algorithm, we set the damping factor to 0.95 after several adjustments.In our experiment, for LDA model, we set the topic number Our LDAP framework adopts the LDA model to process the annual data, and 200 topics are obtained for each year. Then the AP algorithm is employed to cluster the LDA results. We used word clouds to visualize the LDAP results. Word cloud displays different size words where the higher the word weight, the bigger the word. Taking the results of 2016 as an example, as shown in Figure cost is the central word of the first cluster, and the other obvious words such as burden, health, and million are also associated with cost. The caring for persons with AD has caught great attention. AD is considered as one of the most financially costly diseases in developed countries. Moreover, App (Amyloid Precursor Protein) is another central word that can present the whole cluster of protein topics, including beta amyloid protein and bace1 which are directly associated with AD. More research topics from 2007 to 2015 are shown in Supplemental Figure In Figure education, appeared only once in 2015. We found a hotspot on the link between education and dementia in 2015 The topic centers  are presented by years and listed in Table There are 142 clusters for all ten years' data and 20 words in each central topic. We summed up all the words in the central topics and obtained 1,988 unique words. Then we introduced the Mesh terms to classify the categories of these words, and obtained 75 categories in all, which are: 15 diseases, 15 kinds of amino acids, peptides, and proteins, 9 enzymes and coenzymes, 7 hormones, 7 carbohydrates, 5 lipids, 2 organophosphonates, 18 chemicals, 11 types of compounds, 13 different symptoms, 20 different phenomena, and other categories. In our experiments, we focused our research on 11 categories and recorded the trends and hotspots that the center topics words represent. In the following section, we analyzed trends and hotspots on terminology pertaining to diseases, proteins, symptoms and phenomena, and gene pathways.Figure Figure For the 15 diseases identified from the hotspots on Alzheimer's disease, we downloaded the abstracts for each disease from 2007 to 2016 to double check the relation between AD and these diseases. We used the same model to process these 15 diseases to find whether AD appeared in the results. The result is shown in Figure Besides diseases, other entities related to Alzheimer's disease are also found in the results, such as enzymes, hormones, and lipids. The distribution of proteins in each year is shown in Figure Glycoproteins appeared in all years, and amyloid appeared in 8 years from 2007 to 2016, except for 2008 and 2009. It is well known that amyloid and glycoprotein are important to AD It is well known that AD patients have difficulty in remembering recent events and have problems with language. However, other symptoms and phenomena are also common, such as aphasia and telomere. The complete list of symptoms and phenomena from our data is shown in Figure Apart from the decline of memory, the cognition, attention, memory recall, learning and speech abilities of AD patients are also declined. In addition, inflammation, neuroprotection, telomere, syndrome, synapsis also appeared, which could potentially suggest a composite screening or early warning of Alzheimer's disease based on these symptoms.We identified all the genes appeared in the topics, and 556 genes were found in total. To analyze these genes, we uploaded these genes to the David database As a result, 12 pathways were achieved; two of them are the Alzheimer's disease pathway and the nitrogen metabolism pathway. In the AD's pathway, 12 genes were taken from our results, including the ADAM metallopeptidase domain 10 (ADAM10), LDL receptor related protein 1 (LRP1), amyloid beta precursor protein (APP), apolipoprotein E (APOE), beta-secretase 1 (BACE1), cyclin dependent kinase 5 (CDK5), microtubule associated protein tau (MAPT), insulin degrading enzyme (IDE), presenilin 1 (PSEN1), presenilin 2 (PSEN2), synuclein alpha (SNCA) and tumor necrosis factor (TNF). The P-value of the pathway is 4.20E-07, and the false discovery rate (FDR) is 5.08E-04 according to our uploaded genes. Both P-value and FDR are less than 0.01, indicating that the pathway of our uploaded genes is statistically significant.The pathway of Alzheimer's disease is shown in Figure Alzheimer, disease, time, brain, app, cost, and cell are the most noticeable words represented the research hotspots in 2016. The other figures from 2007 to 2015 are shown in the We analyzed the trends and hotspots on center topics and three other aspects based on center topic words. AD research continues to evolve year after year, and there are some latent tendencies in the past years. To capture these tendencies, we merged all the central topics of the whole year into a word cloud. Taking 2016 as an example, the visualization of the result is shown in Figure Parkinson, Huntington, palsy, Neurodegenerative Diseases, encephalopathy, and sclerosis appear in 2017 and hypertension appears in 2018. (2) From Figure Combined with the results of the 10-year (from 2007 to 2016) word clouds, we can infer that (1) phenomena, amino acids, peptides, and proteins appeared in each year and the disease category appeared in 9 years (except 2009). These categories are considered as long-term research hotspots and directly related to AD. Therefore, we can predict that these categories would be the hotspots topics in 2017 and 2018. To validate the prediction, we generated AD-research hotspots in 2017 and 2018 using LDAP and achieved the validation. We found these three categories in Figure Nature in September 2015 and November 2016 In addition, the proportion of MeSH category about the hotspots from 2007 to 2018 is shown in Figure To reveal the hotspots and trends of AD, we proposed a novel LDAP framework which combines topics model and AP algorithm. From about 100,000 AD papers, we found the spotlights of AD research including 556 genes, 15 diseases, 15 amino acids, peptides, and proteins, 19 chemicals and compounds, 33 symptoms and phenomena, and 12 related pathways. We studied the research hotspots via a visualization method to find the regularities, and reveal the research hotspots on AD research. It should be noted that there is no unique criteria to explain the trends of AD. In this paper, we mainly use the information of PubMed abstracts through machine learning models. This may have some bias for completely understanding the trends of AD. However, the discovery of the research trends and hotspots evolution on AD are supposed to provide some guidance for further research and might to useful to drug study. For example, a drug used to treat a disease associated with the 556 AD related genes may treat or alleviate some of the symptoms of AD. In addition, the 15 amino acids, peptides, and proteins, 9 enzymes and coenzymes, 7 hormones, 7 carbohydrates, 5 lipids, 2 organophosphonates, 18 chemicals, 11 compounds can be made into corresponding inhibitors, which may be used to treat AD. These results are required further experimental verification. In addition to the research manuscripts, patents about AD also can provide the hotspots from different views, especially for the drug development. We will introduce this data in future research.Supplementary figures and tables.Click here for additional data file."}
+{"text": "Cell therapy is a promising new treatment option for cancer. In particular, mesenchymal stem cells (MSCs) have shown potential in delivering therapeutic genes in various tumour models and are now on the verge of being tested in the clinic. A number of therapeutic genes have been examined in this context, including the death ligand TRAIL. For cell therapy, it can be used in its natural form as a full-length and membrane-bound protein (FL-TRAIL) or as an engineered version commonly referred to as soluble TRAIL (sTRAIL). As to which is more therapeutically efficacious, contradicting results have been reported. We discovered that MSCs producing sTRAIL have significantly higher apoptosis-inducing activity than cells expressing FL-TRAIL and found that FL-TRAIL, in contrast to sTRAIL, is not secreted. We also demonstrated that TRAIL does induce the expression of pro-metastatic cytokines in prostate cancer cells, but that this effect could be overcome through combination with an AKT inhibitor. Thus, a combination consisting of small-molecule drugs specifically targeting tumour cells in combination with MSC.sTRAIL, not only provides a way of sensitising cancer cells to TRAIL, but also reduces the issue of side-effect-causing cytokine production. This therapeutic strategy therefore represents a novel targeted treatment option for advanced prostate cancer and other difficult to treat tumours. The tumour necrosis factor related apoptosis-inducing ligand (TRAIL), also known as Apo2L, CD253 or TNFSF10, can induce apoptosis in cancer cells while sparing normal cells. The molecular basis for the tumour-selective activity of TRAIL remains to be fully defined ,5,6. UnlTRAIL is a member of the TNF superfamily, which forms multimers that interact with cognate receptors on the cell surface ,9,10. ItIn the DISC, procaspase-8 is activated by a mechanism that involves dimerisation and proteolytic cleavage ,13,14. IActivated caspase-8 sets in motion the apoptosis cascade via a number of mechanisms; firstly, it can directly cleave executioner caspases such as procaspase-3 to the active form of caspase-3. This leads to apoptosis by proteolytic cleavage of a series of cellular targets giving rise to hallmarks of apoptosis such as cellular shrinkage, chromatin condensation and nuclear fragmentation . AlternaTNFSF10 gene, or as an engineered version including the ectodomain of TRAIL (aa114\u2013281) and a strong signal peptide that ensures effective secretion [Even though clinical studies with recombinant TRAIL (rTRAIL) and agonistic antibodies to TRAIL-receptors have shown that they are safe, only moderate evidence of therapeutic efficacy has been observed ,32,33. Decretion ,46. As MSC-based delivery of TRAIL is about to be tested in clinical trials, it is important to identify optimal versions of TRAIL that have the best potential for therapeutic efficacy. Therefore, we compared cells expressing full-length TRAIL (FL-TRAIL) or soluble TRAIL (sTRAIL) in different experimental systems and approaches to investigate their capacity to induce cancer cell killing. Furthermore, we analysed how different forms of TRAIL affect the production of potentially side-effect-causing cytokines ,48,49, aFibrillin gene to provide effective secretion, and a Furin cleavage site to release the ILZ-sTRAIL protein into the extracellular space  is expressed and purified. In cell therapeutic applications, it is possible to use either the full-length, membrane-bound version (FL-TRAIL) or engineer cells to secrete a smaller, soluble form (sTRAIL). Our goal was to compare the cell death inducing activities of the two TRAIL types in the context of cell therapy, and to investigate how other non-apoptotic TRAIL-signalling pathways and outcomes were affected. The FL-TRAIL expression construct consisted of the TRAIL cDNA (aa1\u2013281) under the control of the CMV promoter a. For thar space a. Both constructs were transfected into HEK293 cells and a western blot with respective whole cell protein extracts showed FL-TRAIL and sTRAIL running at the expected different molecular weights b. These First, we analysed how different methods of clearing the cell supernatant of cell debris affected the results of the TRAIL ELISA. We transfected HEK293 cells with the FL-TRAIL and sTRAIL constructs, respectively. Then, either the unprocessed crude supernatants, cleared supernatants by sedimentation of cell debris through centrifugation, or cleared supernatants by filtration through a 0.45 \u03bcm filter were applied to a TRAIL ELISA. FL-TRAIL gives rise to cellular particles containing TRAIL that can be detected in the supernatant. Centrifugation, to some degree, and filtration completely removes these particles and the TRAIL signal, whereas sTRAIL is not affected by these clearing methods a.It is hypothesised that basal, transfection- or TRAIL-induced apoptosis led to the generation of TRAIL-containing cellular fragments from dead cells that were detected in the FL-TRAIL samples. To test this, TRAIL resistant CHO cells b were usWe used adenoviral vectors expressing FL-TRAIL or sTRAIL to transduce mouse and human MSCs, generating MSC.FL-TRAIL and MSC.sTRAIL. Subsequently, we analysed crude, centrifuged or filtered culture supernatants by TRAIL ELISA. There was no detectable level of TRAIL in any of the supernatants from the FL-TRAIL samples, whereas sTRAIL was secreted to substantial levels a,b. ThisWe went on to further test and compare MSC-produced FL-TRAIL with sTRAIL by testing them on the human colorectal cancer cell line HT-29. The results show that the supernatants of MSC.sTRAIL caused a decrease in survival e and a cProstate cancer cells are resistant to TRAIL-induced apoptosis a, but seWhen we co-treated these prostate cancer cells lines with docetaxel and rTRAIL we found that all three could be sensitised to TRAIL-induced apoptosis b\u2013d. The When we treated PC3 cells with rTRAIL and applied the medium supernatant to a cytokine antibody array, containing antibodies against 42 cytokines, we found CXCL5/ENA-78 and IL-6 to be up-regulated when compared to untreated cells . CXCL5/EGiven the potential problems these cytokines could cause, other combination treatments were investigated that promote significant TRAIL-induced apoptosis and at the same time block or limit the concomitant up-regulation of pro-metastatic factors such as CXCL5/ENA-78 and IL-6.PTEN gene [PTEN is a phosphatase negatively regulating the levels of phosphatidylinositol-3,4,5-trisphosphate thereby dampening the pro-growth PI3K and AKT  signalling pathways [PTEN mutations or deletions have elevated PI3K and AKT/PKB activation levels, which is likely to bestow survival benefits onto these cells when challenged by physiological surveillance mechanisms or treatment with chemotherapeutic agents or biologicals such as TRAIL. Therefore, rTRAIL/PI3K inhibitor (PI3Ki) and rTRAIL/AKT-inhibitor (AKTi) combinations were tested on PC3, LNCaP and C4-2B cells, all bearing mutations/deletions of PTEN [Many prostate cancer cells harbour mutations in the TEN gene . The propathways . Thus, p of PTEN . The resOverall, better results were achieved with AKTi and therefore the AKTi was subsequently tested in combination with MSC.FL-TRAIL and MSC.sTRAIL. We also confirmed that AKTi was functional in PC3 cells at the basal level and also when cells were treated with TRAIL and docetaxel . In the Next, we examined the cell death-inducing activities of MSC.FL-TRAIL and MSC.sTRAIL in two prostate cancer cell 3D models that more closely resemble the three-dimensional nature of a tumour in vivo. Again, the superior effects of MSC.sTRAIL in combination with AKTi was confirmed compared to MSC.FL-TRAIL e,f. Finally, we wanted to know whether AKTi would be able to halt the TRAIL-induced up-regulation of CXCL5/ENA-78 and IL-6. Under conditions that result in apoptosis sensitisation and reduced survival g, AKTi aDespite disappointing results from the initial clinical trials, the death ligand TRAIL remains a promising candidate for use in anti-cancer therapies, as it is able to trigger cancer cell death through its two apoptosis-inducing receptors, TRAIL-R1 and TRAIL-R2 ,41. TargFor therapies that utilise recombinant TRAIL it is clear that only the extracellular domain of the protein is needed. For cell therapeutic approaches TRAIL can be displayed as a full-length membrane protein or produced as an engineered secreted form. Both versions have advantages and disadvantages. Using membrane-bound TRAIL limits the apoptosis induction to the immediate vicinity of the delivering cells. This restricts detrimental effects arising from high levels of systemic TRAIL, but also the therapeutic impact as highlighted by Luetzkendorff et al. . They foIn the present study, we observed that MSCs producing sTRAIL have more potent effects than cells expressing FL-TRAIL, both in conventional cell culture assays as well as 3D tumour cell spheroids. This is despite membrane-bound FL-TRAIL being a very strong apoptosis inducer during direct cell interactions, but for cell therapeutic applications the wider reaching activities of secreted and diffusing sTRAIL appear to be more important and effective . We founIn relation to the discrepancies in the observed therapeutic effects of MSC.sTRAIL vs. MSC.FL-TRAIL, they are reconcilable by the fact that in lung cancer models MSCs with FL-TRAIL might work very well, as most systemically administered MSCs will infiltrate the lungs in the first 24\u201348 h, before they are cleared and appear in other tissues including tumours, but in far smaller numbers . Thus, MIrrespective of the form of TRAIL used, the fact that not all tumours are naturally sensitive to TRAIL remains an issue. This not only limits the therapeutic effects, but also allows cancer cells to respond with induced cytokine production. Some of these factors are known to have pro-metastatic activity and could cause serious side effects. It was shown that TRAIL-triggered cytokine secretion from TRAIL resistant cancer cells is FADD- and caspase-8 dependent. CXCL1, CXCL5, CCL2, and IL-8 were significantly induced by TRAIL in lung, colorectal and pancreatic cancer cell lines ,77. In pWe found that rTRAIL and sTRAIL induced CXCL5/ENA-78 and IL-6 expression in TRAIL-resistant prostate cancer cells. CXCL5/ENA-78 is a member of the CXC chemokine family and is known to be activated by inflammatory cytokines such as interleukin-1 or tumour necrosis factor-alpha . It is aPTEN that lead to high constitutive PI3K and AKT activation, providing a major therapeutic opportunity in the form of PI3Ki and AKTi [The prostate cancer cells utilised in this study, as with many other tumour types, were resistant to TRAIL and therefore needed a sensitising treatment. This provides the opportunity to combine a TRAIL-based therapy with standard chemotherapy, which would be good practice for testing a new experimental cell therapy such as MSC.sTRAIL. Docetaxel is the most commonly used chemotherapy for men with advanced prostate cancer and synergies with TRAIL have been reported . In thisand AKTi . Indeed,and AKTi . While bThus, MSC-delivered sTRAIL in combination with AKTi (or other appropriate co-treatments) is a potential future therapeutic approach for advanced prostate cancer and other difficult to treat tumour types, as it enables TRAIL to induce apoptosis and prevents or reduces the production of harmful cytokines at the same time.All chemicals, unless otherwise stated, were purchased from Sigma . Recombinant TRAIL (rTRAIL) was from R&D Systems . Docetaxel, AKTi (MK-2206) and PI3Ki (LY294002) were from Stratech .The colorectal carcinoma cell lines Colo205 (obtained from Dr. Eva Szegezdi) and HT-29  were cultured in RPMI  and in McCoy\u2019s 5A (Lonza), respectively. HEK293 cells were grown in DMEM (Lonza) and CHO cells (ATCC) in Ham\u2019s F12. The prostate cancer cell lines, LNCaP (ATCC), C4-2B  and PC3 (ATCC), were cultured in RPMI medium (Lonza). All media were supplemented with 10% fetal bovine serum (FBS) , 100 U/mL penicillin and 100 \u03bcg/mL streptomycin.Human bone marrow derived MSCs were from Lonza, human adipose derived stem cells were from Amsbio  and human umbilical cord derived MSCs were from Promocell . The different human MSCs were cultured in StemMACS MSC Expansion Medium . Murine MSCs were isolated and cultured as previously described . In expeThe TRAIL full-length (FL-TRAIL) cDNA was cloned by RT-PCR from Jurkat cell RNA . The sTRTRAIL expressing MSCs were co-cultured with HT-29 cells in 24-well plates, where HT-29 cells in the lower chamber are separated from the MSCs in the upper chamber by a semipermeable membrane (0.4 \u03bcm pore size). For this, MSCs were seeded in a 6-well plate and transduced with Ad.sTRAIL or Ad.FL-TRAIL respectively. 48 h later, the MSCs were harvested and transferred into the chamber of the transwell-insert . For further analyses the transwell-inserts were removed.3D Spheroid Colorimetric Proliferation/Viability Assay  was performed according to the manufacturer\u2019s instructions. Briefly, upon completion of spheroid formation, the LNCaP spheroids were co-treated with 1 \u03bcM AKTi, 5 ng sTRAIL or an equal volume of FL-TRAIL secreting cell supernatant for 3 days before cell viability was assessed. 500 cells were resuspended in 40 \u03bcL Matrigel . The 3D culture was established for 5 days before the tumoroids were co-treated with 1 \u03bcM AKTi and TRAIL expressing MSCs. Control vector transduced MSCs served as control. Images were taken after 3 days, before cell viability was determined by resazurin reduction.4 cells in a 24-well plate. After 24 h the cells were treated for 3 days. Crystal violet staining was performed as previously described [Cells were seeded at a density of 1 \u00d7 10escribed . The cryCHO and 293 cells were Fugene-HD  transfected with pcDNA.FL-TRAIL and pcDNA.sTRAIL. 48 h post-transfection, the supernatants were removed and filtered through a 0.45 \u03bcm syringe filter (Sarstedt) unless otherwise stated. Secreted TRAIL levels were determined by DuoSet ELISA , according to the manufacturer\u2019s instructions. 1 ng sTRAIL, or an equal volume of FL-TRAIL cell supernatant, was used in further assays unless otherwise stated.For the IL-6  and CXCL5/ENA-78 ELISA , PC3 cells were seeded and treated with 5 ng TRAIL or 1 ng sTRAIL unless otherwise stated. For combination treatments, the cells were pre-treated with 10 \u03bcM AKTi for 10 min and with 12.5 nM Docetaxel for 6 h before TRAIL was added for a further 72 h. CHO cells were transfected with an empty pcDNA3 vector (ctrl), pcDNA.FL-TRAIL and pcDNA.sTRAIL. After 24 h, cells were washed and trypsinised, then counted and mixed with PC3 cells at a ratio of 1:5 for 72 h. The resulting supernatants were analysed for CXCL5/ENA-78 and IL-6 by ELISA.6 cells per test were washed with PBS supplemented with 2% bovine serum albumin. Anti-human TRAIL antibody , anti-human TRAIL-R1 (Diaclone) and anti-human TRAIL-R2 (Diaclone) were added for 20 min at 4 \u00b0C. Following a washing step, the cells were incubated with rat anti-mouse IgG1 PE . An isotype-matched immunoglobulin was used as control. After fixation with 2% paraformaldehyde samples were immediately analysed.Cells were analysed for TRAIL and TRAIL-receptor expression on their cell surface by flow cytometry. 1 \u00d7 10For apoptosis measurements, cells were treated 24 h with TRAIL, unless otherwise stated. In combination treatments with docetaxel, cells were pre-treated with docetaxel for 48 h before TRAIL was added. Apoptosis was determined 24 h after TRAIL was added. In combination treatments with AKTi, cells were co-treated with 1 \u03bcM AKTi and TRAIL for 24 h. TRAIL expressing cells were harvested 48 h post transfection/transduction and added to the tumour cells at the indicated ratios. Apoptosis was determined 24 h later.w/v) containing 0.1% Triton X-100 (w/v) and propidium iodide 50 \u03bcg/mL) [DNA hypodiploidy staining: cells including their medium supernatant and PBS wash were harvested and centrifuged at 1300 rpm for 7 min at 4 \u00b0C. After washing with PBS, Nicoletti buffer (Sodium citrate 0.1% (0 \u03bcg/mL) ,98 was aProteins were separated by SDS-PAGE and transferred onto PVDF membranes . Primary and secondary antibodies were diluted in TBS, 0.1% Tween and 3% BSA. Bands were visualised with ECL Western blotting substrate (Thermo Fisher Scientific). Rabbit anti-TRAIL , rabbit anti-phospho AKT and rabbit anti-AKT  were used as primary antibodies and peroxidase-conjugated anti-rabbit antibody  was used as secondary antibody.For these studies, supernatants from PC3 cells treated with 5 ng rTRAIL were used. They were compared to the cytokine levels of untreated PC3 cells. The respective supernatants were diluted 10-fold and applied to human cytokine antibody arrays III  according to manufacturer\u2019s instructions. Each cytokine signal was normalised to the internal control and the fold upregulation in response to rTRAIL was calculated.Experimental values are expressed as mean value \u00b1 standard error (SE). For significance analyses, analysis of variance (ANOVA) between groups was used.PTEN mutations/deletions, but other combinations may be necessary in a cancer-specific, or even patient-specific, manner.Our results demonstrate that in the context of cell therapy an engineered, secreted version of TRAIL has more anti-cancer potency than full-length TRAIL that remains membrane-bound and is not secreted into the extracellular space. Therefore, in most cases the use of sTRAIL will be more beneficial. Furthermore, TRAIL resistant prostate cancer cells can be sensitised by AKTi co-treatment, which also prevents the TRAIL-induced induction of tumour promoting and pro-metastatic cytokines from the cancer cells, providing an added advantage. This response is specific, as it cannot be afforded by docetaxel co-treatment, despite being able to sensitise prostate cancer cells to TRAIL-induced apoptosis. Thus, MSC-delivered sTRAIL in combination with AKTi is a potential option for advanced prostate cancer with"}
+{"text": "TRAIL specifically induces apoptosis in cancer cells without affecting healthy cells. However, TRAIL\u2019s cancer cytotoxicity was insufficient in clinical trials. Circulatory-shear stress is known to sensitize cancer cells to TRAIL. In this study, we examine the mechanism of this TRAIL sensitization with the goal of translating it to static conditions. GsMTx-4, a Piezo1 inhibitor, was found to reduce shear stress-related TRAIL sensitization, implicating Piezo1 activation as a potential TRAIL-sensitizer. The Piezo1 agonist Yoda1 recreated shear stress-induced TRAIL sensitization under static conditions. A significant increase in apoptosis occurred when PC3, COLO 205, or MDA-MB-231 cells were treated with Yoda1 and TRAIL in combination, but not in Bax-deficient DU145 cells. Calpastatin inhibited apoptosis in Yoda1-TRAIL treated cells, indicating that calpain activation is necessary for apoptosis by Yoda1 and TRAIL. Yoda1 and TRAIL treated PC3 cells showed increased mitochondrial outer membrane permeability (MOMP), mitochondrial depolarization, and activated Bax. This implies that Piezo1 activation sensitizes cancer cells to TRAIL through a calcium influx that activates calpains. The Calpains then induce MOMP by enhancing Bax activation. From these experiments a computational model was developed to simulate apoptosis for cells treated with TRAIL and increased calcium. The computational model elucidated the proapoptotic or antiapoptotic roles of Bax, Bcl-2, XIAP, and other proteins important in the mitochondrial-apoptotic signaling pathway. Tumor-necrosis-factor-\u03b1 (TNF-\u03b1)-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy discovered by Wiley et al. in 19952. TRAIL induces apoptosis specifically in cancer cells, while sparing healthy cells thus minimizing side effects3. This prompted multiple clinical trials using TRAIL7. The clinical trials showed that TRAIL lacked the necessary cytotoxicity for clinical relevance. Thus, focus has shifted to discovering compounds that enhance TRAIL\u2019s cytotoxicity while maintaining its specificity8.With 8 million cancer-related deaths per year, major breakthroughs in cancer therapy are needed3. Cancer cells will undergo different forms of TRAIL-mediated apoptosis dependent on whether they are type I or II cells9. Type I cells follow the extrinsic pathway. When TRAIL binds to DR4/5, the death-induced signaling complex (DISC) is formed, activating caspase 8. Caspase 8 activates caspase 3, which cleaves functional proteins needed for cell survival10. In type II cells the extrinsic pathway cannot commit a cell to apoptosis. Caspase 8 additionally cleaves Bid to truncated Bid (tBid) leading to activation of the intrinsic pathway11. TBid activates this pathway by inhibiting Bcl-2 and activating Bax to form pores in the mitochondria. These pores lead to mitochondrial outer membrane permeability (MOMP) and the release of the apoptogenic proteins cytochrome c and Smac15.TRAIL induces apoptosis in cancer cells by binding to death receptors 4 and 5 (DR4/5)16. One potential explanation for this shear stress mechanism is the activation of mechanosensitive ion channels (MSCs), specifically the MSC Piezo1. Piezo1 is an MSC that opens in response to mechanical stimuli, such as shear stress and like other MSCs has been previously associated with proapoptotic effects21. Furthermore, Piezo1 has a small molecule agonist known as Yoda1, meaning Piezo1\u2019s activity can be translated to static conditons22.Previously cancer cells have been sensitized to TRAIL-mediated apoptosis when exposed to circulatory levels of shear stress20. One pathway by which calcium induces apoptosis is by causing mitochondrial dysfunction. Calcium influx can cause mitochondrial dysfunction by activating calpains, proteolytic enzymes that cleave Bcl-2 and process Bid to tBid, inducing intrinsic apoptosis25.The proapoptotic effects of Piezo1 and other MSCs have primarily been associated with calcium influxThe mechanism through which shear stress sensitizes cancer cells to TRAIL-mediated apoptosis has not yet been elucidated, nor has a method of exploiting shear stress TRAIL sensitization within tumors been identified. In this study, we demonstrate the role of Piezo1 in shear stress-induced TRAIL sensitization of cancer cells, translate Piezo1\u2019s TRAIL-sensitizing role to static conditions using Yoda1, and explore the mechanism of Piezo1 and TRAIL\u2019s apoptotic synergy using Yoda1 experiments and a new computational model.2, and 10\u2009\u00b5M GsMTx-4 for 4\u2009h  cells were treated with 250\u2009ng/mL TRAIL, shear stress of 2.0\u2009dyn/cm4\u2009h Fig. . The perund Fig. . Shear sund Fig. . This inund Fig. . Shear sund Fig. . These rTo determine if Piezo1 specifically plays a role in this shear stress sensitization, Piezo1 expression was confirmed in PC3 cells via flow cytometry  and bongkrekic acid (BKA), or the Bax channel inhibitor, Bax channel blocker (BCB). CsA and BCB enhanced TRAIL-sensitization by Yoda1 and BKA had no effect  opening or Bax activationCytochrome c (CYCS) and Smac were inhibited by siRNA to determine if MOMP was necessary for Yoda1-TRAIL sensitization. Knockdown of these proteins reduced TRAIL sensitization from 42.7% for the scrambled-siRNA treated cells to 27.2% and 15.8% for siCYCS and siSmac, respectively Fig. . The red30. The threshold for a cell to be considered apoptotic was if cPARP concentration reached 5*105 molecules per cell. MOMP was modeled using the concentration of cytosolic Smac.Figure The computational model was used to determine how Yoda1 and TRAIL act synergistically to induce apoptosis. When TRAIL was used as a monotherapy, cPARP was generated, but the concentration did not reach a critical level Fig. . When YoThe TRAIL initial condition was varied from 0\u2009ng/mL to 500\u2009ng/mL. For TRAIL concentrations greater than 0, cells underwent apoptosis in 24\u2009h, but the time of apoptosis varied Fig. . The 0.031. When caspase 3 expression was reduced, cPARP was lowered to a negligible level because there was not enough active caspase 3 to cleave PARP and induce MOMP , prostate adenocarcinoma cell lines PC3 (ATCC #CRL-1435) and DU145 (ATCC #HTB-81), and breast adenocarcinoma cell line MDA-MB-231 (ATCC #HTB-26), were purchased from American Type Culture Collection . COLO 205 and PC3 cells were cultured in RPMI 1640 cell culture medium , DU145 cells were cultured in EMEM cell culture medium (Invitrogen), and MDA-MB-231 cells were cultured in DMEM cell culture medium (Invitrogen). Media was supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) PenStrep, all purchased from Invitrogen. HUVECs were purchased from Lonza  and used from passages 4\u20135. HUVECs were cultured in Medium 199 (Invitrogen) and supplemented with EGM endothelial cell growth medium SingleQuots kit (Lonza). COLO 205, PC3, DU145, MDA-MB-231, and HUVECs cells were incubated under humidified conditions at 37\u2009\u00b0C and 5% CO2+ and Mg2+ free DPBS  and then treated with Accutase  for 5\u201310\u2009min at 37\u2009\u00b0C before handling. PC3 cells were washed with Ca2+ and Mg2+ free DPBS at 300\u2009\u00d7\u2009g for 5\u2009min. Cells were resuspended in media at a concentration of 0.5\u2009\u00d7\u2009106 cells/mL prior to performing fluid shear stress studies.PC3 cells were washed in CaFor TRAIL studies, cells were treated with 0.250\u2009\u00b5g/mL recombinant human TRAIL  and 10\u2009\u00b5M GsMTx-4  prior to the application of fluid shear stress.16. The design of the cone-and plate-viscometer allows a uniform shear rate to be applied to the cell suspension volume. PC3 cells were treated with 2.0\u2009dyn/cm2, 10\u2009\u00b5M GsMTx-4, and 250\u2009ng/mL TRAIL for 4\u2009h. TRAIL sensitization due to shear stress was calculated under GsMTx-4 treatment and GsMTx-4 treatment conditions using the following equations:To study the fluid shear stress response of PC3 cells in a controlled, uniform environment, studies were conducted using a cone-and-plate device consisting of a stationary plate underneath a rotating cone maintained at room temperature (RT) as described previouslyor for GsMTx-4 treatment2+ and Mg2+ free DPBS and analyzed for cell death using an Annexin-V assay.After treatment, cells were washed thoroughly in CaCancer cells with Piezo1 knockdown were also assessed for shear stress-induced TRAIL sensitization.COLO 205, PC3, DU145, MDA-MB-231, and HUVECs were seeded on 12 well plates and incubated overnight at 37\u2009\u00b0C to allow cells to adhere to the plate surface. Cells were then treated with 1\u201350\u2009\u00b5M Yoda1  or 0.01\u20130.5% DMSO (vehicle control), and TRAIL  for 24\u2009h to identify an EC curve for Yoda1. PC3 cells were treated with 10\u2009\u00b5M Yoda1 or 0.1% DMSO, and 50\u2009ng/mL TRAIL for 1\u201324\u2009h. PC3 cells were also transfected with siRNA against Smac, cytochrome c, and a nontargeting negative control. After transfection, the cells were treated with 10\u2009\u00b5M Yoda1 or 0.1% DMSO, and 50\u2009ng/mL TRAIL. Cancer cell TRAIL sensitization responses were determined by comparing samples exposed to DMSO and Yoda1 conditions using the following equation:The sensitization equation applies to all cancer cell lines labeled for apoptosis and necrosis for all magnitudes of Yoda1 and DMSO, durations, and transfections.PC3 cells were treated with Piezo1 siRNA  in combination with Yoda1 and TRAIL to inhibit Piezo1 activation in response to Yoda1.PC3 cell death was inhibited using 50\u2009\u00b5M bongkrekic acid (BKA) , 1\u2009\u00b5M cyclosporin a (CsA) (MilliporeSigma), 1\u2009\u00b5M calpeptin (Tocris), and 5\u2009\u00b5M Bax channel blocker (BCB) (Tocris) in addition to Yoda1 and TRAIL.2+ and Mg2+ free DPBS and lifted with Accutase. The lifted cells were then added to cell culture supernatants. The samples were analyzed for cell death using an Annexin-V assay.After treatment, supernatants of the cell cultures were collected. Adherent cells were washed with CaFITC-conjugated Annexin-V  and propidium iodide (PI) (BD Pharmingen) were used to assess cell apoptosis and necrosis. The manufacturer\u2019s instructions were followed to prepare samples for flow cytometric analysis. Viable cells were identified as being negative for both Annexin-V and PI, early apoptotic cells as positive for Annexin-V only, late apoptotic cells were positive for both Annexin-V and PI, and necrotic cells were positive for PI only.Cells were incubated for 15\u2009min with Annexin-V reagents at RT in the absence of light and immediately analyzed using a Guava easyCyte 12HT benchtop flow cytometer (MilliporeSigma). Flow cytometry plots were analyzed using FlowJo software . The following control samples were used to calibrate the instrument: unlabeled cell samples to evaluate the level of autofluorescence and adjust the instrument accordingly, and cell samples labeled individually with Annexin-V and PI to define the boundaries of each cell population.2+ and Mg2+ free DPBS and JC-1 fluorescence was assessed via flow cytometry. Cells with depolarized mitochondria were identified as having low JC-1 red fluorescence and cells with healthy mitochondria were identified as having high red fluorescence.PC3 cells were seeded onto 12 well plates and incubated overnight at 37\u2009\u00b0C to allow cells to adhere. Cells were then treated with 10\u2009\u00b5M Yoda1 or 0.1% DMSO, and 50\u2009ng/mL TRAIL. After treatment, the cells were collected and incubated for 20\u2009min at 37\u2009\u00b0C with JC-1 dye (Invitrogen) according to the manufacturer\u2019s directions. The cells were then thoroughly washed with Ca5 PC3 cells were collected and incubated for 30\u2009min with 1\u2009\u00b5M Fluo-4 and 2\u2009\u00b5M Fura Red (Invitrogen) at 37\u2009\u00b0C. The cells were washed in Ca2+ and Mg2+ free DPBS and resuspended in HBSS with Ca2+ and Mg2+ and allowed to incubate for 30\u2009min. PC3 cells were then treated with varying concentrations of Yoda1 and immediately analyzed via flow cytometry to assess real time influx of calcium. Ratiometric fluorescence was calculated by dividing Fluo-4 fluorescence by Fura Red fluorescence. Cells with higher ratiometric fluorescence were identified as having increased intracellular calcium.2\u2009\u00d7\u200910siRNA oligonucleotides against human Smac (siSmac), Cytochrome C (siCYCS), Piezo1 (siPiezo1), and a nontargeting negative control (Scr) were purchased from Thermo Fisher Scientific. PC3 cells were plated on 12 well plates with mixtures of siRNA, Lipofectamine RNAiMAX reagent (Invitrogen), and Opti-MEM added to each well for a 30\u2009nM siRNA solution. After 48\u2009h of transfection, the culture media was changed to RPMI 1640.After PC3 cells were transfected with siRNA oligonucleotides, they were lysed with laemmli buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)  and transferred to PVDF membrane. After transfer, membranes were blocked with 5% bovine serum albumin (Millipore Sigma) in Tris-buffered saline supplied with 0.1% Tween (Thermo Fisher Scientific). Primary antibodies were prepared at 1:1000 dilution at 5% bovine serum albumin in the case of Piezo1 (Abcam ab128245) and Smac  antibody, or at 1:5000 dilution in 5% bovine serum albumin in the case of GAPDH (Millipore MAB374) and cytochrome c (Abcam ab133504). Anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase  were prepared at 1:2000 dilution in 5% bovine serum albumin. Membranes were imaged with West Pico or Dura (Thermo Fisher Scientific) per their respective protocols, using an ImageQuant LAS-4000 system .COLO 205, PC3, DU145, and MDA-MB-231 cells were stained with a rabbit polyclonal human Piezo1 IgG antibody  or a rabbit polyclonal Isotype IgG antibody . Cells were then stained using a secondary antibody goat anti-rabbit IgG Alexa Fluor 633 (Invitrogen A-21070). Piezo1 expression was analyzed using flow cytometry.PC3 cells were seeded onto glass coverslips that were previously coated with poly-L-lysine (MilliporeSigma). Cells were allowed to grow for 48\u2009h and then treated with TRAIL at a concentration of 50\u2009ng/mL. Additionally, the test group was treated with 10\u2009\u00b5M of Yoda1 and the vehicle control group received DMSO. Cells were treated for 24\u2009h. Cells were fixed for 15\u2009min with 4% paraformaldehyde in PBS , and then permeabilized using 1% Triton X-100 (MilliporeSigma T8787) in PBS. Blocking was done for 2\u2009h with 5% goat serum (Thermo Fisher Scientific) and 5% bovine serum albumin in PBS. Cell staining was performed in the blocking serum. Slides were stained with a Bax antibody (6A7) (Invitrogen MA5-14003). Secondary staining was carried out with an Alexa Fluor 488 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific A11029), in addition to ActinRed 555 ReadyProbes reagent (Invitrogen R37112). All antibodies were diluted in the blocking solution at a ratio of 1:100. Primary staining was done overnight at 4\u2009\u00b0C. Secondary staining was performed for 30\u2009min at room temperature. All slides were stained with DAPI (Invitrogen D1306) for 30\u2009min at room temperature in the blocking solution at 1:500. Washes were done twice between each step for 5\u2009min each using 0.02% Tween20 in PBS. Slides were assembled using 10\u2009\u03bcL of Vectrashield antifade mounting media . Confocal imaging was performed using an LSM 880  with a 63x/1.40 Plan-Apochromat Oil, WD\u2009=\u20090.19\u2009mm objective (Carl Zeiss). Image analysis was performed using FIJI. Intensity measurements in the green channel (active Bax) were quantified for each cell. This was then divided by the corresponding blue channel (DAPI) intensities for each cell.2 was added to quench cytosolic calcein, or calcein in permeabilized mitochondria. The cells were allowed to incubate for 20 minutes. The cells were then washed once and analyzed via flow cytometry. Reduced calcein fluorescence was considered to be indicative of mitochondrial permeability.MOMP was measured using the MitoProbe Transition Pore Assay Kit (Thermo Fisher Scientific) and flow cytometry as the kit directed. 2 \u00b5M Calcein-am was added to cells treated with combinations of DMSO, Yoda1, and TRAIL. Next, CoClt-test was used for comparisons between two groups with p\u2009<\u20090.05 considered significant. One-way ANOVA was used for comparing multiple groups with p\u2009<\u20090.05 considered significant. At least three independent replicates were used for each experiment.Data sets were plotted and analyzed using Prism 8 . Two-tailed unpaired Apoptosis modeling was carried out in MATLAB. The systems of ODEs were solved using the \u2018ode15s\u2019 function of MATLAB. All model figures were prepared using MATLAB.44) of TRAIL-mediated apoptosis44. The Albeck-Sorger model was updated to include calcium, calpain, and calpastatin to model Yoda1\u2019s sensitizing effects. Calcium-activated calpain activity, which was inhibited by calpastatin. Calpastatin was degraded by caspase 3, releasing calpain to degrade Bcl-2 and cleave Bid to its active form. Figure 5 or half the initial condition of PARP.The apoptosis model was modified from Albeck-Sorger\u2019s model (For each given reaction, the biochemical equation is represented by one of the following general mass-action paradigms:E represents an enzyme or other protein that reacts with its substrate or binding partner S to form E:S or to form product P, depending on the reaction. ki+, ki\u2212, and Ki+ represent forward, backward, and catalytic rate constants, respectively.x] represents the number of molecules in each compartment44.The cytosolic and mitochondrial compartments are assumed to be well mixed. The transport of molecules between the two compartments is represented by the differential equation:To generate a random population of cells treated with TRAIL and increased calcium, the expression of cytosolic Bcl-2 was modeled as a random-normal distribution.Supplementary Table 1Supplementary Table 2find_Td.mTRAIL_init_calcium.mtestPiezo1.mDuration.mcellDeathPopulation.mSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure 7Supplementary Figure 8Supplementary Figure 9Author contributions document"}
+{"text": "Gastrointestinal stromal tumors are the most prevalent mesenchymal tumors of the gastrointestinal tract. Distant metastases are most often found in the liver or peritoneum with surgery being the preferred treatment option. In our center, fluorescence-guided surgery with indocyanine green is used as standard-of-care for hepatic metastases in colorectal cancer. This case report describes fluorescence-guided metastasectomy for a hepatic gastrointestinal stromal tumor in two patients undergoing open liver resection and radiofrequency ablation.A 69-year old women was seen during follow-up after laparoscopic resection of a GIST in the lesser curvature of the stomach. Contrast-enhanced computed tomography imaging showed two suspicious lesions in liver segment VI and VIII. Intraoperative near-infrared fluorescence imaging of the liver clearly revealed the lesion in segment VIII, and an additional lesion in segment V \u2013 which was not seen on preoperative CT-imaging, neither on intraoperative ultrasonography. The lesion in segment VI was not seen with NIRF imaging due to its deeper location in the liver parenchyma. The second case is an 82-year old man who was also diagnosed with liver metastases from a GIST in the stomach and was scheduled for near-infrared fluorescence-guided liver resection and radio frequency ablation.In this case report we demonstrated the feasibility of fluorescence-guided surgery in detection of liver metastases and treatment planning of two patients with hepatic GIST metastases using indocyanine green.NIRF-imaging with ICG is useful for identification of preoperatively discovered lesions, surgical resection planning and margin evaluation, and for detection of additional hepatic GIST metastases. OverallIndocyanine green (ICG) has been used as fluorescent dye for determining liver function, liver anatomy and for guiding liver resections, with increased utilization over the past decade. ICG binds to plasma proteins and is rapidly taken up by hepatocytes before its excretion into the biliary system. Near-infrared fluorescence (NIRF) imaging shows a variability in distribution of ICG between different tumor types . PreviouHere we report on two cases of open liver resection and radiofrequency ablation (RFA) of hepatic GIST metastases. Informed consent for publication was obtained from both patients and this work is reported according to the SCARE criteria ,9.22.1According to the hospital\u2019s standard-of-care protocol for secondary liver tumors, both patients were admitted to the hospital and were intravenously injected with 10 mg (5 mg/mL) ICG  24 h prior to surgery. During the surgical procedure, after access to the abdominal cavity was acquired, a visual inspection of the abdomen was performed. After the liver was fully mobilized, intraoperative open-field NIRF-imaging of the liver was performed, prior to intraoperative ultrasonography (IOUS), using the Quest Spectrum system  to localize the metastases, and identify possible additional lesions. This was followed by resection and/or RFA of the lesions. The surgical procedures were performed by HH and JM, having 12 years of experience with NIRF-guided liver surgery. After surgery the resected specimens were assessed by a pathologist, and closed-field NIRF-imaging was performed with the Pearl Trilogy Imaging System  for further inspection of the tumor bread loaves.2.2A 69-year-old female was seen at the outpatient clinic for follow-up after resection of a GIST of the stomach (mitotic count 10/50 HPF). Other medical history was notable for laparoscopic cholecystectomy, pulmonary embolism, cerebral infarction, and pyramidal tract syndrome. The CT-scan revealed a suspicious superficial hypodense lesion of 34 \u00d7 44 mm in segment VIII and a 4 \u00d7 5 mm suspicious lesion in segment VI. In segments V and VIII two preexistent calcifications were found, thought to originate from intraoperative spillage of bile stones during previous cholecystectomy. There was no sign of recurrence of the primary tumor, and no lymph nodes or other distant metastases were found. The primary tumor was a \u2018wild-type\u2019 GIST and therefore neoadjuvant chemotherapy was not indicated [Intraoperative NIRF-imaging clearly revealed the lesion in segment VIII . The lesAfter completion of the resection, all resected specimens were cut into 5 mm bread loaves by the pathologist, followed by consecutive NIRF bread loaf imaging. The bread loaves were further processed for histopathological analysis and to determine resection margins. Immunohistochemical staining with Discovered on GIST 1 (DOG1), a gene encoding anoctamin 1 \u2013 a chloride channel protein  \u2013 was peFive months post-surgery a new hypodense lesion was found on CT during regular follow-up. After a multidisciplinary team meeting the patient was scheduled for percutaneous RFA. The first post-RFA scan showed no residual disease after RFA. However, two new liver lesions were found, for which the patient was referred to the oncologist for systemic therapy.2.3The second case represents an 82-year-old male with a solitary suspicious hypodense lesion in segment III of 21 mm, discovered on routine CT-scan during follow-up after previous partial gastrectomy for GIST (mitotic count 2/50 HPF). Previous medical history reported tuberculosis, acute myocardial infarctions, and a GIST in the stomach. After a multidisciplinary team meeting, the patient was planned for percutaneous, CT-guided RFA. However, the metastasis was located in close relation to the intestine and the RFA procedure was aborted. Instead a biopsy of the lesion was taken, confirming a metastasis from a primary GIST. The patient was planned for open resection of the metastasis. Prior to surgery, another CT-scan was performed, revealing a second 4 mm suspicious lesion in segment VIII. Because of its deeper location, the new lesion was considered to be better suitable for RFA.After mobilizing the liver, NIRF-imaging revealed the lesion in segment III a. The seClosed-field NIRF-imaging confirmed the intraoperative findings and showed tumor-free resection margins on the bread loaves b. Immuno3To the best of our knowledge, this is the first case report solely describing the use of NIRF-imaging with indocyanine green in patients with hepatic metastases from GIST. Positive resection margins and missed tumors following surgery are directly related to a decrease in disease free survival and 5-year survival rates in CRLM ,12, we eWe described the use of ICG in hepatic metastases from GIST in two patients in our institution. Intraoperative NIRF-imaging showed to be feasible and effective in both localizing preoperatively found superficial metastases as well as finding additional superficial lesions. ICG is currently used as standard-of-care as intraoperative fluorescent contrast agent in our center for liver metastases.NIRF light penetrates tissue up to 8 mm, allowing the surgeon to identify subcapsular lesions invisible for the naked eye. However, the limited penetration depth remains an important limitation for deeper located lesions. Therefore, IOUS is still required for the identification of deeper (>8 mm) lesions. NIRF-imaging with ICG has proven to be useful for surgical planning and evaluation of resection margins . ImagingNIRF-imaging has shown to be an effective imaging technique in the surgical treatment of CRLM, and now in hepatic GIST metastases as well.4NIRF-imaging with ICG is useful for identification of preoperatively discovered lesions, surgical resection planning and margin evaluation, and for detection of additional hepatic GIST metastases for capsular lesions and subcapsular lesions up to 8 mm below the liver surface. Because of the limited penetration depth of NIRF-imaging should be used as an additional imaging technique next to IOUS. Development of tumor-specific fluorophores for primary GIST and metastases can aid in more accurate detection of these tumors and may lead to lower false-positive rates.The authors report no declarations of interest.No funding was received.According to the local medical ethics committee (METC-LDD) no formal ethical approval was required.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Not applicableOkker D. Bijlstra and Alexander L. Vahrmeijer.Not commissioned, externally peer-reviewed.O.D. Bijlstra: Conceptualization, Methodology, Validation, Formal analysis, Writing - original draft. F.B. Achterberg: Methodology, Writing - original draft. Q.R.J.G. Tummers: Writing - review & editing. J.S.D. Mieog: Writing - review & editing. H.H. Hartgrink: Writing - review & editing. A.L. Vahrmeijer: Writing - review & editing, Supervision."}
+{"text": "Midwifery-led care is recognised as the best choice of maternity care for low-risk women. Robson\u2019s Ten Group Classification System (TGCS) is an internationally recognised audit tool, however there is no midwifery-led service presenting their statistics in this way. The objective of this study was to analyse caesarean section rates for the women attending midwifery-led care at the National Maternity Hospital Dublin, Ireland, using the Robson TGCS.This is a retrospective study of electronic records for a total of 1097 women who were booked to attend the community midwife team in the National Maternity Hospital, during 2016 and 2017.The rate of caesarean section in low-risk nulliparous women (Robson Group 1) was under 6%, without affecting the perinatal outcome. The induction rate in nulliparous women (Group 2) was 36%, less than the national average the cesarean rates were quadruple in this group.Low-risk women who attend midwifery-led services, have a low caesarean section rate in this study. This was achieved with continuity of care, good antenatal preparation, and support throughout labour and birth by a dedicated team of midwives. Outcomes can only be truly compared if we use the same criteria to measure them. The TGCS demonstrates the effectiveness of midwifery-led care. Over the last 20 years, the caesarean section (C/S) rate has increased worldwide2, leading to severe maternal complications including placenta accrete and placenta percreta.Although childbirth is not without risk, most women will have a safe birth and a healthy baby3, attend obstetric-led care for birth. However, midwives provide the care for women in labour and assist at a normal birth. Midwifery-led care has been defined as care where a midwife, in partnership with the woman, is develops a relationship of trust. They are the lead professional with responsibility for assessment of her needs, planning her care, providing continuity of care, making referrals to other professionals as appropriate, and ensuring provision of maternity services5. There is no doubt that some women's pregnancies and births have become more complicated due to many reasons including maternal age, fertility treatment, and obesity6, but the majority of pregnant women are still deemed to be low-risk and should have a straightforward birth under the care of a midwife7.The majority of the 62053 women giving birth in Ireland8 have confirmed that midwifery-led care is very cost-effective, as it reduces labour costs and reduces medical intervention for women9. Fetal outcomes including preterm labour, neonatal death and admission to intensive care are lower for women attending midwifery-led care4.Fawsitt et al.10. Antenatal classes are offered to all women, with particular emphasis on first-time mothers. The classes are honest, promote active birth, use hypnobirthing techniques, use visualization, include a visit to the labour room and may have a birth story from a mother who has given birth. The labour hopscotch11 is taught and used to encourage active labour. Nulliparous women are visited at home when contractions commence to encourage them to remain out of the hospital until labour is established.The first Community Midwifery Service (CMS) was established in Ireland in 1999, at The National Maternity Hospital, Dublin, Ireland. The service is led and managed by a team of 14 midwives and is divided into two geographical locations. Women who attend midwifery-led services have continuity of care and a choice of homebirth or hospital birth. A room designated to the community midwives in the hospital labour ward offers a home-from-home atmosphere for women choosing to give birth in hospital. The team of midwives cares for women from their booking visit at approximately twelve weeks gestation up to seven days after birth. Clinics take place in 5 urban and rural locations, up to 30 km from the hospital. This includes a first booking visit, antenatal visits, antenatal classes, labour, and birth. Postnatal care takes place in the woman\u2019s home. The low intervention labour policy was developed by the midwives, based on the NICE guidelines revised in 2014However, the midwives must also adhere to hospital policy in relation to monitoring and induction of labour. The Community Midwifery birthing suite is not a standalone unit so if a woman requests an epidural, has been induced or requires monitoring, she remains in the birthing room with the community midwife and any further treatment, which may be deemed necessary for the woman, can be carried out there. Two midwives attend all hospital and homebirths. All assessments and any procedures are carried out by the Community Midwives while they offer one-to-one care throughout labour and birth. Women remain with the CMS for care, even if they have developed complications in pregnancy. A consultant obstetrician assists the scheme by reviewing women who are reffered by midwives, with concern in pregnancy. A senior registar is available to them for emergency consultations.Prior to 2015, there was no standard measurement tool that could inform maternity-care health professionals in which group of women the rise of a caesarean section rates were occurring. The Robson\u2019s Ten Group Classification System (TGCS) was recommended by the World Health Organisation (WHO) in 2015 to classify caesarean section. The time is right for midwives to also consider using the TGCS to assist audit in the reporting of midwifery-led care.This study aims to examine the rates of C/S for women attending the Community Midwifery Services, by using the TGCS. This retrospective study applies a clinical audit tool to review the cases of 1097 pregnant women attending Community Midwifery Services in a Tertiary Maternity Care hospital during 2016 and 2017. Information already collected from all women who booked into the Community Midwives Service (CMS) was used anonymously to create a Report Table using TGCS (2001).10. This included no previous caesarean section, no fertility treatment, and a body mass index  under 35 for hospital birth or 30 for homebirth. Women were advised that the community midwives promote active labour and birth. A total of 1102 women booked with the CMS over the two years. There were five women not included in the study as they had developed diabetes or had a twin pregnancy diagnosed after the initial booking visit with the midwives at 12\u201314 weeks pregnant. It is important to emphasize that all the remaining women were included in the study even if complications developed in the pregnancy. In this study, there were no ethical dilemmas as the study did not involve intervention or manipulation of participants. The electronic information used in this study, was previously in existance and the data were collated to create this research. Ethical approval was obtained from the National Maternity Hospital Ethics Committee on 8 January 2018 and an Ethical exemption was also obtained from the UCD Ethics Committee in March 2018. No charges were requested by the NMH ethics Board.This is a retrospective study of electronic records for a total of 1097 women who were booked to attend the community midwife team in the National Maternity Hospital during 2016 and 2017. Women were accepted in the low-risk scheme using a simple inclusion\\exclusion criterion: \u2018Women who have no medical/surgical history that might affect pregnancy\u201912. Since 2015, Robson\u2019s (2001) TGCS has been the internationally recognized tool for the Classification of Caesarean Section by the World Health Organisation (WHO); the WHO statement on the caesarean section13 notes: \u2018\u2026 Let's start to collect data uniformly so that in the near future we will be able to move our focus from C/S rate at the population level to monitoring and discussing C/S rates and outcomes in each group of the Robson classification \u2026\u2019.The TGCS is a standardized objective classification system where events and outcomes of labour and delivery can be incorporated14 reported that using an internationally applicable C/S classification would facilitate auditing, analysing and comparing C/S rates across different settings globally.In 2011, Torloni et al.This classification system is not just the study of C/S but is a platform for other research. Robson and the WHO recommend that any research being carried out in relation to labour or birth should be presented in a 10-group format that will allow for easier comparison, nationally and worldwide. The TGCS divides all women into ten basic groups according to parity, gestation, fetal presentation, and whether labour was induced or was spontaneous. All women fall into one of the ten groups, which is totally inclusive, mutually exclusive.15.The TGCS involves the use of a simple table to gain greater understanding of clinical practice and assists in the comparison and contrast of clinical outcomes. The classification is not a criticism of practice but a data collection tool to count outcomes and improve the quality of careData were collected including the electronic charts of women. The participants needed to be identified as attending the community midwives. The data input was examined and cross-checked using the registration documents collected at each woman\u2019s booking visit to ensure all women were recorded. Using this information, all women identified were placed in one of the ten groups, according to parity, gestation, fetal presentation and induced or spontaneous labour to produce the TGCS table. Community midwives do not take care of women with previous C/S. Therefore, these women (corresponding to Group 5) are omitted, and women with a multiple pregnancy (Group 8) are cared for by the fetal assessment unit and leave the care of the community midwives. An excel spreadsheet was developed to allow any non-computerized maternity unit to collect similar data.17. Therefore, a woman in spontaneous labour at term with her first baby in a head-down position, had a 94.1% change of a vaginal birth with the community midwives.17. Women attending the Community Midwives Services strive to have normal labour but the midwifery team must adhere to criteria for induction within the hospital. The induction rate for first-time mothers was 36%, compared to the hospital rate of 43.4%.It is very evident that the C/S rate rose rapidly to 30% if a nulliparous woman had an induced labour or pre-labour C/S. This group of women also contributes the most substantial proportion of women having C/S, in fact 4% of the total 9.75% C/S rate. Four women had a pre-labour C/S for a placenta previa or PET18. One woman achieved a vaginal breech birth. Although most women have an elective C/S in this group, it still only contributes 2.57% to the total C/S rate.The largest group of women cared for by the community midwives had a previous vaginal birth and were in spontaneous labour. These women had the best outcomes with a very low C/S rate of 0.72%. These women had a high homebirth success rate, with 85% achieving a homebirth. The induction rate was 14.6% for multiparous women and the C/S rate in this group was 4.2%. The C/S rate of 9.1% in this group includes the 5 women who had a pre-labour C/S for placenta previa, IUGR or a previous 3rd degree tear. Breech C/S rate is high as all women who were diagnosed with a breech presentation were booked for an elective C/S as recommended by the Term Breech Trial12 refers to this group as the \u2018quality assurance\u2019 group as the C/S rate should always be 100% and the occurrence is always similar. Preterm labour had a relatively high C/S rate of 12.5% as women usually present with a pregnancy induced problem such as pre-eclampsia or fetal compromise. In line with the Sandall et al.4 study, this group of women was unexplainably small at 1.5% compared with the hospital preterm rate of 4.3%.Women giving birth with an abnormal lie over 37 weeks is a small group with only 3 women recorded. Globally, this figure is 0.3\u20130.5% or 3\u20135 per 1000 births. RobsonIn this study, further research was completed on Group 1 (low-risk nulliparous women). One infant had an apgar score <7 (0.4%) and 26 babies (10.3%) were admitted to the neonatal unit. In the same group, 18.5% of the hospital babies were admitted to the neonatal unit.19. According to Dabrowski20, when the woman has a relationship with her midwife there is greater trust and the woman is more likely to share information with the midwife that could be important in her care and pregnancy.Normal-risk women should be encouraged to attend a midwife for their maternity care. There is very strong evidence that better continuity of care and provider of care lead to better outcomes for the mother and baby21, shows that care and safety of mothers and newborn babies should be at the very heart of maternity services. Women who received continued care throughout pregnancy and birth from a small team of midwives were less likely to give birth prematurely and required fewer interventions during labour and birth. Brocklehurst et al.22 reviewed 64538 eligible women showing fewer interventions with no impact on perinatal outcomes.Cochrane Review into Midwifery-led CareThe findings of the current study show that the necessary data collection and application of the Robson TGCS can be carried out quite simply and effectively, and in a range of settings.23. The ultimate aim of any clinical audit should lead to improvements in women\u2019s care24. The use of audit helps raise the quality of healthcare, and highlights the most essential concern of any health professional: to optimize clinical performance and provide the best possible care for women. Maternity care providers can give better care with better data25. The examination of the data collected by the CMS in the current study can help improve quality of care by examining and discussing the C/S carried out in each group. While every service produces data that can be analysed, the current study findings would support the view that all professionals offering maternity care should use the same tool, the TGCS, as recommended by the WHO26. The TGCS tables from the community midwifery service can then be compared with those of other services, hospitals and countries around the world.The increasing C/S rate worldwide has led to an increase in maternal mortality26 recommended TGCS tool. On the other hand, the use of a pre-determined structured data set means that the level of detailed data is a limiting factor. That said, the use of the TGCS as part of a clinical audit offers a basic framework for interesting research. It must be commented that the women who attended the community midwifery service had chosen that model of care and may have sought a low-intervention birth.This is the first study to examine C/S rates in a Community Midwifery setting by applying the WHOThe TGCS is a starting point to review many areas of research for midwives. Community Midwifery Services can demonstrate reductions in interventions in labour for low-risk women without affecting maternal or fetal outcomes. These statistics when delivered appropriately can help women make the choice between maternity services based on the outcomes of labour.28 assists audit by allowing a comparison and analysis of similar groups of women availing themselves of midwifery-led care. Over time, this system can be used and shared with units around the world. This quality improvement initiative may help us learn from other midwifery-led practices how to maintain or reduce interventions and C/S rates. Midwives need to focus on the care of low-risk, first-time mothers, as reducing the C/S rate in this group will influence C/S rates in the future.This study successfully demonstrates the effectiveness of midwifery-led care in maintaining a reasonable C/S rate for low-risk women without affecting fetal or maternal outcomes. The use of the Robson Ten Group Classification System"}
+{"text": "This work furthers our understanding of how these phases originate in many natural and synthetic systems.Han  This is a geometric relation involving the volume (V) occupied by the amphiphilic molecule divided by its cross-sectional area (a) and its hydro\u00adphobic alkyl chain length (l) , NMR, calorimetric analysis, optical textures et al. relies on a detailed study by state-of-the-art high-resolution transmission electron microscopy and electron crystallography of bicontinuous mesoporous silica crystals, which enable the relevant bicontinuous mesophase to be frozen in a stable silica replica of the structure. The mesoporous crystals were prepared to produce particles rich in contact reflection twin defects, possessing a twin boundary plane, which separates the two identical crystalline domains (Fig. 1The approach of Han ns Fig. 1.et al. describe how the formation of G-twin boundaries in the {211} plane may be related to structural transformations between hexagonal and lamellar phases. These may give rise to the G structure, supported by observed epitaxial {211} relationships between the structures involved and the generation of the essential chiral surface of the G-twin boundary. The authors also treat the D-twin as a stacking fault, which is neatly used to postulate potential stacking orders and new surface and channel motives that may arise through twinning defects in bicontinuous minimal surfaces.Two bicontinuous cubic phases, the G and D phases, and their corresponding G-twin and D-twin boundaries are structurally assessed revealing a detailed model of the connectivity of the surface and its curvature fluctuations at the twin position itself, as well as 3D reconstructions of the silica network. Han This work opens a new avenues to study defect formation and changes to the surface geometry of bicontinuous minimal surfaces, and will be especially useful if it can be applied to analogous biological systems. Knowledge of the structural transformations may also help in directing the design and application of mesoporous silica particles."}
+{"text": "Atrial fibrillation (AF) is at least partially heritable, affecting 2\u20133% of the population in Europe and the USA. However, a substantial proportion of heritability is still lacking. In the present study, we aim to identify potential crucial genes associated with AF through bioinformatic analyses of public datasets.Microarray data sets of GSE115574, GSE31821, GSE79768, GSE41177 and GSE14975 from the Gene Expression Omnibus (GEO) database were retrieved. After merging all microarray data and adjusting batch effect, differentially expressed genes (DEGs) were identified. Functional enrichment analyses based on Gene Ontology (GO) resource, Kyoto Encyclopedia of Genes and Genomes (KEGG) resource, Gene Set Enrichment Analysis (GSEA), Reactome Pathway Database and Disease Ontology (DO) were carried out. Protein-protein interaction (PPI) network was constructed using the STRING database. Combined with aforementioned significant bioinformatics information, potential crucial genes were subsequently selected. The comparative toxicogenomics database (CTD) was used to explore the interaction between potential crucial genes and AF.IGFBP2, C1orf105, FHL2, CHGB, ATP1B4, IGFBP3, SLC26A9, CXCR4 and HTR2B were considered the potential crucial genes. CTD showed CXCR4, IGFBP2, IGFBP3 and FHL2 had higher scores with AF.We identified 27 of DEGs with gene expression fold change (FC)\u2009\u2265\u20091.5 or\u2009\u2264\u20092/3 (|log2 FC|\u2009\u2265\u20090.58) and 5 with FC\u2009\u2265\u20092 or\u2009\u2264\u20090.5 (|log2 FC|\u2009\u2265\u20091) of AF patients compared with sinus rhythm controls. The most significantly enriched pathway was regulation of insulin-like growth factor transport and uptake by insulin-like growth factor binding proteins. CXCR4, IGFBP2, IGFBP3 and FHL2, may be associated with risk of AF. Our study provided new insights of AF into genetics, molecular pathogenesis and new therapeutic targets.The 9 potential crucial genes, especially  Atrial fibrillation (AF) is the most common sustained arrhythmia and is one of the major causes of stroke, heart failure, sudden death, and cardiovascular morbidity in the world . The estPITX2, ZFHX3, and PRRX1, associated with AF . In each data set, only human left atrial appendage (LAA) samples from AF and sinus rhythm (SR) subjects were selected, and finally 46 AF and 31 SR samples were included for subsequent analyses.Raw files of 5 registered microarray data sets, including GSE115574, GSE31821, GSE79768, GSE41177 and GSE14975 (Table\u00a0https://www.activestate.com/ products/perl/) to convert the gene probe IDs to gene symbol codes. Because GSE14975 was extracted using the Affymetrix Microarray Suite 5 (MAS5) algorithm, it was log base 2 transformed. After merging all microarray data, batch effects were adjusted by the \u201ccombat\u201d function of \u201csva\u201d package of R software using empirical Bayes frameworks [Series matrix files were processed with ActivePerl 5.24.2 software  value was \u22651.5 or\u2009\u2264\u20092/3 (|log2 FC|\u2009\u2265\u20090.58), which were visualized as Volcano plots and heat map plots. \u201cggplot\u201d packages of R software was applied to generate box plots for genes which have the adjusted P value <\u20090.05 and the gene expression FC value \u22652 or\u2009\u2264\u20090.5 (|log2 FC|\u2009\u2265\u20091).To assess differential expression, using the \u201climma\u201d package of R software, a linear model was fitted and a simple empirical Bayes model was used to moderate standard errors . A moderhttp://geneontology.org/) is a bioinformatics tool providing a framework and set of concepts for describing the functions of gene products from all organisms [https://www.kegg.jp/) is a database resource integrated the information of genomes, biological pathways, diseases and chemicals [http://software.broadinstitute.org/gsea/index.jsp) is a computational method for interpreting gene expression data based on molecular signature database [https://reactome.org/) is pathway annotation database collecting the biological pathways and processes in the human [http://disease-ontology.org) represents a comprehensive knowledge base of 8043 inherited, developmental and acquired human disease [org.Hs.eg.db\u201d was used to convert gene symbol codes to Entrez ID. To better understand the biological function and characteristics, R software was used to perform enrichment analysis, with the \u201cclusterProfiler\u201d package for Go and KEGG enrichment analyses, the \u201cReactomePA\u201d package for Reactome pathway analysis and the \u201cDOSE\u201d package for DO enrichment analysis. The \u201cggplot2\u201d, \u201cpathview\u201d and \u201cgraphite\u201d packages of R software were used to visualize the plots. GO terms and KEGG maps of biological functions associated with an adjusted P value <\u20090.05 and Q value <\u20090.05 was considered to be significantly enriched. GSEA v4.0.3 was applied for GSEA analysis. Using a permutation test 1000 times, the cutoff of the significance level nominal P-value, false discovery rate (FDR) Q-value and family wise-error rate (FWER) P-value were all chosen as 0.05 for the most significant pathways related to AF.The Gene Ontology (GO) Resource  was performed to construct a PPI network to reveal the generic organization principles of functional cell systems and to predict protein-protein interactions [P value <\u20090.05 and |log2FC|\u2009\u2265\u20091; (2) P value <\u20090.05, |log2FC|\u2009\u2265\u20090.58 and enriched in biological functions. In addition, genes with the degree of connectivity larger than 5 in the PPI network were also included.The STRING database (http:/ /ractions . The Molhttp://ctdbase.org/) integrated information including chemical-gene/protein interactions, chemical-disease and gene-disease relationships to develop hypotheses related to the mechanisms of disease [The comparative toxicogenomics database  were enriched in insulin-like growth factor I binding process  and insulin-like growth factor binding process . Using screening criteria of adjusted P value <\u20090.05 and Q value <\u20090.05, no pathway was enriched in KEGG. The \u2018mineral absorption\u2019, \u2018calcium signaling pathway\u2019 and \u2018proximal tubule bicarbonate reclamation\u2019 pathways were enriched . Specifically, SLC26A9 and ATP1B4 genes were enriched in mineral absorption, while two up-regulated genes (CXCR4 and HTR2B) were correlated with calcium signaling pathway. Only up-regulated ATP1B4 gene was enriched in proximal tubule bicarbonate reclamation pathway. However, these enrichments did not remain significant after multiplicity adjustment by BH.To further investigate the biological functions of the 27 DEGs, functional enrichment analyses were performed and results were shown in Table\u00a0IGFBP2, IGFBP3 and CHGB) were enriched in Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) , but not in AF associated process . The sets were (1) regulation of cell growth involved in cardiac muscle cell development, (2) regulation of cardiac muscle cell differentiation, (3) cardiac muscle cell differentiation, (4) positive regulation of cardiac muscle cell differentiation, (5) physiological cardiac muscle hypertrophy. To explore whether these sets contained DEGs, we examined the leading-edge subsets for each gene set (defined above). In five leading-edge subsets, only DEG FHL2 was involved in cardiac muscle cell differentiation.GSEA was applied to test merged GEO dataset to identify functional gene sets correlated with heart. The SR\u2009>\u2009AF analysis identified five sets whose expression was correlated with AF , and several important pathways, which were associated with AF risk, were identified, suggesting these may play important role in the mechanism of AF.In the present study, we integrated gene expression profiles of 46 AF samples and 31 SR samples from 5 GEO datasets and analyzed the data using bioinformatics tools. A total of 27 DEGs with |log2 FC|\u2009\u2265\u20090.58 and 5 with |log2 FC|\u2009\u2265\u20091 in AF compared with SR samples were selected. Furthermore, 9 potential crucial genes  in the circulation. IGFBP3 is involved in oxidative stress, atherosclerosis and left ventricular hypertrophy [IGFBP2 and IGFBP3 were enriched in GO term of insulin-like growth factor binding and pathway of IGF transport and uptake by IGFBPs. Thus, IGFBP2 and IGFBP3 might bind and regulate the functions of IGFs through IGF transport and uptake by IGFBPs pathway, and then affect the susceptibility of AF [The functional gene ertrophy , 22. Morertrophy . Enrichmty of AF .CHGB was involved in IGF transport and uptake by IGFBPs pathway including IGFBP2 and IGFBP3, and the gene expression of CHGB was higher in AF patients. Chromogranin B (CHGB) is an emerging cardiovascular biomarker, which is encoded by CHGB [IGFBP2, IGFBP3 and CHGB may participate in the occurrence and development of AF. A more thorough understanding of IGF transport and uptake by IGFBPs pathway in AF is necessary.Our study revealed that  by CHGB . CHGB ca by CHGB , 24. CHG by CHGB . These fIGFBP2, IGFBP3 and CXCR4 were divided into a group according to protein-protein interactions. There was evidence that CXCR4 was overexpressed in chronic AF patients, and might contribute to the process of AF through regulating atrial fibrosis and structural remodeling [CXCR4 was also found to be potential crucial gene related to AF. KEGG pathway enrichment analysis showed that the CXCR4 and HTR2B were enriched in calcium signaling pathway, which had been extensively characterized in the role in cardiac hypertrophy and remodeling processes [HTR2B is located in chromosome 2q37.1. 5-HT2B (5-hydroxytryptamine receptor 2B) receptor coded by HTR2B, is presented in the cardiovascular system, and may indirectly produce life-threatening arrhythmias and cardiodepression [2B enhances gap junctional intercellular communication (GJIC) in a receptor subtype-specific manner, and prolongs 5-HT exposure to alter the Cx expression pattern which associated with AF [With PPI analysis, modeling . In the rocesses . HTR2B ipression , 28. In FHL2 and its protein product has a function in cardiovascular disease [FHL2 was up-regulated in AF samples compared to SR, and was involved in cardiac muscle cell differentiation. These results indicated that FHL2 might be a potential biomarker of AF.Increasing evidence demonstrated that  disease , 30. FHL disease , 32. We C1orf105 and ATP1B4 had 2 fold lower and higher gene expression, respectively in AF patients than SR control. SLC26A9 and ATP1B4 were enriched in KEGG pathways of mineral absorption and proximal tubule bicarbonate reclamation. Previous study showed that a SNP on C1orf105 was associated with remodeling response to atherosclerosis [Slc26a9 encodes transporters with diverse functional attributes and RT-PCR showed that Slc26a9 is detectable in heart [C1orf105, SLC26A9 and ATP1B4 were related with cardiovascular disease and might have a function in AF.In this study, clerosis . Slc26a9in heart . The aboIn current study, we have discussed that 9 potential crucial genes are involved in the occurrence and development of AF, suggesting these genes may serve as potential biomarkers and therapeutic targets for AF. However, the limitations of this study should be considered. Firstly, it is difficult to consider some important factors such as regions, races and age. Considering that the development of AF results from various environmental and genetic factors, some unmeasured factors including region, family history and risk factors of AF should be evaluated in further research. In addition, the potential crucial genes need further validation by RT-qPCR in clinical samples. Finally, the mechanisms in which these genes play are not completely clear. More evidence is required to find out the biological foundation.IGFBP2, IGFBP3, CHGB, CXCR4, HTR2B, FHL2, C1orf105, ATP1B4 and SLC26A9), and pathways using bioinformatic analyses. The exploration of potential crucial genes of AF may provide some potential aid in further identification of new biomarkers for the susceptibility of AF and useful treatment targets.Our study integrated data with relative larger sample size from multiple GEO datasets and identified 9 potential crucial genes  Data standardization. Pre-standardization gene expression levels of each data set are presented as blue boxplots; (b) Data standardization. Post-standardization gene expression levels of each data set are presented as red boxplots.Additional file 2: Figure S2. Volcano plot of DEGs in AF samples compared to SR samples. Red indicates the gene expression was up-regulated in AF samples compared to primary samples ; Green indicates the gene expression was down-regulated in AF samples compared with primary samples ; Black indicates the adjusted P value was >\u20090.05.Additional file 3: Figure S3. Pathway view of Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) using the REACTOME database. IGFBP2, IGFBP3 and CHGB were enriched in the pathway.Additional file 4: Figure S4. Bar plot of DO enrichment of DEGs. The X-axis indicates the number of genes represented in the disease.Additional file 5: Figure S5. (a) PPI network of the DEGs and modular analysis. Yellow nodes represent DEGs in the same module. (b) Bar plot for the interaction numbers of each gene in PPI network.Additional file 6: Figure S6. Relationship to AF diseases related to potential crucial genes based on the CTD database.Additional file 7: Table S1. The DEGs of merged data set with the use of criteria of adjust P value <\u20090.05 and |log2FC|\u2009\u2265\u20090.58. Table S2. Summary of GSEA result with FDR Q-value <\u20090.05 and FWER P-value <\u20090.05."}
+{"text": "Complex network infrastructure systems for power supply, communication, and transportation support our economic and social activities; however, they are extremely vulnerable to frequently increasing large disasters or attacks. Thus, the reconstruction of a damaged network is more advisable than an empirically performed recovery of the original vulnerable one. To reconstruct a sustainable network, we focus on enhancing loops so that they are not trees, which is made possible by node removal. Although this optimization corresponds with an intractable combinatorial problem, we propose self-healing methods based on enhancing loops when applying an approximate calculation inspired by statistical physics. We show that both higher robustness and efficiency are obtained in our proposed methods by saving the resources of links and ports when compared to ones in conventional healing methods. Moreover, the reconstructed network can become more tolerant than the original when some damaged links are reusable or compensated for as an investment of resource. These results present the potential of network reconstruction using self-healing with adaptive capacity in terms of resilience. Unfortunately, the frequency of large disasters or malicious attacks is increasing day by day due to climate change, crustal movements, military conflicts, cyber-terrorism, and mega-urbanization. For example, it is well known that a small accident involved a large-area power collapse in North America  and the Therefore, when large-scale failures or attacks occur, the recovery of the original vulnerable network is inadvisable. Instead, reconstruction by healing is required. By changing the structure instead of recovering the original one, a question arises as to how a sustainable network should be reconstructed to maintain the network function. However, because the linking resources  and ports  are usually limited, allocation should be controlled at the same time as the rewiring or making additional investment for healing. Such a reconstruction conforms with the concept of resilience in system engineering or ecology as a new supple approach to sustaining basic objectives and integrity, even when encountering extreme changes in situations or environments  for technological systems, organizations, or individuals ,8,9.In this paper, through numerical simulation, we study how to reconstruct a sustainable network under limited-resource conditions, and propose effective self-healing methods based on enhancing loops through a local process around damaged parts. In addition, we show a significant improvement from the previous study  by reducq and N denotes the total number of nodes (as the network size). Some of disconnected links may be reusable on neighboring sides depending on the damage level. Although we call the reuse \u201crewiring\u201d, removal of nodes is a different problem to that in the so-called \u201cusual\u201d rewiring methods [Step 0:\u00a0Detection and initiationAfter detecting a removal as a malfunction at the nearest neighbor of the attacked node, the healing process is initiated autonomously.Step 1:\u00a0Selection of two nodesSince the neighbor loses links at least temporarily before rewiring, the damaged ones become an attached candidate for healing. Thus, two unconnected nodes are chosen from the neighbors of the nodes removed by attacks. The selections are different in our proposed and the conventional healing methods. Moreover, neighbors are extended in our proposed methods.Step 2:\u00a0Rewiring for healingThe chosen two nodes are connected as rewiring for healing. The above process is repeated for Here, Almost simultaneously attacked nodes are not recoverable immediately. Therefore, they are removed from network function for an amount of time. In such a case of emergency for healing, two unconnected nodes are chosen and rewired, as the reconstruction assistance or reuse of links emanates from the removal of  methods ,13,19. Ti by attacks, where i. Thus, there exist at least as many active (reusable) ports of a node as its degree in the original network before any attacks.In the healing process, rewirings are performed by changing directions and ranges of flight routes or wireless beams. However, we will not discuss the details of realization, which depends on current or future technologies and target systems. Here, we focus on connectivity at the most fundamental level in many network systems for not only communication but also transportation, power supply, and other infrastructures, while our approach may be useful for path control or failure detection, e.g., by software-defined network-based reconfiguration of communication systems with switches in managing reliability, latency, or security at some service levels ,21,22. IRingBP\u00a0i to be necessary for loops is calculated only once, just after an attack. Please note that a ring is the simplest loop when it uses the least number of links.Previously, our combination method of ring formation and enhancing loops was based on . After mRingRecal\u00a0We modified our method with the recalculation of BP. After making rings, a set RingLimit1,5,10\u00a0i to its degree We modified our method with limited rewirings. After making rings, in enhancing loops, the number of rewiring links is limited at node RingLimit5Recal\u00a0i to We modified our method by a combination of RingRecal and RingLimit5. After making rings, a set of In our proposed healing methods, there are two phases: ring formation and enhancing loops by applying BP in the next subsection. Moreover, they  are modified to reduce the additional ports from the previous results [First, in ring formation see , the ordj with the minimum j belongs. For each rewiring, a set of i to Next, when enhancing loops on each ring for the remaining rewirings in i is removed =pj\u2192ij,zi\u2192j(t)=RBR\u00a0Conventional Random Bypass Rewiring (RBR) method  (correspGBR\u00a0Greedy Bypass Rewiring (GBR) method improved from RBR heuristically .SLR\u00a0Conventional Simple Local Repair (SLR) method  with priWe briefly explain the following typical healing methods in network science  and computer science.In network science, a self-healing method that adds new random links to interdependent two-layered networks of square lattices has been proposed, and the effect against node attacks is numerically studied . In parti, only Furthermore, the following self-healing methods, whose effects are investigated for some data of real networks, are worthy to note. One is a distributed SLR  with repr is RBR  on the mver, GBR  is propoIn computer science, the ForgivingTree algorithm has been proposed . Under ri-j nodes in the surviving We evaluate the effect of healing by four measures: the ratio S(q)/Nq  for the networks ,30,31 afIn each of q increases.tion see . The folP method , they arWe have proposed self-healing methods with modifications from the previous one  for recoSimulation results show that our proposed combination methods of ring formation and enhancing loops are better than the conventional SLR , RBR, anD (see However, what structure is the optimally tolerant against further attacks in varying the degree distribution after healing remains an open question. Even if our prediction comes true, it is not yet known what approach is more effective and practical for approximately maximizing the FVS. These challenging problems are beyond the discussion of onion-like structure under a given degree distribution ,12. In aD see . The dev"}
+{"text": "Calciphylaxis is a rare but highly fatal vascular calcification disorder with a predilection for patients with end stage renal disease (ESRD). The pathogenesis of calciphylaxis is unknown, however, several risk factors have been identified such as hypercalcemia, hyperphosphatemia, hyperparathyroidism, low serum albumin, and history of warfarin therapy. This article presents a case of calciphylaxis induced by warfarin in a COVID-19 patient. Calciphylaxis also termed calcific uremic arteriolopathy (CUA) in patients with end-stage renal disease (ESRD) is a rare and life-threatening vascular calcification disorder with unclear pathogenesis. A number of studies suggest that the main pathology is hypercoagulability status which causes occlusion of small blood vessels in the subcutaneous adipose tissue and dermis. This results in painful and ischemic skin lesions\u00a0-4.There are few reported cases of calciphylaxis, and most of these cases were elderly patients and shared one or more of the following: female sex, obesity, impaired renal function, diabetes mellitus, hypertension, hyperparathyroidism, and use of Warfarin or calcium binders\u00a0-8.Patients with calciphylaxis initially present with a painful skin lesion that was described as plaque, purpura, or livedo, then rapidly progress to the stellate, malodorous ulcer with black eschars. Laboratory tests in the case of calciphylaxis are usually nonspecific, while histopathology test remains the gold standard test for definitive diagnosis. However, in cases where there is a high clinical suspicion of calciphylaxis, prompt aggressive treatment should be initiated, and histological confirmation can be reserved\u00a0-4.A 66-year-old Saudi female presented to the ED on the 24th\u00a0of October 2020 complaining of generalized abdominal pain and multiple ulcers in the left breast, lower abdomen, and right thigh. The pain started four months before the presentation; she described it as a burning sensation, and there were no aggravating or relieving factors. She had a history of COVID-19 pneumonia four months back, and after one to two weeks (the patient cannot remember exactly) these ulcers started to appear as red painful lesions, then became black with yellow to green discharge.\u00a0She has known a case of a pulmonary embolism on warfarin for one year, which was stopped one week before the presentation at another healthcare facility. She is also a known case of uncontrolled type II diabetes mellitus on insulin for three years, ESRD on hemodialysis three times per week in the past two years. Past medical history: stroke five years back, and past surgical history: hemithyroidectomy 10 years ago and since then she was kept on thyroxin.Upon general examination, the patient was hemodynamically stable, conscious, alert, and oriented, and the systemic examination was unremarkable. There were multiple skin ulcerations in the left breast, right lower abdomen  P. aeruginosa from tissue culture, but there was no growth from stool culture. For initial imaging, enhanced CT was done and revealed an osteoporotic fracture of L1, and skin thickening, and subcutaneous fat at the right lower abdomen with no intra-abdominal collection. Moreover, a pulmonary angiography scan was done and excluded pulmonary embolism. Tissue biopsy confirmed the diagnosis of calciphylaxis as it showed markedly necrotic connective and fibrofatty tissue with dystrophic calcification and autolytic changes without signs of malignancy. Figures\u00a0Initial laboratory tests were done to investigate other differential diagnoses Table\u00a0. MicrobiThe patient was admitted to the general ward and managed primarily by supportive measures while waiting for the tissue biopsy results. These measures are as follows: regular hemodialysis, diabetic diet, daily wound care, pain management, empirical antibiotic, and adjustment of home medications. Following the culture sensitivity, the patient was shifted to a culture-sensitive antibiotic. After that, she was clinically stable, and her pain subsides, but the skin lesions persist. Therefore, she was discharged on the 1st of December 2020 with outpatient clinic follow-up.\u00a0Based on the\u00a0clinical data, and the histopathology findings this patient was diagnosed with warfarin-induced calciphylaxis, which was precipitated by COVID-19. This suspicion was raised at the beginning, because of the patient\u2019s risk factors, which were as follows: female sex, obesity (BMI 45.8), thrombophilia, ESRD on hemodialysis, type II diabetes mellitus, and use of warfarin\u00a0-13.Other risk factors that are frequently implicated in patients with calciphylaxis include rapid weight loss, hepatobiliary disease, hypoalbuminemia, vitamin K deficiency, hypercalcemia, hyperphosphatemia, and hyperparathyroidism or medical treatment such as calcium-based phosphorus binder and vitamin D\u00a0-3, 12-13Clinical presentation of calciphylaxis can be classified into nonulcerated lesions (early-stage) and ulcerated lesions (late-stage). The affected areas can be peripheral adipose tissues  or central  which are more common in patients with high body mass index (BMI) and ESRD. This patient had a late-stage presentation with central lesions mainly, this indicated poor prognosis\u00a0-2.Skin biopsy is considered the gold standard diagnostic test for calciphylaxis, which demonstrates calcified blood vessels with or without fibrosis. CT scans and laboratory tests are also helpful for an initial assessment to exclude other differential diagnoses as mentioned earlier\u00a0, 14.Calciphylaxis is treated supportively by the elimination of risk factors, relieving of the pain, adequate protein intake, and wound care. Intravenous sodium thiosulfate is one of the most common therapeutic interventions for calciphylaxis. It is an antioxidant and vasodilator medication that is used to inhibit calcification of the vascular wall. Some medications have been described in case reports that may have potential benefit in treating calciphylaxis such as vitamin K, a low dose of tissue plasminogen activator infusion, bisphosphonates, low-density lipoprotein apheresis, and kidney transplantation\u00a0, 15-17.This case is reported because calciphylaxis remains a relevant subject for medical research as the pathogenesis is not yet clear, and the mortality rate reaches up to 80%. Besides, this case concurrence with COVID-19 raised questions for further research about skin manifestation in COVID-19 infection.\u00a0Understanding the risk factors of calciphylaxis is necessary both in the diagnosis and management of this rare and life-threatening condition. Given its high mortality and poor prognosis, early diagnosis and comprehensive management by a multidisciplinary team are important."}
+{"text": "Observational studies have suggested that better protein nutritional status may contribute to prevention of frailty.We sought to examine this hypothesis using a Mendelian randomization (MR) analysis.We conducted a two-sample MR study using GWAS summary statistics data of the UK Biobank. We applied genetically predicted serum albumin as a primary exposure measure and serum total protein as a secondary exposure measure. The outcome measure was the Rockwood frailty index (FI) based on 49 deficits from 356,432 individuals . In the full sample, genetically predicted serum total protein was inversely associated with FI . In both women and men, higher serum total protein was significantly inversely associated with FI; regression coefficients were\u00a0\u22120.148 per g/L  for women, \u22120.154 per g/L  for men.A genetically predicted serum albumin concentration was not statistically significantly associated with FI in the full sample. However, in women, we observed a preventive association between genetically predicted serum albumin and FI (\u03b2 = \u22120.172 per g/L; 95% CI: \u22120.336, \u22120.007; The present MR study implies that better protein nutritional status modestly contributes to reducing the risk of frailty. With a rapidly aging global population, frailty has become a major health issue , 2. FraiAlthough the prevalence of frailty is higher in older persons , 6, fraiProtein nutritional status is considered a preventive factor for frailty , 10. To Observational studies are generally susceptible to residual confounding and reverse causation. Indeed, a systematic review highlighted the influence of confounders on the association between protein intake and frailty . TherefoTo overcome the problem of confounding and reverse causation in observational studies, the Mendelian randomization (MR) approach is proposed . MR is aMoreover, the MR study design examines the association of lifelong exposure because the genetic variants influence traits across the whole life course . HoweverWe conducted two-sample MR analyses using summary statistics of a genome-wide association study (GWAS). As an exposure measure of genetically predicted protein nutritional status by using established SNPs in the previous GWAS analysis, we applied serum albumin as a primary exposure measure and serum total protein as a secondary exposure measure. Serum albumin and serum total protein are widely used as indicators of protein nutritional status , 14, andhttps://www.ukbiobank.ac.uk/).The primary analysis was based on data from the UK Biobank . The UK https://github.com/Nealelab/UK_Biobank_GWAS). For a quality control of the samples, the GWAS summary statistics were considered using the following filter parameters: a principal components analysis calculation filter for selection of unrelated samples, a sex chromosome filter for removal of aneuploidy, a filter of principal components for European sample selection to determine British ancestry; and filters for selection of self-reported \u201cwhite British\u201d, \u201cIrish\u201d, and \u201cwhite\u201d. This GWAS included 315,268 individuals (white-British ancestry). We obtained 3 kinds of summary statistics data, for the full sample including both sexes , women , and men . The details can be found at https://github.com/Nealelab/UK_Biobank_GWAS.For data on the association between genetic variants and serum proteins, we used GWAS summary statistics from the UK Biobank (regression coefficients for serum proteins in g/L) released by the Neale Lab  for serum albumin and 25,539 individuals (from 6 GWASs) for total protein .1) genotyped, 2) of European ancestry, 3) had complete information on variables included in the FI, and 4) not randomly dropped with a kinship threshold of 0.1768 for considering relatedness .For estimating the genotype-FI associations, we analyzed individual-level data from participants enrolled in the UK Biobank. Among the 500,336 participants who had the information about FI available , we inclP\u00a0<\u00a05\u00a0\u00d7\u00a010\u20138) in the previous GWAS meta-analysis in individuals of European ancestry (P <\u00a05\u00a0\u00d7 10\u20138) associated with serum proteins concentrations in the UK Biobank study  for serum proteins that were defined as \u201cestablished loci\u201d in the transethnic meta-analysis of European ancestry and Japanese GWASs . These SThe Rockwood FI, based on the accumulation of deficits model, was used as an outcome measure of frailty. The FI is a continuous measure that also has high sensitivity at the lower end of the frailty continuum. According to the principles of this model , the FI n\u00a0=\u00a0160,666 women) for the other stratified MR analyses.We estimated the association of each genetic variant with the continuous FI using linear regression analyses and assumed an additive model to obtain summary statistics for the two-sample MR analyses. All the regression analyses were adjusted for age (continuous variable) and sex by using SAS version 9.4 (SAS Inc.). To obtain sex-specific summary statistics of frailty for sex-stratified MR analyses, we also conducted linear regression analyses stratified by sex (adjusted for age). We also conducted stratified analyses by age (<60 y or \u226560 y) and menopausal status  and 95% CIs for FI. All MR analyses were performed by using the \u201cmrrobust\u201d package in STATA version 15 (StataCorp LLC) and the \u201cMendelianRandomization\u201d package for R version 3.4.3 . We primarily used the inverse variance weighted method with fixed effect standard errors . We alsoSupplemental Tables 1\u20133). The analytical sample for genotype\u2013FI associations  comprised 189,949 women (53.3%) and 166,483 men (46.7%), with a mean\u00a0\u00b1\u00a0SD age of 56.7\u00a0\u00b1 8.0 y and with a mean\u00a0\u00b1\u00a0SD FI of 12.0\u00a0\u00b1 7.4%.By using the linear regression analyses, we obtained summary statistics for the associations between the instrumental variables (genetic variants) and FI that were needed to conduct the MR analysis (P\u00a0=\u00a00.694). Results obtained by the weighted median method and MR Egger method were not essentially different from the result based on the inverse variance weighted method. There was no evidence for pleiotropy based on the MR-Egger regression analysis (P-intercept\u00a0=\u00a00.957). However, in women, a genetically-predicted serum albumin concentration was significantly associated with FI . Even when the penalized weights were applied to downweigh the contribution of genetic variants with outlying ratio estimates, a significant result in women was observed . Although point estimates obtained by the weighted median method or MR Egger method were not attenuated compared with the result in the inverse variance weighted method, they were not statistically significant. A plot to visualize the result of the MR Egger method in women is shown in\u00a0 \u00a0P-heterogeneity\u00a0=\u00a00.013).The results of the MR analyses of serum albumin are shown in\u00a0P\u00a0=\u00a00.001).The MR-Lasso method identified all 6 of the SNPs as valid instruments except for the MR results in women. In the MR results in women, the MR-Lasso method identified only 5 SNPs as valid instruments . The inverse variance weighted method using these 5 valid instruments showed that a genetically predicted serum albumin concentration was significantly associated with FI . In both women and men, higher serum total protein concentrations were significantly inversely associated with FI, and no significant sex difference was observed (P-heterogeneity\u00a0=\u00a00.952).The results of the MR analyses (inverse variance weighted method) of serum total protein are shown in\u00a0We also conducted the following 4 types of sensitivity analyses.P\u00a0=\u00a00.002).First, we checked the results of the MR analyses using other summary statistics data of protein markers . These results were also consistent with the main findings in P\u00a0=\u00a00.013). In addition, a genetically predicted serum total protein concentration was significantly associated with FI in the full sample  and in women , but not in men.Second, we checked the results of the MR analyses (the inverse variance weighted method) using another set of SNPs (P-heterogeneity\u00a0=\u00a00.811) and serum total protein (P-heterogeneity\u00a0=\u00a00.688).Third, we checked the difference in the MR results of serum proteins and FI when stratified by menopausal status . For serum albumin, we did not observe statistically significant results in the inverse variance weighted method regardless age groups, and no significant difference by age was observed (P-heterogeneity\u00a0=\u00a00.921). On the other hand, a genetically predicted serum total protein concentration was significantly associated with FI  in the younger age group (<60 y), but not in the older age group (\u226560 y), and significant difference by age was observed (P-heterogeneity\u00a0=\u00a00.006).Fourth, we checked the difference in the MR results of serum proteins and FI when stratified by age groups , they did not impose problems regarding data harmonization in the MR analysis. In addition, even when the summary statistics data of serum proteins were used, the results were not essentially different from the main findings .Although a systematic review of intervention studies has concluded that \u201ca combination of muscle strength training and protein supplementation was the most effective intervention to delay or reverse frailty\u201d , investiOne of the key methodological issues of MR analysis is the generalizability of genotype\u2013exposure associations. An underestimation of the causal relation by the \u201cwinners\u2019 curse\u201d is suggested as a disadvantage of overlapping samples (one-sample MR study), because some statistically significant genotype\u2013exposure associations could be cohort specific , 33. HowAnother key methodological issue in MR analysis is biased estimation due to overlapping samples . AlthougWe also confirmed the results of our MR analyses using another set of SNPs as instrumental variables that were based on transethnic GWAS meta-analysis of European and Japanese ancestry [SNPs were associated with serum protein concentrations in both European and Japanese ancestry ], and thr2 >0.9) in the PhenoScanner GWAS database  to assess any previous associations (P\u2009< 1\u00a0\u00d7\u00a010\u22128) with these traits. As a result, among 6 serum protein SNPs, rs1260326 was reported for 137 traits, including an inflammatory marker (C-reactive protein) . This SNP was also identified as an invalid instrument by the MR-Lasso method in women. However, even when we removed rs1260326 from the instrumental SNPs in our MR, the result was not essentially different from the main result . Therefore, the main result in the present study is unlikely to be explained by potential confounding traits.In addition, MR has the advantage that it is less likely to be affected by confounding and reverse causation than conventional observational studies . This adP\u00a0=\u00a00.027 in Interestingly, our results suggest that the inverse associations between serum albumin and FI are more pronounced in women than in men. A previous cross-sectional study has reported that an inverse association between protein intake and prefrailty is seen in women but not in men, although higher protein intake was associated with lower prevalence of frailty in both sexes . BecauseThe present study has several limitations. First, we only used a relatively small number of SNPs as instrumental variables, especially for serum total protein (only 2 SNPs in the main exposure data). Therefore, we could not conduct MR-Egger regression for serum total protein. Future GWAS studies are thus warranted to explore more SNPs associated with serum protein traits. Second, in the analyses stratified by menopausal status or age, we applied the summary statistics data of genotype\u2013frailty associations by menopausal status or age but not for genotype\u2013exposure associations. These were estimates made under assumptions in which genotype\u2013exposure associations were completely the same regardless of menopausal status or age. Stratified analyses based on one-sample MR analysis would be desirable. Third, because the present analysis was basically restricted to populations of European ancestry, it was unclear that the present findings can be adapted to non-European populations. Fourth, the exposure measures of the present study  are markers of protein nutritional status rather than indicators of dietary protein intake. To directly evaluate the effect of protein supplementation on frailty, clinical trials of protein supplementation would be ultimately necessary.In conclusion, the results of the present MR study indicate that higher protein nutritional status modestly contributes to reducing the risk of frailty. Diet is a common modifiable factor for most people in daily life. Therefore, even though the preventive effect of maintaining better protein nutritional status on frailty was modest, the public health impact may be considerable in the context of population aging worldwide.nxab348_Supplemental_FileClick here for additional data file."}
+{"text": "We aimed to analyse the oral health status of adolescents in Shandong province, including dental caries and gingivitis, and their associated factors.Adolescents aged 12\u201315-years in Shandong province were recruited. Caries and gingival status were assessed following the World Health Organisation diagnostic criteria. Information including the sociodemographic, oral hygiene knowledge, attitudes and practices were collected through the questionnaire. Chi-square test and multivariate logistic regression analysis were used to investigate the oral diseases associated factors.P\u2009<\u20090.024). A low-frequency of brushing, high sugar consumption and no flossing were more associated with calculus formation and gingival bleeding (P\u2009<\u20090.008).In total, 3868 students  were enrolled. Of these, 39.9% of the participants experienced caries, and 81.7% and 31.3% had calculus and bleeding gingival, respectively. Multivariate logistic regression analysis revealed that there was an association between dental caries and toothaches, dental visits and sleeping troubles caused by oral problems (Compared to caries, worse gingival condition was more prevalent among adolescents in Shandong province. Brushing behaviour is associated with gingivitis, while dental visits and toothaches are associated with caries. Hence, prevention-oriented dental visits and oral hygiene training are strongly recommended to improve oral health status.The online version contains supplementary material available at 10.1186/s12903-021-01640-x. Dental caries and gingivitis are the most prevalent oral diseases and bring a huge economic burden , 2. The Compared to early childhood caries, limited data are available on the assessment of oral health status in the adolescents aged 12\u201315. Importantly, the oral habits and ideology formed by adolescents are related to their current and future oral health , 7. PoorHowever, no data is available for the adolescents in Shandong province. Shandong, as one of the birthplaces of the ancient Chinese civilisation, has nearly 100 million inhabitants, and they have a special dietary habit (the major staple food is flour). Hence, this study aimed (1) to describe the prevalence of dental caries and gingivitis in 12\u201315-year-old adolescents in Shandong province and (2) to identify the oral diseases associated factors to provide reference for the improvement of oral health education.This cross-sectional study uses a part of the 4th National Oral Health Survey conducted in China. All parents gave their informed consent for inclusion before their children participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of\u00a0the Chinese Stomatological Association (No. 2014-003).The sample size was calculated based on the data of the 3rd National Oral Health Survey in 2005, in which the prevalence of dental caries for those aged 12\u00a0years was 28.9%. The design effect (deff\u2009=\u20094.5), significance level (\u03b1\u2009=\u20095%), margin of error (\u03b4\u2009=\u200910%) and non-response rate (20%) were also included in the following formula:In total, 28,365 12\u201315-year-old adolescents should be recruited in 31 provinces across the country . Hence, Oral hygiene knowledge, including the impact of brushing, bacteria, sugar, fluoride, pit and fissure sealing, and other factors that affect the teeth and gingiva.Oral hygiene attitude, mainly to evaluate whether they believe that oral health is important.Oral hygiene behaviours, including brushing habits, frequency of snacking, smoking, dentist visits, and trauma.Troubles caused by oral problems, including eating, talking, brushing, working, schooling, sleeping, smiling, easily troubled, and communicating.After giving their informed consent, the students completed the questionnaire survey and underwent an oral examination. The questionnaire collected information on the students\u2019 personal and family demographics, including age, gender, region , father/mother\u2019s education level, whether they were an only child or had siblings, and the following:Please refer to the reference  for detaThree questionnaire interviewers who underwent the screening, training and certification by the nation were arranged to collect the above information as efficiently and unbiasedly as possible. The coincidence rate of the questionnaire answers between each interviewers and trainer must exceed 95%. The questionnaires were completed independently by students in the classroom. During this process, one staff was arranged to explain each question publicly to all adolescents in the same classroom to guild them give as the most reliable answers as possible. Parallelly, two other staffs were arranged to clarify personal doubts. At last, each questionnaire would be rechecked by corresponding staff to avoid omissions and vacancies. All procedures were carefully conducted to minimize potential bias.All clinical examinations were implemented at schools with external equipment  . The folz tests for post hoc comparisons, univariable logistic analysis were conducted to explore the relationship between these oral indicators and the sociodemographic and questionnaire variables. To evaluate the caries and gingivitis associated factors in adolescents, a multivariate logistic regression model (method: backward logistic regression) was used. Those variables with P\u2009\u2264\u20090.10 obtained through bivariate analysis were included in the final models. A P value of\u2009<\u20090.05 was considered statistically significant.Data were entered and statistically analysed using IBM SPSS Statistics version 21.0. The percentage of dental caries (DMFT), calculus and gingival bleeding were calculated and statistical analysis. The indicators were all binary variables, and the other independent variables were also categorical. All the variables were independent of each other. Hence, Chi-square, Fisher\u2019s exact and A total of 3868 students aged 12\u201315\u00a0years were enrolled. The mean age of the participants was 13.95\u2009\u00b1\u20091.11 and 50.2% were males. As shown in Table\u00a0P\u2009=\u20090.041), gender (P\u2009<\u20090.001), living region (P\u2009<\u20090.001), with or without siblings and mother\u2019s education levels (P\u2009=\u20090.01 and 0.005). There was no association with urban/rural region and father's education level. As for dental calculus, age (P\u2009<\u20090.001), gender (P\u2009<\u20090.001) and living region (P\u2009=\u20090.032 and 0.001) were significantly associated with dental calculus, while with and without siblings and parent education level were not associated. As for gingivitis, there was a significative association between bleeding and age (P\u2009=\u20090.01), living region (P\u2009<\u20090.001). However, gender, with or without siblings and parents\u2019 education levels were not associated with bleeding.Table\u00a0P\u2009=\u20090.001 and 0.035), higher frequency of sugar consumption , poorer self-evaluation of oral condition , toothaches and dentist visits . Furthermore, those students who did not believe that regular oral examinations are necessary  and who thought that caries had a negative impact on eating, schooling, sleeping and easily troubled  were associated with a higher prevalence of dental caries. Those with a higher frequency of brushing , higher sugar consumption , dental visits , and believed that regular oral examinations are necessary  were associated with a lower prevalence of dental calculus. In addition, a high prevalence of GB was associated with a lower frequency of brushing and flossing , poorer self-evaluation of oral condition , and the belief that gingival bleeding is normal when brushing . The univariable logistic regression analysis showed similar results  and sugar consumption  were also found to contribute to a higher prevalence of calculus. The GB model  and do not use dental floss  generally experienced worse gingival bleeding. Moreover, age, living region, and the belief that gingival bleeding when brushing is normal were also associated with GB.The multivariate logistic regression analysis revealed that adolescents who were older, female, living in an urban environment, had siblings, a poorer self-evaluation of oral condition, experienced toothaches, visited a dentist, and had sleeping disturbance caused by oral problems had a statistically higher prevalence of DMFT Table\u00a0. InteresThis study evaluated the sociodemographic, clinical, oral health knowledge, attitudes, and behavioural factors among 12\u201315-year-old students in Shandong province and analysed the association between non-clinical variables and the DMFT, CI, and GB indices . The results show that the prevalence of caries in Shandong province is similar to the national level 31.9% vs 41.9%) [1.9% [4].P\u2009<\u20090.0001. data not shown), the hormonal fluctuations in puberty and menstruation [P\u2009<\u20090.0001; Q5, frequency of brushing: twice daily: male vs female: 38.5% vs 61.5%, P\u2009<\u20090.0001. data not shown) may be contribute to this result [Dental caries are an age-related disease \u201322, whictruation , or eventruation . Howevertruation , 27. In truation \u201330. Boy\u2019In addition, region is also an associated factor related to caries, calculus and gingival bleeding. Especially adolescents living in Weifang and Weihai are more likely to experience dental caries and gingival bleeding than those living in Linyi. This may be results from the special dietary habits in Linyi area. Residents living in Linyi take a hard and tough pancake as their staple food, which is made from coarse grains. The low adhesion and easy friction properties of this food may hinder the accumulation of dental plaque, thereby reducing the prevalence of dental caries in this area.Unfortunately, these associated factors, including age, gender, and region are all uncontrollable factors, even if there are significant differences. Therefore, stratification is important for the analysis and assessment of the risk factors at different levels. Moreover, an analysis of controllable oral diseases associated variables is more important to provide robust guidance for oral health education content.The contribution of toothbrushing to remove dental plaque and maintain oral health has been proven \u201333. HowePreventative dental visits are highly recommended . HoweverThe sufficient sample size and the comprehensive collection of various potential associated factor information enabled this study to reliably evaluate the oral health status of adolescents in Shandong province. However, the present study also had some limitations. First, cross-sectional studies are unable to prove causal inference. Second, the correlation between toothbrushing and caries could not be accurately evaluated because toothbrushing efficiency was not assessed. Third, even though the region that the adolescents lived in was an associated factor that correlated with DMFT, CI, and GB, no information on the familial socioeconomic status was recorded. It is known that familial socioeconomic status is closely linked with oral health status \u201344. Due In conclusion, compared to caries, worse gingival condition is more prevalent among adolescents in Shandong and its signs are not taken seriously. Age, gender, region, toothaches, dental visits and sleeping troubles caused by oral problems were found to be associated with dental caries in adolescents. A low-frequency of brushing and sugar consumption were associated with the formation of calculus. A low-frequency of brushing and no flossing were associated with gingival bleeding. Preventative dental visits and oral hygiene training are strongly recommended to improve oral health status.Additional file 1: Table S1. Bivariate analysis of potential variables related to the prevalence of dental caries. N (%). Table S2. Bivariate analysis of potential variables related to the prevalence of calculus. N (%). Table S3. Bivariate analysis of potential variables related to the prevalence of gingival bleeding. N (%). Table S4. Binary logistic regression analysis for dental caries (unadjusted). Table S5. Binary logistic regression analysis for calculus (unadjusted). Table S6. Binary logistic regression analysis for gingival bleeding (unadjusted)"}
+{"text": "Griffonia simplicifolia isolectin B4 (IB4); wheat germ agglutinin (WGA), and Lycopersicon esculentum agglutinin (LEA). The LEA lectin was identified as being far superior to the IB4 and WGA lectins in histological labeling of blood vessels in brain sections. A similar signal-to-noise ratio was achieved with high concentrations of the WGA lectin injected during intracardial perfusion. Lectins were also suitable for labeling vasculature in other tissues, including spinal cord, dura mater, heart, skeletal muscle, kidney, and liver tissues. In uninjured tissues, the LEA lectin was as accurate as the Tie2\u2013eGFP reporter mice and GLUT-1 immunohistochemistry for labeling the cerebral vasculature, validating its specificity and sensitivity. However, in pathological situations, e.g., in stroke, the sensitivity of the LEA lectin decreases dramatically, limiting its applicability in such studies. This work can be used for selecting the type of lectin and labeling method for various tissues.The vascular system is vital for all tissues and the interest in its visualization spans many fields. A number of different plant-derived lectins are used for detection of vasculature; however, studies performing direct comparison of the labeling efficacy of different lectins and techniques are lacking. In this study, we compared the labeling efficacy of three lectins:  The first accurate description of the cardiovascular system dates back to 1628 when the pioneering work of William Harvey revolutionized Galen\u2019s theory by centering the movement of blood around the heart as a central pump that pushes blood through a network of conduit vessels, nourishing tissues and organs [The early stages of cardiovascular development begin in the embryonic mesoderm when angiocysts start forming a primitive endothelial heart tube, preceding the formation of blood vessels through substantial embryonic angiogenesis, followed by pruning and remodeling of the vessels. Although the vasculature develops as one of the first organ systems, reorganization of the vascular system still occurs in adults during different physiological and pathological situations, such as the ovarian cycle and the formation of tumors . FurtherTo visualize blood vessels, immunohistochemical techniques targeting endothelial markers such as cluster of differentiation (CD) 31 ,12,13 orInterestingly, lectins have been used to label vasculature through immunohistochemical techniques ,22,23, aGriffonia simplicifolia isolectin B4 (IB4); wheat germ agglutinin (WGA); Lycopersicon esculentum agglutinin (LEA)), and compared the techniques, histological application vs. intracardial perfusion, for labeling blood vessels in different tissues with a focus on the cerebral vasculature. We also applied lectin labeling to a stroke model.Here, we conducted a comprehensive analysis of the labeling efficacy of three of the most commonly used lectins , but also the presence of two distinct clusters representing IB4 and LEA staining. We also confirmed that autofluorescence did not alter the LEA lectin\u2019s signal-to-noise ratio in comparison with the signal from a tissue that was not labeled with the lectin (The lowest lectin concentration (5 \u00b5g/mL) revealed the lowest background labeling in all lectin stainings S\u2013U and te lectin . Using te lectin , we coule lectin .Taken together, these results indicate that the LEA lectin is preferable over the IB4 and WGA lectins when used to label blood vessels in free-floating brain sections with histological approaches.With a molecular weight of 35 kDa, the WGA lectin does not cross the blood\u2013brain barrier and can therefore be used to label the cerebral vasculature when administered intravascularly during terminal transcardial perfusion , eliminaDifferent concentrations of the WGA lectin solution . The second group was intracardially perfused with the WGA lectin (50 \u00b5g/mL) to allow labeling of the blood vessels through direct binding of the WGA lectin to the luminal part of the endothelial cells before the organs of interest were harvested.The labeling efficacy of histological staining with the LEA lectin and intracardial WGA (50 \u00b5g/mL) lectin perfusion was comparable in blood vessels of the majority of the organs analyzed, i.e., the dura mater, skeletal muscle, kidney, spinal cord, and heart AA, LEA lInterestingly, we detected that both the sagittal and the transverse sinuses in the dura mater were stained with the LEA lectin after histological staining of the tissue , as wellTo assess whether lectins could label lymphatic vessels as well, we used the LEA lectin conjugated to AlexaFluor-647  to stainIn light of our results, albeit seemingly universally applicable, the unique characteristics of the organ of interest used for lectin-based labeling of blood vessels should be considered.We then performed a more detailed investigation of the specificity of lectin labeling of blood vessels by comparing it to immunostaining against glucose transporter-1 (GLUT-1), a protein expressed at the blood\u2013brain barrier site by endothelial cells and astrocytes. GLUT-1 is widely used to label the vasculature in the brain . We alsoIn each section, blood vessels were identified by the presence of GLUT-1 staining and Tie2\u2013eGFP expression. The presence of lectin co-staining was then quantified for the identified vessels. The application of Cy3-conjugated streptavidin to brain tissue sections from the mice intracardially perfused with biotinylated lectin revealed an extensive pattern of arterioles and capillaries C. The peTherefore, lectin labeling of blood vessels in the brain is as reliable as labeling with commonly used endothelial cell markers. However, the intrinsic structure of some organs, e.g., of the kidney or the spleen, may represent a limitation to the ability of lectin to label specifically blood vessels.Lastly, we tested whether lectin-based blood vessel labeling may be affected by disease in a mouse model of ischemic stroke where transient occlusion of the middle cerebral artery causes the formation of a necrotic core that is surrounded by a penumbra region  and the astrocytic marker glial fibrillary acidic protein (GFAP) as both cell types undergo massive activation during brain ischemia ,44. Our Danio rerio) and giant danio (Devario aequipinnatus) [Lectins have been widely used to label blood vessels in experimental animal models ,28. Howeinnatus) , such a Our findings also underline the importance of establishing a reliable and consistent method to implement lectin use in histological staining of blood vessels. Indeed, several recently published studies relied on the IB4 lectin to stain blood vessels in embryos , in the Our study also showed that the lectin with the lowest specificity, the WGA lectin, exhibited a concentration-dependent efficacy in labeling blood vessels in the brain when intracardially injected; furthermore, the highest concentrations used in this study (50 \u00b5g/mL and 125 \u00b5g/mL) showed similar results to the LEA lectin histological staining in labeling blood vessels in the brain. The evidence that WGA efficacy in labeling blood vessels varies depending on the labeling method  points out that the labeling efficacy of the LEA and IB4 lectins may improve upon direct injection in the bloodstream. However, a previous study where perfusion with the LEA lectin was found to be superior to IB4 to label choroidal and retinal vasculature  suggestsSince lectins are able to specifically recognize and bind to different carbohydrates through their carbohydrate-binding domains  has been widely used to stain microglia in rodent [The previous studies reported that lectins are able to label macrophages , thus itn rodent  and human rodent . Interesn rodent , suggestIn the initial stages of ischemic stroke, microglia are known to polarize towards the M2 neuroprotective phenotype and switch later on towards the M1 neurotoxic phenotype, especially in the peri-infarct region ; this prThe fact that most of our studies were conducted with lectins conjugated to a 488 or FITC dye may raise concerns regarding autofluorescence phenomena. Indeed, autofluorescence of the brain tissue has already been used to assess tridimensional brain morphology and structural organization with high resolution through optical projection tomography . To addrFinally, lectins might present some limitations when applied for in vivo labeling of the vasculature; indeed, lectins are known to cause coagulation of red blood cells with potentially fatal consequences. Therefore, lectins are likely limited to sectioned tissues or in vivo delivery shortly before sacrificing the animal, while fluorescent dyes without specific binding capabilities are suitable for imaging over extended periods of time ,18.In light of the topics discussed above, we want to encourage the use of the LEA lectin as a reliable marker for labeling blood vessels in immunohistochemical approaches. If the experimental design allows it, direct injection of the WGA lectin at the medium concentration (50 \u00b5g/mL) in the bloodstream during terminal intracardial perfusion provides results that are comparable to histochemical techniques, whilst higher doses (125 \u00b5g/mL) are able to produce an even better signal-to-noise ratio. Indeed, lectins show specificity towards blood vessels, as suggested by overlapping signals with GLUT-1 staining and Tie2\u2013eGFP reporter mice. However, it has to be noted that the physiological characteristics of the tissue of interest can interfere with the binding of lectins to the carbohydrate residues on cellular surfaces such as in stroke mouse models, therefore limiting their applicability in some experimental settings.Adult male C57BL/6 mice (12-week-old) were purchased from Janvier Laboratories and Charles Rivers (for the tMCAo studies) and were used in this study. The mice were kept in the standard laboratory conditions, with a 12 h dark\u2013light cycle and ad libitum access to water and food. The experiments were conducted according to ethics permit Nos. 5.8.18-08269/2019 and 5.8.18-08160/2021 approved by the Malm\u00f6\u2013Lund Ethics Committee on Animal Research and Landesamt f\u00fcr Natur-, Umwelt- und Verbraucherschutz (81-02.04.2019.A214/01).Tie2\u2013eGFP mice purchased from The Jackson Laboratory (stock No. 003658) were bred in the University of Rochester vivarium and used for experiments in adult age after approval of the University of Rochester Medical Center Committee on Animal Resources.Prox1-eGFP mice , back-crAll the supplies, including lectins, were purchased from Sigma-Aldrich unless otherwise stated. The primary antibodies were purchased from Merck Millipore , WAKO , Serotec , DAKO , and Abcam . The secondary antibodies were purchased from Invitrogen . Cy3-conjugated streptavidin was purchased from Jackson ImmunoResearch .2O, 30% O2). The monofilament  was inserted through microincision in the external carotid artery and further advanced to the internal carotid artery. The body temperature was maintained at 37 \u00b1 0.5 \u00b0C with a heating pad. The cerebral blood flow (CBF) was monitored with laser Doppler flowmetry . Reperfusion was initiated after 60 min by removing the filament. The mice were sacrificed 1 (n = 5 mice) or 7 (n = 5 mice) days after tMCAo.The left middle cerebral artery (MCA) was occluded under isoflurane anesthesia  was used. After washing the sections in dH2O to remove the excess stain, they were dehydrated through immersion to the increasing concentrations of ethanol. Finally, the sections were immersed in xylene and covered using Eukitt . Infarct size was determined from Nissl-stained brain sections using the ImageJ software . The areas were integrated to calculate the infarct volume. In order to correct for tissue swelling, the infarct volume was expressed as the percentage of the non-infarcted  hemisphere.The mice were intracardially perfused with phosphate-buffered saline (1\u00d7 PBS) followed by perfusion with 4% paraformaldehyde (4% PFA) for fixation of the tissues 1 or 7 days after tMCAo. Brains were harvested and post-fixed overnight in 4% PFA before they were sectioned with a vibratome (100 \u00b5m thickness). Every 300 \u00b5m, a 20 \u00b5m section was collected to perform Nissl staining for infarct size determination. Briefly, the selected sections were mounted on a slide and allowed to dry before they were washed in dHn = 6) were anesthetized with a ketamine\u2013xylazine mix  perfused with phosphate-buffered saline (1\u00d7 PBS) followed by a perfusion with 4% PFA for fixation of the tissues. The brain, skeletal muscles, the heart, the kidney, and the liver were harvested and post-fixed overnight in 4% PFA before they were sliced with a vibratome (100 \u00b5m thickness). The vertebral column containing the spinal cord was also harvested and post-fixed overnight in 4% PFA before the spinal cord was dissected and processed like the other harvested tissues. The skull cap containing the dura mater was also collected and post-fixed overnight in 4% PFA, followed by dissection. Solutions of the IB4 , LEA-488 , or LEA-649  and WGA-FITC  lectins were prepared at concentrations of 5 \u00b5g/mL, 10 \u00b5g/mL, and 20 \u00b5g/mL and used to histochemically stain coronal sections of the organs and the whole-mount dura mater for one hour before the sections were washed three times with 1\u00d7 PBS and mounted for imaging.The C57BL/6 mice  and then intracardially perfused with 1\u00d7 PBS. Immediately after that, 5 mL of the WGA lectin solution were injected during the perfusion and allowed to bind to blood vessels for two minutes before further perfusion with 4% PFA for fixation of the tissues. The organs were harvested and post-fixed overnight in 4% PFA before they were sliced with a vibratome (100 \u00b5m thickness) and mounted on glass slides for imaging; n = 4) were used in order to assess the specificity of lectin labeling of blood vessels. Briefly, the Tie2\u2013eGFP reporter mice were intravenously injected with biotinylated lectin (125 \u00b5g/mL) and intracardially perfused with 1\u00d7 PBS and 4% PFA thereafter. Brain sections obtained from the Tie2\u2013eGFP reporter mice were histochemically stained with Cy3-conjugated streptavidin and the GLUT-1 antibody. Briefly, a blocking solution (0.5% Triton X-100 and 5% serum) was applied for one hour at room temperature (RT) to minimize unspecific antibody binding. Then, the primary antibody \u03b1-GLUT-1  was allowed to label the tissue overnight at 4 \u00b0C under gentle shaking. The day after that, the sections were rinsed three times with 1\u00d7 PBS before the secondary antibody  and Cy3-conjugated streptavidin (1:500) were allowed to label the brain sections for 90 min at RT under gentle shaking. Following three rinses with 1\u00d7 PBS, 4\u2032,6-diamidin-2-fenilindolo  was applied before the sections were mounted.Tie2-eGFP reporter mice  after the secondary antibody incubation and before the DAPI incubation. Co-labeling with the GFAP  and Iba1  primary antibodies was performed in some samples to immunolabel astrocytes and microglia.CD31 and \u03b1-SMA immunostaining was used to assess lectin co-labeling in coronal sections of the brain, kidney, and spleen of wild-type mice. The abovementioned procedure was used for the staining, with the following exceptions for the CD31 staining: (i) the sections were preincubated in a sodium citrate buffer (pH 6.0) to allow antigen retrieval; (ii) a blocking solution  was also used for primary antibody incubation.Mounted organ sections and the whole-mount dura mater were imaged using a confocal scanning microscope (Nikon A1RHD) at 10\u00d7 magnification for whole section visualization and 20\u00d7 Nyquist magnification for detailed imaging of the stained blood vessels. The acquired confocal images were analyzed using the Fiji software . Specifically, the labeling efficacy of the lectins used for histological staining as well as intracardial perfusion was calculated as the ratio of the averaged peak signal at the vascular walls to the background signal  obtained by drawing a cross-section of the vessels using the line plot tool in Fiji .2, the vessels were counted manually in each image ; for the analysis of the percentage of the area over the threshold, the Fiji software was used to set a manual threshold for each image , and the area covered by the stained vessels over the total area was automatically calculated by the software.For the analysis of the number of vessels/mmFor the co-localization analysis of the LEA lectin and GLUT-1 staining, Tie2-labeled vessels were identified and the presence of lectin and GLUT-1 staining in each Tie2-labeled vessel was assessed as 1 (stained) or 0 (non-stained) . The same analysis was used to assess the co-localization of the LEA lectin with CD31 and \u03b1-SMA staining, with the exception that in this case blood vessels were identified by CD31 or \u03b1-SMA labeling.The LEA lectin\u2019s specificity in infarcted brains was calculated as the percentage of the difference between the total number of elements labeled by the lectin and the number of blood vessels identified by the positivity to GLUT-1 immunostaining in each image .Three-dimensional reconstructions and volumetric projections were generated using the Arivis Vision4D software.t-test and Welch\u2019s t-test or paired Student\u2019s t-test according to the experimental design; the nonparametric Mann\u2013Whitney U test was used when the unpaired data followed non-normal distribution, and the nonparametric Wilcoxon test was used when the paired data followed non-normal distribution. For comparison of more than two groups, one-way ANOVA followed by Tukey\u2019s multiple comparisons post hoc test was used. To compare the differences between the intact hemisphere and the infarcted hemisphere between the na\u00efve and tMCAo mice, a two-way ANOVA followed by Tukey\u2019s and Sidak\u2019s multiple comparisons post hoc tests was performed.All the statistical testing was performed in GraphPad Prism 9 . The Shapiro\u2013Wilk test was used to test the normal distribution of the data. Two groups were compared using unpaired Student\u2019s n represents the number of animals; p < 0.05 was accepted as statistically significant.All the values are expressed as the means \u00b1 SEM;"}
+{"text": "The purpose of this study was to characterize the nasopharyngeal microbiota of infants with possible and confirmed pertussis compared to healthy controls.This prospective study included all infants <1 year with microbiologically confirmed diagnosis of pertussis attended at a University Hospital over a 12-month period. For each confirmed case, up to 2 consecutive patients within the same age range and meeting the clinical case definition of pertussis but testing PCR-negative were included as possible cases. A third group of asymptomatic infants  were also included. Nasopharyngeal microbiota was characterized by sequencing the V3-V4 region of the 16S rRNA gene. Common respiratory DNA/RNA viral co-infection was tested by multiplex PCR.Bordetella with no other major changes on nasopharyngeal microbiota. Possible cases had limited abundance or absence of Bordetella and a distinctive microbiota with lower bacterial richness and diversity and higher rates of viral co-infection than both confirmed cases and healthy controls. Bordetella reads determined by 16S rRNA gene sequencing were found in all 12 confirmed cases (100%), 3 out of the 21 possible cases (14.3%) but in any healthy control.Twelve confirmed cases, 21 possible cases and 9 healthy controls were included. Confirmed whooping cough was primarily driven by detection of This study supports the usefulness of 16S rRNA gene sequencing for improved sensitivity on pertussis diagnosis compared to real-time PCR and to understand other microbial changes occurring in the nasopharynx in children <1 year old with suspected whooping cough compared to healthy controls. Bordetella pertussis. Other Bordetella species such as B. parapertussis, B. holmesii and B. bronchiseptica have also been associated with pertussis-like illness  and 66.7% of them were female. The three study groups were similar in age and gender. Clinical symptoms of whooping cough were also similar between confirmed and possible cases . Proportion of viral respiratory infections was 86%, 67% and 22% among possible cases, confirmed cases and healthy controls, respectively .Nineteen pertussis confirmed cases less than 1 year old were attended at HSJD during the study period and 12 out of the 19 (63.2%) accepted to be included in the study  and trends for lower bacterial genus diversity than healthy controls (Shannon: 0.77 [IQR: 0.29\u20131.37] vs 1.06 [IQR: 0.93\u20131.55] / Inverse Simpson: 1.67 [IQR: 1.17\u20132.38] vs 2.6 [IQR: 1.88\u20133.07])  . Among i8\u20133.07]) . DiffereBordetella was only found within the top-ten in confirmed cases. Specifically, it was the second most abundant bacterial genus with a mean \u00b1 SD abundance of 17.44 \u00b1 30.93 , 3 out of the 21 possible cases (14.3%) but not in healthy controls, hence increasing diagnosis sensitivity from 36.4% (12/33) to 45.5% (15/33) in suspected cases  and Bordetella relative abundance  detected by 16S rRNA gene sequencing.Significant differences were observed according to ed cases . OverallBordetella ASVs, a BLAST search identified 54 as B. pertussis  and 1 as B. holmesii . Bordetella genus abundance in all confirmed cases was dominated by either a unique ASVs (ASV45 in yellow) or by two ASVs (ASV45 in yellow and ASV48 in light green)  . Among tect C2_1 . After tBordetella genus had the highest potential for classification with a mean decrease accuracy value up to 4 times higher any other taxa  of the random forest model showed a good discriminatory power to discern between confirmed and possible cases  . At the her taxa . Other rBordetella along with reduced abundance of beneficial bacteria. On the other hand, in possible cases, pertussis symptoms may not be directly related to Bordetella presence but to a less rich and diverse nasopharyngeal microbiota along with higher prevalence of viral coinfection.Changes in the nasopharyngeal microbiota have been previously described in the context of respiratory diseases, but, to date, the composition of the whooping cough-associated microbiota and whether and to what extent it plays a role in disease outcome are unexplored. To the best of our knowledge, this study addresses for the first time the characterization of the nasopharyngeal microbiota composition in infants with possible and confirmed pertussis compared to healthy controls. In the present study we hypothesize that, on the one hand, confirmed whooping cough may not be related to profound changes on overall nasopharyngeal microbiota composition but to the appearance of Staphylococcus and Corynebacterium are expected to be replaced by other more unstable genera including Moraxella, Streptococcus and Haemophilus [Moraxella and Streptococcus as the major bacterial genera in the nasopharyngeal microbiota of infants regardless of whooping cough symptomatology. While these taxa were accompanied by Bordetella in the confirmed cases, Haemophilus and Dolosigranulum were found other major players in possible cases and healthy controls.Diverse longitudinal studies performed in newborns during the first year of life show how the nasopharyngeal microbiota changes over time and can either become a healthier or more aberrant community. In healthy infant populations similar in age to ours (5 months old), initial colonizers such as mophilus , 27. Ouret al. reported higher rates of respiratory tract infections in children younger than 1 year old with an altered nasopharyngeal microbiota including decreased microbial community stability, prolonged decreases of Corynebacterium and Dolosigranulum, and an enrichment of Moraxella [Moraxella and Haemophilus, both members of the phylum Proteobacteria, have not only been related to asthma and bronchiolitis development, but also to frequent acquisition of respiratory viruses [Dolosigranulum as a main component of the healthy nasopharyngeal microbiota in children as well as negatively associated to inflammatory states [Moraxella as well as lower Corynebacterium compared to healthy controls. Despite differences were not statistically significant, less Dolosigranulum was also found in the former. Healthy controls also showed higher abundance of Actinomyces. This finding agrees with results from Pettigrew et al. that reported Actinomyces being associated with better clinical course of pneumonia [Bordetella, possible cases of whooping cough showed an interesting and more complex profile including lower alpha diversity, lower Capnocytophaga, Prevotella and Neisseria abundance, and higher viral co-infection rate than both confirmed cases and healthy controls. Despite there is no consistency in literature about whether respiratory disease states relate to lower or higher alpha diversity [Bosch oraxella . Moreove viruses , 30. In y states . In the neumonia . Hence, iversity , this reiversity .Ulrich et al. reported in 2011 that viral co-infections were rare in B. pertussis infection [et al. that found viral co-infection in 47.2% of children younger than 6 months diagnosed of pertussis [Interestingly, we found higher rates of respiratory viral co-infection among suspected whooping cough infants (confirmed and possible) compared to healthy controls, with the highest positivity in possible cases. nfection , but ourertussis .et al., who found the highest positive rate in whooping cough diagnosis by 16S rRNA gene sequencing data (66.7%) compared to either real-time PCR (59.8%) and microbiological culture (14.3%) [B. pertussis, but also present in different copy number in other non-pertussis species as B. holmesii and B. bronchiseptica [Bordetella species in clinical specimens. Moreover, a real-time PCR positive result is subjected to an arbitrary decision on the resulting Ct value and up to date there is some controversy on what Ct values >35 really mean [pertussis species can lead to whooping cough characteristic symptomatology [B. holmesii in one possible case, non-targeted 16S rRNA gene sequencing, may bring advantages not only in terms of sensitivity (any detected read assigned to Bordetella will be reported as positive) but also of specificity (any species will be detected), at least, in young children (<1 year old).We included 33 children clinically suspected of whooping cough, but only 12 of them were confirmed by real-time PCR. Using 16S rRNA gene sequencing, diagnostic yield increased from 36.4% to 45.5% compared to real-time PCR. Our results are similar to those previously reported by Ding  (14.3%) . While Niseptica . Conseqully mean , 40. SinIn addition to improve pertussis diagnostic, the benefit of running a non-targeted 16S rRNA gene sequencing analysis over directed NAATs is the amount of valuable information on all other bacterial species present that could be playing a role in the development of whooping cough symptomatology and could be useful for improved future therapeutic treatments. The main disadvantages of using a non-targeted microbiological diagnostic based on Illumina sequencing of a fraction of the 16S rRNA gene, are the long wet-lab procedures for library preparation and sequencing, as well as the limited taxonomic resolution reached at the species level. Both limitations could be overcome sequencing the entire 16S rRNA gene with portable sequencers such as MinION B. pertussis vaccination information for all children, but mother\u2019s vaccination state could not be collected for the healthy controls. Nonetheless since 88.9% of the healthy children\u2019s mothers were from Spain, it is plausible to assume they would have been vaccinated being the target group of the Spanish vaccination program for pregnant women [Findings reported in this study are subject to limitations derived from the reduced sample size and the poor taxonomic resolution reached at the bacterial species level with the available taxonomic reference databases. However, we believe our results are representative since they include more than 60% of all whooping cough confirmed cases attended in the biggest pediatric Hospital in our country. In addition, while our targeted virome analysis included the major respiratory viruses known to infect and cause disease among children <1 year old, we are aware that a metagenomic sequencing approach would have covered a broader viral spectrum as well as other taxonomic groups such as fungi and other minor eukaryotes. Last, we were able to collect nt women .Bordetella detection is the primary change on the nasopharyngeal microbiota of children with confirmed pertussis. Non-confirmed children with whooping cough symptomatology appear to have a differential nasopharyngeal microbiota composition with a potentially relevant role of respiratory viruses. NGS of the 16S rRNA gene has potential for improved whooping cough diagnosis in young children and opens a new avenue for microbiological diagnosis of other diseases with a suspected infections origin but with an undefined causative agent.In conclusion,"}
+{"text": "RSC Advances article due to concerns with the reliability of the data. The images in the article, and the raw data provided by the authors, were screened by an image integrity specialist.The Royal Society of Chemistry hereby wholly retracts this The western blots in this paper all have the same look, the bands have a very generic shape on very similar-looking backgrounds, and do not look genuine. The raw data provided had no background apart from light reflexes that may have been added artificially. Further analysis of the raw data provided for Fig. 5F and G, revealed that the GAPDH control panels in both figures were identical. Furthermore, the background to the TAGLN2 panels were identical, but the bands are not. The raw data, therefore, does not appear to be genuine and cannot be used to validate the published data.Given the significance of the concerns about the validity of both the data in the article and the raw data provided by the authors, the findings presented in this article are not reliable.Fujun Wang opposes the retraction. The other authors have been informed but have not responded to any correspondence regarding the retraction.RSC Advances.Signed: Laura Fisher, Executive Editor, th January 2021.Date: 19"}
+{"text": "Thr654 mediated activation of DNA-PK by Cetuximab in cervical cancer cells\u2019 by Yunxiang Qi et al., RSC Adv., 2020, 10, 1132\u20131141, DOI: 10.1039/C9RA04962B.Retraction of \u2018Down-regulation of the radiation-induced pEGFR RSC Advances article due to concerns with the reliability of the data. The images in the article were screened by an image integrity expert. There are inconsistencies in the appearance of a number of panels, especially in Fig. 2, which are reported to be from the same experiments. There are also many instances where control panels have been duplicated across different figures and experiments, for example:The Royal Society of Chemistry hereby wholly retracts this The Lamin B1 (HeLa) control panels in Fig. 1, Fig. 2B and C are all identical.The Lamin B1 (CaSki) control panels in Fig. 2B and C are also identical.The Lamin B1 panels in Fig. 3 and 4 appear to be identical images.The authors were asked to provide the raw data for this article, but did not respond. Given the significance of the concerns about the validity of the data, and the lack of raw data, the findings in this paper are not reliable.The authors have been informed but have not responded to any correspondence regarding the retraction.RSC Advances.Signed: Laura Fisher, Executive Editor, th January 2021.Date: 15"}
+{"text": "Idiopathic trigeminal neuralgia (ITN) is a common pain disease in elderly people. Many methods have been used to alleviate the pain of patients, but few studies in the literature have compared the effect of nerve combing and percutaneous radiofrequency thermocoagulation.The purpose of this study was to describe and evaluate the clinical outcome of idiopathic trigeminal neuralgia after nerve combing (NC) and compare them with those obtained using percutaneous radiofrequency thermocoagulation (RF).The study included 105 idiopathic trigeminal neuralgia patients with similar symptom, age and underlying disease, which were divided into two groups. One group was treated by nerve combing (50 patients), the other by RF (55 cases). All patients were considered medical failures prior to the surgeries. A questionnaire was used to assess the long-term outcomes: pain relief, recurrence, complication and need for additional treatment.p\u00a0>\u00a00.05). Postoperative morbidity included dysesthesia, diplopia, partial facial nerve palsy, hearing loss, tinnitus, cerebrospinal fluid leak, meningitis and mortality.The median duration of follow-up in both groups was 90 months. Satisfactory relief was noted in 41 patients (82%), 5 patients (10%) initially experienced pain relief, then recurred, and four patients (8%) were designated poor among the group NC. In the group RF, satisfactory relief was noted in 42 patients (76.4%). There were eight \u201cpain free with recurrence patients (14.5%) and 5 poor cases (9.1%). No statistically significant differences existed in the outcomes between both groups (Nerve combing and RF are both satisfactory treatment strategies for patients with ITN. Because of the higher risk of sensory morbidity and surgical risk as open surgery, RF is preferred as the recommended procedure for patients with ITN. Idiopathic trigeminal neuralgia (ITN) is the most common cephalic neuralgia afflicting the middle-aged and elderly people. It is a chronic pain syndrome characterized by paroxysmal, shock-like, stabbing, recurrent episodes of pain localized in the distribution area of one or more branches of trigeminal nerve.Up to now, there are no medications that completely alleviate pain caused by trigeminal neuralgia. For ITN, surgical treatments in current use include microvascular decompression (MVD), percutaneous radiofrequency thermocoagulation (RF), nerve combing (NC), percutaneous balloon compression, Gamma knife surgery, percutaneous retrogasserian glycerol rhizotomy, sensory root section of the trigeminal nerve and avulsion of peripheral branch of trigeminal nerve.Our previous paper has reported its reliable efficacy in patients with ITN.Our retrospective study including 105 patients was approved by the local Ethics Institutional Committee. Each individual involved in the study signed an informed consent form authorizing the Institute to utilize their information for research purposes.All of 105 patients had complete medical records insuring the correct diagnosis and cooperation with our follow-up postoperatively. Each patient suffered from severe, recurrent, unilateral pain and demonstrated an evident \u201ctrigger point\u201d on the spat, upper lip or wing of nose. Although all the patients took carbamazepine as pain-controlling medication, the final pain relief was judged insufficient. They required surgical procedure for alleviating their refractory pain. A 3.0-Tesla Magnetic Resonance Imaging (MRI) scanner  was employed to determine if there was tumor, hemangioma, multiple sclerosis or other occupying lesions compressing the root entry zone of trigeminal nerve in each patient preoperatively. In 61 cases blood vessels were found at the root of the trigeminal nerve by MRI. A total of 105 patients were divided into group NC and group RF, each group with 55 cases. Those patients with psychiatric disorders, significant cardiovascular disease, serious infectious diseases and severe endocrine diseases were been excluded. Treatment was chosen according to the patient's option. People who were unwilling to accept the increased risk of a posterior fossa craniotomy were advised to select an RF procedure. All the operations were performed by surgeons at Department of Otorhinolaryngology Head and Neck Surgery; Provincial Hospital affiliated to Shandong University, Jinan, China.Our study data that included patient demographics, details of technical procedure, pain relief, recurrence, complications and need for additional treatment were collected by a physician who was not directly involved in the patients\u2019 care. A questionnaire was designed for evaluation of the long-term outcome and was completed at the time of the last follow-up examination or by mail. Postoperative relief was graded according to the criteria established by University of California at San Francisco (UCSF).A retrosigmoid craniotomy cerebellopontine angle exploration was performed in 50 patients under general anesthesia. Patients were placed in the lateral decubitus position with the affected side upward. A vertical skin incision about 4\u00a0cm posteriorly from the internal acoustic meatus was made, which began above the ear pinna and extended to 2\u00a0cm below the mastoid level. The soft tissues were dissected at the junction of parietal, occipital and temporal bone. A 3\u20134\u00a0cm retrosigmoid craniectomy with exposing of the crest of the transverse and sigmoid sinuses was performed.The opening was shaped with superior and anterior borders constituted by the inferior margin of the transverse sinus and the posterior margin of the sigmoid sinus. The dura mater was opened with U curve and reflected against the sinuses; the cerebellar hemisphere was exposed. A gentle traction of cerebellum with spatula and draining of cerebrospinal fluid were performed, so that the cisterna was opened. Under a 10\u00d7 microscope, the trigeminal nerve was identified at the posterior surface of the petrous vein below the tentorium.The circumference of the nerve was carefully inspected to determine if the compressive vessel was present, especially at the REZ. The affected branches of trigeminal nerve, according to preoperative pain locations and intraoperative findings, were longitudinally split by a special fiber knife (manufactured by our institution). Every branch was split 2\u20136 fascicles from entrance to pons, depending on the size of branches. When the combing was performed under microscopic control, the special fiber knife was maintained ahead of microscope so that it would be constantly visual, avoiding possible interaction with other brain tissue. Closure was carried out by standard surgical techniques.55 patients underwent this procedure under local or general anesthesia in the supine position. A special needle was introduced at a cutaneous puncture site 1.5\u00a0cm from the corner of mouth, through the cheek and into the foramen ovale. The trigeminal ganglion is heated to 70 for 100\u00a0s with a radiofrequency probe, producing a partial lesion.t test, Pearson Chi-Square, Continuity Correction or Fisher's test using commercially available software (IBM SPSS version 19.0). A p-value of <0.05 or <0.01 was considered as statistically significant.Values are expressed as mean\u00a0\u00b1\u00a0SD and significance was evaluated by Wilcoxon Rank Sum Test, independent Student p\u00a0>\u00a00.05, A total of 105 patients underwent surgical treatments. Patients\u2019 characteristics including sex, median age, median duration, and affected side are similar between the groups (The median age of Group NC was 48.9\u00a0\u00b1\u00a08.6 years (range 34\u201372 years); 28 patients were male and 22 were female. The average age of diagnosis was 54.8\u00a0\u00b1\u00a010.4. Their duration of symptoms had lasted for a median of 5.8 years (range 1\u201314 years). 23 patients felt pain on the left side and 27 on the right side.p\u00a0>\u00a00.05) (The median age in Group RF was 49.3\u00a0\u00b1\u00a08.7 years (range 36\u201373 years); 30 patients were male and 25 were female. The average age of diagnosis was 56.1\u00a0\u00b1\u00a011.0. Their duration of symptoms had lasted for a median of 6.1 years (range 1\u201315 years). Twenty-six patients had pain on the left side and 29 on the right side. The details of trigeminal roots affected as well as a comparison of demographic and clinical data in both groups of patients are displayed in \u00a0>\u00a00.05) .At the end of the follow-up, satisfactory relief were noted in 41 patients (82%), 5 patients (10%) initially experienced pain relief, then recurred, and 4 patients (8%) were judged to have a poor outcome among the group NC. Thirteen patients with recurrence had to repeat the same procedure and found satisfactory relief.p\u00a0>\u00a00.05). Both procedures were highly effective and had similar efficacy.In the group RF, at the end of the follow-up, satisfactory relief were noted in 42 patients (76.4%). There were eight \u201cPFR\u201d patients (14.5%) and 5 poor cases (9.1%). In patients of group NC , the posIn patients of group RF, the most frequent complications were dysesthesia and tinnitus, which developed in 2 patients (3.6%). Diplopia, meningitis and mortality were all recorded in only 1 patient (1.8%). This patient suffered from coma because of older age (69 yrs), hypertension and diabetes, and died ultimately of pulmonary infection. This patient was classified into \u201cpoor\u201d.p\u00a0>\u00a00.05), except for dysesthesia and facial nerve palsy (p\u00a0<\u00a00.05).The statistical analysis showed that no significant differences existed between the patients in the 2 groups regarding the surgery-related complications , except for dysesthesia and facial nerve palsy (p\u00a0<\u00a00.05). The former may be associated with longer procedure duration in the nerve combing surgery and the latter may relate to damaging of the facial nerve in the open surgery. Dysesthesia was a common side effect after the RF surgery. Erdine et al. reported a prospective, randomized study about RF and described that all of the 20 patients suffered from mild hypoesthesia and paresthesia after the procedure.The morbidity rate and the surgery-related complication rate in the current series seemed to be similar in both group NC and RF (p\u00a0>\u00a00.05).This series involved fewer patients than in our study, but dysesthesia was still the most frequent complication among all the side effects in both groups. It is difficult to avoid injuring the normal trigeminal function from heat. Hearing loss developed in 1 patient of group NC. The reason may be damaging the cochlear nerves, which also lead to tinnitus. One mortality was recorded in the group RF, because of concomitant serious conditions, such as diabetes and hypertension, and not the surgery itself. No doubt that NC as an open procedure has led to more CSF and meningitis morbidity than RF, but there was not significant difference observed . In addition, NC is an open retrogasserian surgery with much higher surgical risk, compared to minimally invasive RF. Some elderly patients may be not suitable for this type of surgical intervention. Therefore, RF is preferable to NC in patients with ITN.Nerve combing and RF are both satisfactory treatment strategies for patients with ITN. From our analysis above, both NC and RF have similar pain relief rates, recurrent rates and complications. But NC carries higher sensory and facial palsy morbidity than RF (The authors declare no conflicts of interest."}
+{"text": "To explore the factors influencing Taiwanese adolescents\u2019 consumption of sugar-sweetened beverages (SSB) and sugary snacks from a socio-ecological perspective.This study adopted a qualitative design by using face-to-face, in-depth interviews guided by a semistructured questionnaire.Eight junior high schools in New Taipei City and Changhua County, Taiwan, September to November 2018.Fifty-nine participants aged 12\u201314 years participated in this study.Reflexive thematic analysis was used to analyse the data. This study identified four themes to address the multifaceted factors that influence adolescents\u2019 consumption of SSB and sugary snacks. At the intrapersonal level, physiological factors, psychological factors, individual economic factors and taste preferences were mentioned in connection with people\u2019s consumption of SSB and sugary snacks. Positive or negative influences of parents, siblings, peers and teachers on SSB and sugary snack intake were identified at the interpersonal level. The availability of SSB and sugary snacks at home, their availability in vending machines or in school stores in the school environment and participants\u2019 access to convenience stores and hand-shaken drink shops in the broader community influenced SSB and sugary snack consumption. Additionally, food culture and food advertising were identified as influencing societal factors.Overall, this qualitative study determined not only that the consumption of SSB and sugary snacks is influenced by intrapersonal factors but also that interpersonal, environmental and societal factors affect adolescents\u2019 increased sugar intake. The findings are helpful to broaden the options for designing and developing interventions to decrease SSB and sugary snack consumption by adolescents. Overweight and obesity in children and adolescents are associated with adverse health consequences, such as early-onset diabetes, sleep problems and depression. Increased intake of added sugar, particularly from sugar-sweetened beverages (SSB) and sugary snacks, is a major health concern related to an increased risk of obesity in children and adolescents. Hence, the WHO suggests that free sugar intake should be less than 10 % of the total energy intake to reduce the risk of non-communicable diseases in children and adults.Over the last four decades, the number of children and adolescents with obesity has dramatically increased worldwide, from 11 million in 1975 to 124 million in 2016. Taiwan, located in East Asia, has a unique food culture and food environment, and eating out is a common dietary habit. There are a wide variety of available options for eating out. The ubiquitous breakfast shops, convenience stores and street food vendors contribute to the overconsumption of daily added sugar among Taiwanese people.Globally, the regions with the highest SSB consumption are the Caribbean, central Latin America and North America; however, the young generation in Taiwan has the highest daily intake of SSB in East Asian countries. Hand-shaken beverages are drinks consisting of tea, tapioca , ice and added sugar, all shaken together. In North American and European countries, frequently consumed SSB are regular sodas, sports drinks, energy drinks, fruit drinks and coffee and tea beverages with added sugar. However, hand-shaken beverages are a typical source of daily SSB intake in Taiwan because they represent an important local food tradition (boba). Given the convenience and accessibility of SSB and sugary snacks in Taiwan, adolescents have increased their intake of added sugar, posing an alarming threat to their well-being. The national nutrition and health survey reported that 49\u00b70 % of Taiwanese adolescents aged 13\u201315 consumed SSB daily. Furthermore, 10\u00b78 % of these adolescents had overweight, and 13\u00b75 % had obesity.Well-known hand-shaken drink shops are widespread throughout Taiwan, with more than 20 000 such shops countrywide,13. Adolescents are particularly vulnerable to consuming energy-dense, low-nutrient foods and beverages due to complex determinants. The socio-ecological approach,14 offers a comprehensive conceptual framework to understand the multifaceted factors that influence unhealthy diets. SSB and sugary snack consumption are determined by intrapersonal-level influences such as nutritional knowledge, self-efficacy and taste and preference. Additionally, interpersonal factors , environmental influences,18,21,22 and macrolevel and policy environments,24 are key drivers of SSB and sugary snack intake. Therefore, multifaceted strategies are needed to reduce added sugar intake in adolescents. In this vein, interventions at the intrapersonal level  and interpersonal level (e.g. education campaigns tailored to caregivers or peers), a food environment that supports health , and food policies  are all crucial for supporting reduced SSB and sugary snack intake by adolescents in daily life.Adolescents today experience challenges in adopting healthy eating habits because food systems need to be transformed to support such habitsThere is a lack of studies that explore the multifaceted influences on SSB and sugary snack consumption to broaden the understanding of this phenomenon in adolescents. Therefore, this qualitative study aimed to explore adolescents\u2019 perspectives and experiences with respect to SSB and sugary snack consumption from a socio-ecological perspective to inform effective multifaceted interventions., respectively). Two schools in New Taipei City were included in this study through the social contacts of members of the research team, and six schools in Changhua County were included in this study through the Changhua County Public Health Bureau. In total, eight schools agreed to participate in our research. The target participants were students in grades 7\u20139 who were 12\u201314 years old. The schools varied in size, ranging from as few as seventy-five students in rural areas to as many as 1799 students in urban areas. Two schools in rural areas had vending machines that sold beverages as well as sugary snacks.This qualitative study was conducted from September to November 2018 in New Taipei City and Changhua County in Taiwan using a convenience sampling method. New Taipei City and Changhua County were selected as typical urban and rural settings, respectively .Additionally, two schools in rural areas and two schools in urban areas had one store. Each school was equipped with water dispensers (machines for cooling/heating and providing drinking water); the number of water dispensers in each school ranged from as few as 6  to 61 (urban areas). The characteristics of the eight schools included in this study are described in the online supplementary material  as well as snacks or lunch boxes as incentives for participation in the face-to-face, in-depth interviews.2). The interviews were conducted by two research assistants who were trained by D.R.C. before the interviews to minimise potential bias and ensure the research quality. The interviews were conducted in Chinese in a designated room (occupied only by one research assistant and one student) in each school, and each interview lasted 30 to 60 min. After the interviews, demographic information, such as age, sex, weight and height, was collected by administering a short-structured questionnaire. We recruited the participants until data saturation was achieved (no new data and enough information for this study).Data were collected through face-to-face, in-depth interviews guided by a semistructured questionnaire. An interview guide was developed to investigate various factors influencing SSB and sugary snack consumption, including intrapersonal, interpersonal, environmental and societal factors, through a socio-ecological lens. A guide was developed to direct the interviews  read and reread all the transcripts of the interviews and recorded notes and comments until they were thoroughly familiar with the data. All transcripts were imported into NVivo 12 software  for analysis. C.W.W. performed the following coding process. Initial coding incorporating in vivo coding was used for the first cycle of the coding process. Each transcript was entirely coded before moving to another. After the first codes were generated, they were used to code the relevant data. During the coding process, new codes were developed to capture the meaning of data, and existing codes were modified to integrate new material. The process was repeated throughout all the transcripts. Pattern coding was used in the second cycle coding process to group similar codes into categories. These categories were identified based on the different levels of influence on SSB and sugary snack consumption through a socio-ecological analytic lens. Based on the coding analysis, categories were further refined, and themes were generated. To improve the credibility of the data analysis, D.R.C. and C.W.W. discussed and refined the codes, categories and themes until consensus was achieved. All analyses were conducted in Chinese to maintain the intended meaning. For publication purposes, the quotes, codes and categories were translated from Chinese into English by a bilingual researcher to ensure accurate representation.All the interviews were recorded, and the audio recordings were transcribed verbatim in Chinese, including hesitations, laughter and long pauses. Reflexive thematic analysisThe five researchers have doctoral degrees in public health or sociology. C.W.W. , D.R.C. , C.C.C.  and H.H.C  work in academia in public health. Y.P.Y.  is the director of the Changhua County Public Health Bureau and has extensive experience promoting public health in the community. Our backgrounds in public health and/or related work experiences in the community may have an impact on the collection, analysis and interpretation of the data.sd) cm and 52\u00b78 (\u00b113\u00b72 sd) kg, respectively. Of the fifty-nine participants, 25\u00b74 % were overweight or obese  to address the multifaceted factors that influence SSB and sugary snack consumption by adolescents. These themes, categories and codes are presented with supporting quotes below.Most participants (93\u00b73 %) reported drinking SSB at least once per week, and 32\u00b73 % consumed SSB every day. Regarding the types of SSB, participants reported drinking breakfast shop beverages , hand-shaken drinks and packaged beverages, such as beverages in cartons or bottles. In addition, 54\u00b72 % of the participants reported eating sugary snacks at least once per week. The sugary snacks mentioned included chocolates, popsicles, potato chips, shaved ice, cakes, tofu pudding, flavoured gelatine, candy and cookies. This study identified four themes, seventeen categories and thirty-eight codes . I feel refreshed and happy after drinking them. After that, I feel much more motivated to do something that I would like to do.\u2019 Negative psychological states were linked to SSB and sugary snack consumption. Participants stated that they consumed SSB and sugary snacks when they felt bad, angry, stressed, bored or tired. SSB and sugary snack consumption appeared to be a means by which to improve their mental status. In addition, the participants perceived motivation as one of the psychological benefits of SSB and sugary snack intake.\u2018I kept eating and eating (potato chips), and I felt that I couldn\u2019t stop it. I knew I should stop, but I couldn\u2019t control myself. Then, I ate one more pack of potato chips after eating one pack, and then I took another. I couldn\u2019t stop eating.\u2019 \u2018If you get used to drinking them (SSBs), you will probably get addicted to them. Like an older student I know, he drinks bubble tea every day, and he says he has become addicted to it. I feel afraid of that.\u2019 Furthermore, the participants stated that they or their schoolmates seemed to be addicted to SSB and sugary snacks. They knew that these foods were bad for their health, yet they could not stop drinking or eating them.\u2018I got NT 150 in allowance every day. I used it to buy my dinner and sweetened white gourd drink.\u2019 \u2018My mom gives me a limited allowance so that I will save money on my own. Then, I can use my savings to buy beverages\u2026it is painful to use my savings to buy beverages, so I chose MineShine  green tea because it only cost NT 10.\u2019 \u2018If I have money on hand and I feel thirsty, then I will buy the cheaper and bigger one (SSB).\u2019 Participants stated that their purchases of SSB and sugary snacks depended on the allowance their parents provided. In addition, the prices of SSB and sugary snacks affected participants\u2019 purchase of these foods. Some participants mentioned that MineShine, one of the regularly consumed SSB in cartons, was easily affordable for them.\u2018I felt happy because it (an SSB) tasted sweet. It tasted good because it was sweet.\u2019 \u2018I like drinking water\u2026But I will choose to drink sweet beverages if they are available. They taste good and sweet.\u2019 Taste also seemed to be one of the participants\u2019 reasons for consuming SSB and sugary snacks. The participants noted that SSB and sugary snacks taste sweet or good. When asked what they chose when water and SSB were available, participants reported selecting SSB since SSB taste better than water.Participants reported various positive and negative influences of their parents, siblings, peers and teachers that affected SSB and sugary intake at the interpersonal level.\u2018They (parents) say nothing (if they see me drink SSBs or eat sugary snacks). Instead, they buy hand-shaken drinks and sugary snacks for me sometimes.\u2019 A few participants reported that their parents regularly consumed SSB and sugary snacks at home and that they consumed these foods together when they saw their parents doing so. Lack of parental supervision also appeared to influence participants\u2019 SSB or sugary snack consumption. Finally, participants reported that their parents provided SSB and sugary snacks.\u2018My mom would say, \u201cDo not drink too much (beverage).\u201d She recommends that I drink beverages with no sugar; however, I feel they taste bitter with no sugar, so I usually drink beverages with low sugar.\u2019 Parents can be critical positive role models regarding SSB and sugary snack consumption for their children. The majority of the participants noted that their parents consumed fewer SSB and sugary snacks than they did. In addition, participants said that parental supervision and control influenced their consumption of SSB and sugary snacks. Participants told their parents to admonish or stop them when they were consuming too many of these items.\u2018My sister convinces me to go together to buy beverages. However, she always takes one or two sips, and then she gives me the rest of the drink.\u2019 In addition to their parents\u2019 influences, participants mentioned that sibling influences affected their consumption of SSB. For example, they went with their siblings to buy SSB and sugary snacks. The participants also described food-sharing behaviour between siblings.\u2018I went out with my classmates. We went to the hand-shaken drink shop, and they all bought hand-shaken drinks. On that occasion, I felt that I had to buy a beverage with them; it would be very weird if I were the only one who didn\u2019t do that.\u2019 Peers have a significant influence on SSB and sugary snack intake by adolescents. In the interviews, participants expressed the perception that their peers regularly consumed these foods at school. In addition, food sharing behaviour among peers, food recommendations from peers and peer pressure were social forces that prompted participants to consume SSB and sugary snacks.\u2018He/she (the teacher) pretended that he/she didn\u2019t see us drink the beverages.\u2019 \u2018For example, if we got good scores on a test, the teachers would reward us, and they would buy ice cream or beverages to treat us.\u2019 In the interviews, participants mentioned that when their teachers consumed SSB at school, they also wanted to drink SSB. Lack of supervision from the teachers also appeared to influence participants\u2019 SSB or sugary snack consumption. In some instances, SSB and sugary snacks were used by teachers as an incentive or reward to encourage students to perform well.\u2018I always see him (the teacher) drink water\u2026Also, I don\u2019t see him eat sugary snacks.\u2019 Teachers were also crucial positive role models for adolescents\u2019 eating behaviours. For example, teachers who drank beverages with no sugar or water at school were healthy beverage role models for participants.\u2018Our teacher will check whether you bring them (SSBs) into the school. If you do, you will be punished and have to do push-ups.\u2019 In addition, participants mentioned that some of the teachers supervised or regulated their consumption of SSB or sugary snacks. Participants from rural areas reported that teachers assigned them exercises as punishment if they violated these rules.\u2018My parents buy a lot of beverages at one time and then store the beverages in the fridge. If you want to drink one, you can take it yourself. They (my parents) buy one box of black tea or the green tea Yakult.\u2019 Parents are gatekeepers of SSB and sugary snacks at home, and the availability of these items at home might prompt their children to consume them.1). In addition, the field investigation and interviews revealed that two of the rural schools had vending machines that sold beverages and sugary snacks, and four schools  had stores in the school. Participants reported purchasing SSB and sugary snacks from the stores and vending machines found in the schools.\u2018Many of my classmates eat candy, cookies, or crackers. They buy crackers from the vending machine because it sells very yummy crackers, such as cheese- or chicken-flavoured crackers.\u2019 \u2018My classmates drink beverages daily. They drink one cup in the morning, and they go to the store (at school) to buy beverages (juice or black tea).\u2019 The school setting is important for adolescents since they spend a substantial amount of time studying at school. Water dispensers were accessible in the eight schools located in rural and urban settings , and there is only one grocery store nearby. It is a rural area, so there are no convenience stores. It takes five or ten minutes by car to arrive in an urban area.\u2019 However, a few participants from rural settings stated that there were few or no convenience stores or hand-shaken drink shops in the neighbourhoods near their homes; therefore, it was inconvenient for them to buy or obtain SSB or sugary snacks.1.\u2018It is easy to get them (SSBs) because there is a hand-shaken drink shop near our school gate. When I go to my cram school and pass by it, I buy a beverage.\u2019 The food environment in the neighbourhood near school is an important determinant of adolescents\u2019 eating behaviour, particularly in Taiwan. Taiwanese adolescents usually buy breakfast with SSB at breakfast shops in the neighbourhood near school before entering school. After school, they buy or eat dinner with SSB in the neighbourhood near school before entering their cram schools . Overall, participants reported that it was easy and convenient to buy or obtain SSB and sugary snacks in the areas around their schools. The food environment in each neighbourhood near each school is described in detail in Supplemental Table \u2018There is no milk tea and black tea at the store. It sells beverages like soy milk and fruit juice.\u2019 On a large scale, school food policy also has an impact on adolescents\u2019 SSB consumption. There is no specific SSB policy in urban settings. However, the rural setting of Changhua County imposed a regulation banning SSB on school campuses in 2017. This regulation limits some types of SSB, including but not limited to hand-shaken drinks, sports drinks, apple-flavoured milk, cola and non-100 % fruit juice. However, some SSB can be sold in school if they have fewer than 250 calories and have < 30 % added sugar. For instance, beverages such as 100 % fruit juice, buttermilk and soy milk are allowed.\u2018I usually go to 50 Lan  to buy bubble tea with half sugar and no ice.\u2019 Food culture has a significant influence on diet and eating behaviours. In Taiwan, hand-shaken drinks and breakfast are two crucial components of daily life. In the interviews, participants mentioned a popular type of SSB from hand-shaken drink shops, namely, bubble tea. Hand-shaken drinks are commonly consumed beverages in Taiwan.\u2018I usually buy my breakfast at breakfast shops and drink them (SSBs) with my breakfast (sandwich or egg pancake roll) \u2026 It is black tea or soy milk with sugar (500 cubic centimetres).\u2019 In addition, participants reported that they bought SSB  when they purchased breakfast at breakfast shops or convenience stores and took these foods to school to eat. Taiwan is well known for its breakfast culture, with a variety of dining options on the street. It is common to drink SSB with breakfast in Taiwanese society.\u2018Advertisements for sugary snacks are shown on TV or the internet, and they recommend some delicious sugary snacks. If the sugary snack looks cool and looks like it will taste good, then I will go buy it.\u2019 Food advertising may affect the types of foods that adolescents consume. Participants reported that advertising made them want to consume SSB or sugary snacks. The advertising avenues included television, store and shop flyers, YouTube channels and social media, such as Facebook or Instagram.This qualitative study generated some of the first evidence of the multifaceted factors influencing the consumption of SSB and sugary snacks by adolescents from a socio-ecological perspective to highlight intrapersonal, interpersonal, environmental and societal influences. This study also provides an understanding of adolescents\u2019 SSB and sugary snack consumption with complex determinants to inform and develop practical multilevel approaches to reduce such intake. or maintaining healthy diets to encourage students to drink water or eat healthy snacks when they feel thirsty or hungry. Consistent with previous studies,33, the results showed that adolescents\u2019 mental states affected their consumption of SSB and sugary snacks. Individual interventions such as healthy stress or emotion management training by schools can enhance students\u2019 ability to avoid SSB and sugary snacks when they experience a negative psychological state. In addition, the price of SSB and sugary snacks and the amount of students\u2019 allowance could affect their consumption of SSB and sugary snacks. The findings suggested that food prices were considered when adolescents bought SSB and sugary snacks. A previous study demonstrated that excise taxes on SSB to increase SSB retail prices could reduce SSB purchases and consumption. Legislative interventions can introduce taxes on sugary foods to encourage adolescents to choose cheaper and healthier foods.At the intrapersonal level, the results suggested that thirst and hunger were common reasons that students consumed SSB and sugary snacks. Educational interventions can involve programmes such as campaigns about drinking water,36. In addition, the home environment is important in determining the availability of SSB and sugary snacks to adolescents,37,38. Thus, effective interventions tailored to parents include training parents to serve as healthy dietary role models, increasing the availability of healthy snack food and reducing the availability of SSB and sugary snacks at home. The influence of peers on the consumption of SSB and sugary snacks has been investigated in previous studies on topics such as the peer environment, peer pressure, peer norms and friendship group consumption. Consistent with these studies, this study found that peers influenced adolescents\u2019 SSB and sugary snack consumption. Interventions based on peer influence, such as social network-based interventions or peer-led education programmes, can decrease adolescents\u2019 SSB and sugary snack consumption.At the interpersonal level, this study found that adolescents\u2019 families, peers and teachers influence their consumption of SSB and sugary snacks. These people are critical role models for the development of healthy eating behaviour in adolescents throughout their lives. The findings suggested that parental SSB and sugary snack consumption, as well as a lack of parental supervision, can influence adolescents\u2019 SSB and sugary snack intake, which is consistent with previous studies or use nutritious food to reward students.The findings indicated that teachers\u2019 consumption of SSB and sugary snacks might impact adolescents. Additionally, some teachers may use SSB and sugary snacks as incentives/rewards to encourage adolescents to perform well. Interventions tailored to teachers can encourage teachers to frequently drink water in front of their students to demonstrate healthy beverage habits,42. School food environment programmes could, for instance, remove SSB and sugary snacks from vending machines and school stores and provide free or subsidised fruits or vegetables for students as strategies to reduce SSB and sugary snack intake. In the present study, although the students had access to water dispensers at school , the numbers of water dispensers at a few schools were inadequate considering the total numbers of students at these schools. For example, approximately 50 students shared one water dispenser in two schools (schools 3 and 7). Thus, some students might not have enough time to visit the water dispenser during their 10-minute class breaks. Increasing access to water by installing more water dispensers in schools may help to encourage students to drink water instead of SSB. Additionally, schools have an important responsibility to ensure the availability of safe and clean drinking water through the regular maintenance and inspection of water dispensers in schools.The results showed that the school food environment, including vending machines and school stores, also increased the availability of SSB and sugary snacks, contributing to higher consumption by students. These results are consistent with those of prior research1). It is easy and convenient for students to access SSB and sugary snacks, which increases their daily added sugar intake. Previous studies have shown that greater neighbourhood access to convenience stores\u201345, fast-food restaurants and combination grocery and other stores is associated with an alarming increase in the risk of obesity in adolescents. A supportive neighbourhood food environment is essential for successfully reducing sugar intake in adolescents. The school neighbourhood food environment could be improved by implementing related public policy. For example, the government should increase business taxes on hand-shaken drink shops, convenience stores and fast-food restaurants built within 500 m of schools. Additionally, breakfast shops could receive tax reductions if they provide healthy menus and beverages with no added sugar to students.Furthermore, this study found that the food environments of the neighbourhoods near school and home affected SSB and sugary snack consumption. In the present study, there were no notable differences in adolescents\u2019 SSB or sugary snack consumption between New Taipei City and Changhua County. Overall, except for a few students who lived in highly rural communities, adolescents could easily buy SSB or sugary snacks in the neighbourhoods near their schools and homes. This study found that in both rural and urban settings, the school food environment includes many fast-food restaurants, hand-shaken drink shops, convenience stores and breakfast shops  has implemented regulations that ban some SSB on campus, the results showed that SSB were brought into schools by students for consumption with breakfast. Interventions targeting the school food environment can provide free healthy breakfasts for students, facilitating the consumption of nutritious breakfasts instead of breakfasts purchased from outside breakfast shops. Legislative interventions can introduce a sugar tax on SSB and sugary snacks to reduce the intake of these products. Many countries, such as the UK, Finland, the UAE, Mexico and Thailand, have introduced a sugar tax on foods and drinks. Because hand-shaken drink shops, breakfast shops and convenience stores are ubiquitous in Taiwan, legislative/environmental interventions may effectively decrease SSB and sugary intake in adolescents.In general, this qualitative study suggests that SSB and sugary snack consumption are influenced by multifaceted factors from the intrapersonal level to the societal level. The evidence indicates that SSB interventions targeting individuals, the environment or both can effectively decrease SSB consumption; legislative/environmental interventions have had a 90 % success rateAlthough this qualitative study contributes a deeper understanding of the factors that influence SSB and sugary snack consumption in the Taiwanese sociocultural context, it has some potential limitations. First, it relied on voluntary participants, and self-selection bias could not be avoided. Therefore, the results might not sufficiently represent the grade 7\u20139 students in each school. Second, the findings from this study may be biased due to the small sample size. Hence, they are not necessarily representative of all Taiwanese junior high school students. Third, due to the private nature of the topic of dietary intake, this study may have been affected by social desirability bias and failed to capture some critical aspects across intrapersonal, interpersonal, environmental and societal influences. Fourth, although a socio-ecological model was used in this qualitative study, it was challenging to identify the effects on SSB and sugary snack consumption interactions across levels. Thus, crucial interactions across levels may be omitted when designing effective packages of interventions.Overall, this qualitative study determined that intrapersonal factors influence the consumption of SSB and sugary snacks and that interpersonal, environmental and societal factors also facilitate adolescents\u2019 increased sugar intake. The findings can help broaden options for designing and developing interventions to decrease adolescents\u2019 SSB and sugary snack consumption. These findings can have benefits beyond Taiwan as many adolescents worldwide develop unhealthy diets."}
+{"text": "Material classification is similar to texture classification and consists in predicting the material class of a surface in a color image, such as wood, metal, water, wool, or ceramic. It is very challenging because of the intra-class variability. Indeed, the visual appearance of a material is very sensitive to the acquisition conditions such as viewpoint or lighting conditions. Recent studies show that deep convolutional neural networks (CNNs) clearly outperform hand-crafted features in this context but suffer from a lack of data for training the models. In this paper, we propose two contributions to cope with this problem. First, we provide a new material dataset with a large range of acquisition conditions so that CNNs trained on these data can provide features that can adapt to the diverse appearances of the material samples encountered in real-world. Second, we leverage recent advances in multi-view learning methods to propose an original architecture designed to extract and combine features from several views of a single sample. We show that such multi-view CNNs significantly improve the performance of the classical alternatives for material classification. Material classification is a visual recognition task closely related to texture classification and dedicated to classifying input texture/material images into categories such as fabrics, wood, steel, or cotton . It is oHowever, this is still a challenging problem, since material images show a large intra-class variability ,6. FirstRecent studies have shown that deep neural networks clearly outperform many alternatives for material classification, but it is also clear that their performances are highly related to the data on which they are trained and tested ,6,7. ForThen, in order to go a step further towards better generalization of deep features for material classification, we propose exploiting a multi-view learning solution. Indeed, since one image provides a single view of a material sample, we claim that the performance could be significantly improved by considering a set of images for each material sample. Indeed, when a human being tries to determine the material that constitutes an object, they often tend to vary their point of view by moving their head or manipulating the object when possible to vary the viewpoint and light direction. We propose to mimic this natural behavior by taking advantage of the recent advances in multi-view learning , which mWe analyze the current material datasets and show that they do not have enough intra-class diversity for material classification tasks,We provide a new public material dataset with high variations across acquisition conditions (lighting and viewpoint) in order to better represent the multiple appearances of a single real-world material sample,We propose to exploit a multi-view learning approach to extract features from a set of images of the same material sample and to merge them into an accurate material representation,Extensive tests on two material datasets show that exploiting multiple views of the same material sample clearly outperform the single-view alternative.Our contributions are fourfold:In Several categories of methods have been proposed in state-of-the-art studies. The first ones are related to pattern-recognition-based methods, i.e., the computation of image features such as textons ,12. NextSome recent papers have demonstrated the efficiency of CNN methods for material recognition  and theUntil recently, most material classification methods used only single-view image as input or combined few single view image features as input. For example, in , the autIn ,27, the In the ideal case, the user would like to predict the appearance of a material regardless of the viewing direction and other factors that could have an impact on the capturing process. This is a quite challenging, ill-posed, and under-constrained problem that remains hard to solve for the general case .The aim of multi-view learning is to extract accurate features from data of different modalities , or representing different views of the same sample  .Features can be extracted from images very accurately with convolutional neural networks (CNN), and many approaches have integrated multi-view learning into CNN ,30,31,32Finally, some approaches have also combined Siamese networks with multi-view learning for person re-identification  or imageEven though each element of our designed network has been carefully selected, the contribution of this paper is not in the definition of a new architecture for a general multi-view CNN. The main aim is rather to show that multi-view learning is an appropriate solution to tackle material classification. To the best of our knowledge, this is the first time that a multi-view CNN has been used for this task.Several categories of texture/material datasets have been introduced over the years. Some image sets were collected in lab settings from cropped stand-alone samples  who takes care to perform the best acquisition  with the available setup system. However, for some materials, such as aluminum foil samples, this is very challenging as this kind of material is very reflective.Our aim was therefore to create a new dataset giving greater flexibility to the user in the image-acquisition process. Our main objective was to perform image acquisition under various lighting and viewing directions, rather than under very strict and well-controlled (and limited) lighting and viewing conditions. We assume that from one viewing direction to another one, the average lightness of the sample may differ, as illustrated in The fabric dataset introduced in  illustraBy playing with lighting and viewing conditions, we can increase the difference in the visual appearance for a material sample. In this paper, we claim that the diversity of the visual appearances of a material sample over variations in acquisition conditions should be accounted for in the final feature vector to optimize the classification accuracy. For example, the image differences observed in In next section, we present the details of our new datasets and the way we propose to exploit multiple views of a single material in order to boost the classification performance.The UJM-TIV material dataset consists of images from 11 distinct classes, namely aluminium foil, brown bread, corduroy, cork, cotton, lettuce leaf, linen, white bread, wood, cracker, and wool see .These images were acquired under controlled viewing and lighting conditions. These 11 classes are also included in the KTH-TIPS2  dataset.In the UJM-TIV dataset the variation in appearance between samples is clearly larger for some categories  than in KTH-TIPS2. Furthermore, in UJM-TIV, wool and cotton have the highest variations in appearance, while cork, brown bread, and white bread have the lowest intra-class variations. As an illustration, see the changes in appearance shown in For our dataset, a Canon EOS 5D Mark IV digital camera was used to capture the images of the samples with a resolution of 6720 \u00d7 4480 pixels. The background surrounding each sample was removed using a post-processing step. For each object sample, two object poses were considered, with a 90\u00b0rotation around the surface normal N of the angle denoted The example shown in The image acquisition setup used to capture the images under controlled viewing and lighting conditions is illustrated in The Patchify  library The viewing directions used in UJM-TIV are different from those used in KTH-TIPS2  and with a larger range. The lighting directions used in UJM-TIV are also different from those used in KTH-TIPS2 .All samples captured in the KTH-TIPS2 were acquired under a combination of three viewing directions  and four illumination directions , unlike the ones used in UJM-TIV. They were also captured at different scales, which is the opposite of UJM-TIV.As with KTH-TIPS2, in UJM-TIV, few images of fine-structured materials appear out of focus at working distances due to perspective effects and roughness of materials; see In contrast with other setups, such as the ones described in  or 44],,44], in Multi-view learning is attracting many researchers today  since itHence, we have selected a simple one-view-one-net architecture with a pre-trained network, leaving for future works any improvements related to the architecture choice.Since each view feeds a backbone, we propose sharing the weights between these backbones in order to minimize the number of learned weights and to prevent overfitting. Furthermore, sharing the weights between backbones can also help to improve the generalization power of the model, since the same backbone must extract accurate features from different views (different appearances). A single architecture merging the outputs of two identical branches is a Siamese network ,46,47.The architecture of the proposed network is shown in Before concatenating the features of each view, a global average pooling (GAP) layer is used in order to reduce the number of inputs of the first fully-connected (FC) layer of the architecture. It is known that such pooling helps to prevent overfitting problems .This GAP layer averages all the local neuron activations into a single activation for each channel. One alternative would be to use a global max pooling (GMP) layer that preserves only the highest score over the activation map. Intuitively, GAP is designed to work on repetitive local patterns, where the average of similar features has a meaning and noise is removed, while GMP is designed to pick the most important detail in each map. In this case, we believe that, for texture images (with repetitive patterns), GAP is more appropriate than GMP.Furthermore, in order to regularize the classifier, dropout is applied in the FC layers.The advantage of such an architecture is that it can be easily adapted to more than two views. Indeed, the pre-trained backbone can be used to extract features from any new views, and only the FC layer has to be adapted and retrained to perform classification. In this paper, we have only trained and tested a two-branch architecture.In order to assess the quality of our new dataset and the performance of the proposed multi-view CNN, we have conducted many tests on two datasets. The idea was to compare the advantages of our dataset over the KTH-TIPS2 dataset and to compare the performance of our two-views CNN with a single-view alternative.We have created two architectures for our tests. One is a classical single-branch architecture with a convolutional backbone to extract features and FC layers for classification. The accuracy provided by this network is called single-view accuracy. Then, we used our Siamese architecture with two backbones with shared weights that extract features from two views and FC layers for classification. This architecture provides the so-called multi-view accuracy. As the backbone for these architectures, we selected a residual network ResNet50  pre-traiLikewise, the hyperparameters and optimization algorithms are the same for both networks. We use the Adam optimizer with an initial learning rate of The Keras framework with TensorFlow 2.8.0 backend and Python version 3.9.5 was used to implement both the single-branch and the Siamese network. The models were trained on a high-performance GPU with an NVIDIA RTX 8000 8 GB graphics card, CUDA version 11.2, and RAM of size 16 GB.We ran experimental tests with two different configurations. The first configuration consists in training and testing on the whole considered dataset. Each dataset is randomly split into training and test sets, with Then, in order to test the multi-view learning, we selected some views in both datasets: 12 views in KTH-TIPS2 and 16 views in UJM-TIV. All the images of each selected view were also randomly split at a ratio The results are organized into two sections, depending on which data the networks have been trained and tested. First, we show results for test on the whole datasets and then, results on selected views.First, the idea is to analyze the results of a single-branch network on the whole datasets. The results are provided in works in . Second,In this section, we provide results on both datasets when the networks are trained and tested on the selected views. We consider the views by pairs in order to test our deep architecture for the two views. Thus, we have trained a network (single- or two- views) with the images of the two considered views (the training set) and tested on the same views (the test set).The results are provided in These results clearly show that our dataset is well designed to train networks for material classification and that the proposed Siamese architecture is a relevant solution for two-view learning.To go a step further in the analysis, we propose looking at the confusion matrices for one experiment where the multi-view approach clearly outperforms the single-view, i.e., the tests on views 5 and 6 for the UJM-TIV dataset. These confusion matrices are displayed in These matrices clearly show that many classification failures are avoided when multiple views are considered. Indeed, we can see in The previous experiments have shown that the results of a basic network not specifically designed for material classification can be strongly improved when considering a multi-view approach. As a final experiment, we checked if a very accurate state-of-the-art solution can also benefit from our contribution. Consequently, we have selected an approach adapted to material classification and based on the deep Fisher score . This soThese results confirm that the tested network is relevant for material classification, since it outperforms all the results from ork from  and the In this paper, we have proposed several contributions to material classification. We have introduced a new dataset with large intra-class variability. The variations in appearance within each class are due to large range of acquisition conditions and the selection of diverse material samples. We have shown that classical deep networks cannot easily generalize on such data, demonstrating the need for alternative solutions for this task. In order to exploit the appearance variations across viewing conditions, we have proposed leveraging the strengths of recent solutions in multi-view learning. We have shown that a Siamese architecture significantly outperforms the single-branch alternative by merging features from two views. Obviously, increasing the number of views at the input of the network is a solution that will be investigated in our future works. The challenge here is to extract features from uncontrolled views and to merge them into a general representation of the considered sample. Next, we plan to demonstrate that multi-view learning could also contribute to better reconstructing  complex spatially varying BRDF and to improve the efficiency of single-image SVBRDF-based rendering methods (see ). In thi"}
+{"text": "In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis.Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during  Inosine is most commonly known to arise in RNA as a necessary and developmentally regulated RNA A-to-I editing modification through the action of adenosine deaminase acting on RNA (ADAR) and adenosine deaminase acting on tRNA (ADAT) proteins . HoweverITPA gene encoding ITPase, leading to partial enzyme deficiency, is a relatively common trait which is clinically asymptomatic but can induce sensitivity to thiopurine therapies and modify outcomes to ribavirin therapy  dephosphorylates ITP to prevent its accumulation in the nucleotide pool and the inappropriate incorporation of inosine into RNA during transcription . Variant therapy . More remyopathy ,13  was amplified by polymerase chain reaction (PCR) using the following primers: forward 5\u2032-CATAGGATCCGCCACCATGGCTTCCAAG-3\u2032 and reverse 5\u2032-GCAGGGAGCTCTTACTGCTCGTTCTTC-3\u2032, digested using BamHI and SacI, and cloned in place of firefly luciferase at the BamHI and SacI sites of Promega L4821. The Myc- and Flag-tagged firefly luciferase construct was generated by PCR amplification of Myc-firefly (GenScript) using the following primers: forward 5\u2032-TACGAGGTACCATGAGCGAGCAGAAGCTGATCTCGG-3\u2032 and reverse 5\u2032-TTAACGAATTCTTAAACGGCGATCTTGCCGCCCTTC-3\u2032, digested using KpnI and EcoRI, and cloned into those sites in pcDNA3. The C-terminal Flag tag was inserted by site-directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (NEB) with the following primers: forward 5\u2032-GACGACGACAAGTAAGAATTCTGCAGATATCCATCACACTGG-3\u2032 and reverse 5\u2032-GTCCTTGTAATCAACGGCGATCTTGCCGCC-3\u2032. Restriction digest and gel electrophoresis were performed to confirm insert sequences and sizes, and all plasmids generated were sequence verified.The firefly luciferase construct was purchased from Promega (L4821). To generate a parallel Renilla luciferase plasmids were linearized downstream of the luciferase sequence using AfeI, gel-purified and used as a template for run-off T7 phage RNA polymerase transcription (NEB HiScribe\u2122 Kit E2050S) according to the manufacturer's instructions. Briefly, 100 ng of template was transcribed in a 20 \u03bcl volume in the presence of 10 mM of each canonical nucleotide. ITP (Sigma-Aldrich 10879) was added to a final concentration of 0, 0.1, 1 or 10 mM. Reactions were incubated at 37\u00b0C for 1 h, then treated with DNase I for an additional 15 min. RNAs were column-purified (NEB Monarch\u00ae RNA Cleanup Kit #T2040L), eluted in nuclease-free H2O and stored at \u201380\u00b0C.Firefly and in vitro transcribed RNA from above or RNA extracted from H9c2 cells below  was digested with Nucleoside Digestion Mix (New England BioLabs) according to the manufacturer's instructions. The digested samples were then reconstituted in 100 \u03bcl of RNase-free water, 0.01% formic acid prior to UHPLC-MS/MS analysis. The UHPLC-MS/MS analysis was accomplished on a Waters XEVO TQ-S\u2122  triple quadruple tandem mass spectrometer equipped with an electrospray ionization (ESI) source maintained at 150\u00b0C and a capillary voltage of 1 kV. Nitrogen was used as the nebulizer gas, which was maintained at 7 bars pressure, flow rate of 1000 l/h and at a temperature of 500\u00b0C. UHPLC-MS/MS analysis was performed in ESI positive ion mode using multiple-reaction monitoring (MRM) from ion transitions previously determined for inosine (m/z 269\u00a0>\u00a0137) and adenosine (m/z 268\u00a0>\u00a0136). The transition for the internal standard [guanosine (13C15N)] was m/z 299\u00a0>\u00a0162. A Waters ACQUITY UPLC\u2122 HSS T3 guard column, 2.1\u00a0\u00d7\u00a05 mm, 1.8 \u03bcm, attached to a HSS T3 column, 2.1\u00a0\u00d7\u00a050 mm, 1.7 \u03bcm, was used for the separation. Mobile phases included RNase-free water (18 M\u03a9cm\u20131) containing 0.01% formic acid (Buffer A) and 50% acetonitrile (v/v) in Buffer A (Buffer B). The digested nucleotides were eluted at a flow rate of 0.4 ml/min with a gradient as follows: 0\u20132 min, 0\u201310% B; 2\u20133 min, 10\u201315% B; 3\u20134 min, 15\u2013100% B; 4\u20134.5 min, 100% B. The total run time was 7 min. The column oven temperature was kept at 35\u00b0C and the sample injection volume was 10 \u03bcl. For adenosine measurement, a 500-fold dilution of the sample was used prior to MS analysis. Data acquisition and analysis were performed using MassLynx V4.1 and TargetLynx. Calibration curves were plotted using linear regression with a weight factor of 1/x.Measurements of the level of inosine and adenosine were performed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) using a method similar to that described previously . BrieflyIn vitro transcribed, purified RNA was polyadenylated using the Invitrogen\u2122 Poly(A) Tailing Kit (AM1350) according to the manufacturer's instructions. Briefly, 3 \u03bcg of RNA was incubated in a 100 \u03bcl reaction containing Escherichia coli poly(A) polymerase (E-PAP), 10 mM ATP and 25 mM MnCl2, at 37\u00b0C, for 1 h. Polyadenylated RNA was then column-purified (NEB Monarch\u00ae RNA Cleanup Kit #T2040L), eluted in nuclease-free H2O and stored at \u201380\u00b0C. Samples were electrophoresed on a 1% agarose gel containing Invitrogen\u2122 SYBR\u2122 Safe Gel Stain (S33102) for 1 h at 100 V, and imaged to confirm polyadenylation.Polyadenylated luciferase RNA was used to generate libraries according to the ONT Direct RNA Sequencing (SQK-RNA002) protocol version DRS_9080_v2_revM_14Aug2019. Briefly, a minimum of 500 ng of poly(A)-tailed RNA along with 110 nM RNA CS  was ligated to RT Adapter (RTA) with T4 DNA ligase (NEB M0202S) and reverse-transcribed (Thermo-Fisher SuperScript\u2122 III 18080093), then purified using 1.8\u00d7 RNAClean XP beads (Beckman Coulter\u00ae A63987), with 70% ethanol washes, on a magnetic stand. RNA Adapter (RMX) was ligated and products were purified using 1\u00d7 RNAClean XP beads, with 2\u00d7 WSB wash steps. The final library was eluted (ELB) and mixed with running buffer (RRB). Freshly prepared libraries were immediately loaded onto a FLO-MIN106 (R9.4.1) flow cell and sequenced using a MinION Mk1B sequencer and MinKNOW software version 19.12.5.MinKNOW-generated FAST5 files were base called using Guppy version 4.4.2 using Q-score filtering with a minimum score of 7. Reads were mapped to the Promega T7 luciferase plasmid reference sequence using Minimap2 version 2.17 employing parameters -ax splice -uf -k14. SAMtools version 1.7 was used to convert alignment SAM files to BAM format as well as to sort and index reads using default parameters. Pysamstats version 1.1.2 was used to generate statistics text file outputs with the variation_strand parameter. RStudio version 1.4.1103 was used to calculate the accuracies for each base for reads aligning specifically to the luciferase gene of the Promega T7 luciferase plasmid reference sequence. The R package ggplot2 version 3.3.2 was used to generate the base substitution figures.In vitro translation of luciferase RNA was conducted using a cell-free, nuclease-treated rabbit reticulocyte lysate (RRL) system (Promega L4960). Briefly, 400 ng of RNA was incubated with 7 \u03bcl of RRL, 0.1 \u03bcl of amino acid mix without cysteine (1 mM), 0.1 \u03bcl of amino acid mix without leucine (1 mM), 1 \u03bcl of RNaseOUT\u2122 (Thermo-Fischer 10777019) and nuclease-free H2O to 10 \u03bcl. Samples were incubated at 30\u00b0C for 10\u201360 min. For radiolabelling experiments, the reactions above were modified by using 0.2 \u03bcl of amino acid mix without methionine, and 0.4 \u03bcl of 11 mCi/ml of EasyTag\u2122 EXPRE35S35S Protein Labeling Mix (Perkin Elmer) was added and samples were incubated for 60 min. Samples were resolved by 10% sodium dodecyl sulphate (SDS)\u2013polyacrylamide gel electrophoresis (PAGE), dried, exposed to a phosphor-screen and scanned on a Typhoon scanner (GE Amersham). Data analysis was performed using ImageQuant software (Molecular Dynamics).Luciferase activity was detected using the Promega Luciferase Assay System (E1500), and luminescence was read on a Promega Glomax 96 microplate luminometer. Immediately following translation at the appropriate time point, 2.5 \u03bcl of sample was mixed with the supplied cell lysis reagent. Luminescence in 96-well plates was recorded following injection of 40 \u03bcl of luciferase assay reagent into wells containing 20 \u03bcl of lysate sample.The protocol was carried out as previously described with minor modifications . RNA quaItpa-null library by its counterpart wild-type library.FASTQ files were aligned using STAR alignment tool version 2.7.9a with parameters set to output BAM alignment files. SAMtools version 1.13 and BCFtools version 1.13 were used to create variant call files (VCFs) using the SAMtools mpileup command with the -uf options and the BCFtools call command with -mv parameters. Variants were filtered for Q scores\u00a0\u226520. Pysamstats version 1.1.2 was used to generate statistics text file outputs from BAM files with the variation_strand parameter. RStudio version 1.4.1103 was used to calculate the base substitution frequencies using the statistics text files. The base substitution frequency at any particular position is defined as the number of mismatches divided by the number of matches and mismatches  multiplied by 100. Specific base substitutions (e.g. C>G) were calculated as described above, but specific to a particular reference and a specified alternative base. Fold changes were obtained by dividing the base substitution frequency in the 2. CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated peptide 9 (Cas9)] gene editing was carried out essentially as described in Ran et\u00a0al., 2013 (Itpa (ENSRNOG00000021233), were selected using the online CRISPR design tool (http://crispr.mit.edu/). Oligonucleotide pairs incorporating these sequences (Sigma) were annealed (at 50 mM each) in 10 mM Tris pH 8, 50 mM NaCl and 1 mM EDTA by incubation at 95\u00b0C for 10 min followed by cooling to room temperature. Annealed oligonucleotides were diluted and ligated into BbsI-digested pX461 and pX462 plasmids (Addgene) using HC T4 ligase and rapid ligation buffer (Promega). Sequences of the recombinant plasmids were verified by direct sequencing. Cells were transduced using a Nucleofector II device and Amaxa Cell Line Nucleofector Kit \u2018L\u2019  according to the manufacturer\u2019s instructions. Transduced cells were selected by puromycin resistance (pX462) using 24 h puromycin treatment. Following 12 h recovery, they were selected by green fluorescent protein (GFP) fluorescence (pX461) and cloned using a FACSMelody instrument . Separately cultured wild-type cells were cloned in parallel to serve as controls. After sufficient growth, clones were analysed by PCR of the targeted exon (using primers 5\u2032-AGACAGATCATTTCGCCCTG-3\u2032 and 5\u2032-CAAAGAGGTTGGGTGAGAGG-3\u2032). In order to sequence individual gene-edited alleles, PCR products from each clone were first cloned into ZeroBlunt TOPO vector (ThermoFisher) and then subjected to colony PCR. Clones with apparent loss-of-function alleles were validated by immunoblot analysis of lysates using a rabbit anti-ITPA antibody (Millipore), with anti-\u03b2-actin (ThermoFisher) serving as a loading control.H9c2 Rat cardiomyoblast cells were obtained from the BHF Glasgow Cardiovascular Research Centre (GCRC)  and maintained in Dulbecco\u2019s modified Eagle\u2019s medium  supplemented with 10% foetal calf serum (FCS) and 1% penicillin\u2013streptomycin at 37\u00b0C and 5% COl., 2013 . A pair g for 15 min at 4\u00b0C and the supernatant was collected. The protein lysate concentration was quantified using the Pierce BCA Protein Assay Kit (ThermoScientific), and 10 \u03bcg of soluble protein lysates were separated on a 4\u201312% Bis\u2013Tris gel (BioRad) and transferred to a nitrocellulose membrane (Amersham). Membranes were blocked for 2 h at room temperature in 5% dry milk in 1\u00d7 PBS containing 0.05% Tween-20  and probed with anti-FLAG antibody , anti-myc antibody , anti-Firefly Luciferase antibody  and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody  overnight at 4\u00b0C in blocking solution. Following 3\u00a0\u00d7\u00a05 min washes in 1\u00d7 PBST, membranes were incubated with species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies (ThermoFisher) and IRDye-680 secondary antibodies (Li-Cor) for GAPDH in blocking solution for 1 h at room tempertaure and washed 3\u00a0\u00d7\u00a05 min in 1\u00d7 PBST. Bands were visualized by using ProSignal Pico ECL Spray (Genesee Scientific) and imaging on the ChemiDoc MP Imaging System (Bio-Rad). Quantification of protein expression was performed using Image J. All protein levels are normalized within each lane to GAPDH which served as a loading control.H9c2 cells were washed with 1\u00d7 phosphate-buffered saline (PBS) 72 h post-mRNA transfection and were lysed in 200 \u03bcl of radioimmunoprecipitation assay (RIPA) buffer (ThermoScientific) with 1\u00d7 cOmplete Protease Inhibitors (Roche) for 15 min on ice. DNA was sheared by passage through a 21-gauge needle, lysates were centrifuged at 21 000\u00a0\u00d7\u00a0Itpa-null H9c2 cells were grown to 50% confluence on 245 mm x 245 mm dishes (Corning). Cells were treated with cycloheximide (Sigma) at 100 \u03bcg/ml for 3 min, then washed with 1\u00d7 PBS, 100 \u03bcg/ml cycloheximide, trypsinised and harvested by centrifugation. Washed cell pellets were resuspended in an ice-cold lysis buffer containing 150 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol (DTT), 1% IGEPAL, 100 \u03bcg/ml cycloheximide, Turbo DNase 24 U/\u03bcl (Invitrogen), RNasin Plus RNase Inhibitor 90 U (Promega), cOmplete Protease Inhibitor (Roche) and 50 mM Tris\u2013HCl, pH 8, and incubated on ice for 45 min. Insoluble material was removed by centrifugation at 17 000 x g for 5 min at 4\u00b0C. Cytoplasmic lysates were loaded onto 18\u201360% sucrose gradients and subjected to ultracentrifugation . Gradients were fractionated using a Gradient station instrument (Biocomp) equipped with a detector (Biorad) for the measurement of absorbance at 254 nm, and Gradient Profiler software. Data from three technical replicate experiments were analysed. The 40S, 60S and 80S and disome peaks were defined by the lowest abutting points. For the H9C2 cell lines, clear 80S and disome peaks were not resolvable and so these peaks were considered together. Polysome peaks were defined by a distance fixed proportionate to (1.42\u00d7) the 60S\u2013disome distance in each replicate. Absorbance measurements in each replicate were normalized according to the lowest point in each profile . Polysomes:80S and disome ratios were calculated using areas under the resulting curves (trapezoid rule). *P\u00a0< 0.05, Student's t-test.Wild-type and in vitro system that enables comparisons between the level and distribution of inosine misincorporation into RNA and the production of a functional protein. Transcripts containing misincorporated inosine were generated using T7 phage RNA polymerase in an in vitro run-off transcription assay from linearized firefly and Renilla luciferase-coding plasmids. Reactions contained ITP at increasing concentrations within the nucleotide pool , and a fixed concentration (10 mM) of each of the canonical nucleotides   Figure . In vitrA Figure . Within A Figure . Followis Figure .Renilla RNA preparations both displayed comparable reductions in luciferase activity in the presence of inosine, we opted to focus on firefly luciferase to further probe the molecular basis of the effect of inosine misincorporation on RNA function in detail. To ensure that the reduced luciferase activity was not simply due to general base incorporation errors resulting from elevated nucleotide levels during transcription, firefly luciferase RNA preparations derived from in vitro transcription in the presence of 10 mM excess of each of the canonical nucleotides were evaluated  MinION direct RNA sequencing (SQK-RNA002). While inosine is a distinct purine nucleotide, the ONT base-calling algorithms are not currently configured to directly base call inosine, resulting in base miscalls at inosine positions which has been exploited to detect A-to-I editing sites in RNA . We predFrom the average base substitution frequency at each position of the luciferase RNA sequence, the average accuracy was determined for each base Figure . The avein vitro Oxford Nanopore Direct RNA sequencing data, we hypothesized that inosine misincorporation in place of C, A and U would be detectable as an increase in C>G, A>G and U>G variants in Itpa-null tissue compared with the wild type. To test our hypothesis, we generated and analysed Illumina RNA-seq data from previously established Itpa-null mouse embryonic day 16.5 heart tissue RNA samples, where the genotype was confirmed using Sanger sequencing of Itpa and western blotting for ITPA protein  involved, we attempted to quantify base substitutions from Pol I-, II- and III-derived transcripts. While there was very low read depth in our libraries from Pol III transcripts, we had substantial reads from 45S rRNA despite rRNA depletion, enabling us to quantify base substitutions as a proxy for inosine misincorporation in these RNAs. Using the candidate gene approach measuring C>G substitutions within a 1 kb window with a minimum read depth cut-off of 50\u00d7 from the 5\u2032 end of the gene, we could detect elevated C>G base substitutions at varying levels in Pol II transcripts when comparing Itpa-null and wild-type samples; however, there appeared to be no change in substitutions in 45S rRNA by this approach , corresponding to an incorporation rate of \u223c1 in 9037 bases of RNA  which was sufficient to significantly hinder translation in vitro . These data are consistent with a reduced rate of global translation in the Itpa-null cells.In order to determine whether inosine misincorporation in RNA results in reduced translation he heart . CRISPR l., 2013  using vee Figure . There wA Figure . This rao Figure . Compario Figure , D indicin vitro transcribed luciferase mRNA system with the Itpa-null and wild-type H9c2 cells. Purified, capped and polyadenylated firefly luciferase mRNA containing an N-terminal Myc tag and C-terminal Flag tag that was in vitro transcribed in the presence of increasing levels of ITP was transfected into wild-type and Itpa-null cells, and the resulting luminescence and luciferase protein abundance were evaluated  mouse model, displaying a robust neurological condition including seizures with depolarization of the resting membrane potential and increased frequency of neuronal excitation  and Illumina RNA-seq data (BioProject accession no. PRJNA822480) have been deposited in the Sequence Read Archive (SRA) database, gkac709_Supplemental_FileClick here for additional data file."}
+{"text": "Cxcl9 upregulation and cell activation. These results demonstrate that IFNGR1 plays a major role in autoinflammation and immune dysregulation mediated by STING gain of function.STING gain-of-function mutations cause STING-associated vasculopathy with onset in infancy (SAVI) in humans, a disease characterized by spontaneous lung inflammation and fibrosis. Mice with STING gain-of-function mutations (SAVI mice) develop \u03b1\u03b2 T cell\u2013dependent lung disease and also lack lymph nodes. Although SAVI has been regarded as a type I interferonopathy, the relative contributions of the three interferon receptors are incompletely understood. Here, we show that STING gain of function led to upregulation of IFN-\u03b3\u2013induced chemokines in the lungs of SAVI mice and that deletion of the type II IFN receptor (IFNGR1), but not the type I IFN receptor (IFNAR1) or type III IFN receptor (IFN\u03bbR1), ameliorated lung disease and restored lymph node development in SAVI mice. Furthermore, deletion of IFNGR1, but not IFNAR1 or IFN\u03bbR1, corrected the ratio of effector to Tregs in SAVI mice and in mixed bone marrow chimeric mice. Finally, cultured SAVI mouse macrophages were hyperresponsive to IFN-\u03b3, but not IFN-\u03b2, in terms of   Stimulator of IFN genes (STING) plays a major role in regulating immunity against pathogens, but constitutive activation of STING causes inflammatory lung disease. STING is activated by the cyclic dinucleotide second messenger (2\u20323\u2032 cyclic GMP-AMP [2\u20323\u2032cGAMP]), which is produced by cGAMP synthase in response to double-stranded DNA in the cytosol , 2. SubsGain-of-function mutations in STING lead to autoinflammation in humans and mice , 10, 11.We hypothesized that autoinflammatory lung disease and T cell cytopenia in SAVI mice depends on 1 of the 3 IFN receptors. To test this hypothesis, we crossed our gain-of-function (SAVI) knockin mice to animals lacking IFNAR1, type II IFN receptor (IFNGR1), or type III IFN receptor (IFN\u03bbR1). We discovered that deletion of IFNGR1, but not IFNAR1 or IFN\u03bbR1, ameliorated inflammatory lung disease in SAVI mice. We show that macrophages from SAVI mice were hyperresponsive to IFNGR1. Deletion of IFNGR1, but not the other IFN receptors, completely restored lymph node development in SAVI mice. Amelioration of lung disease and restoration of lymph node development was associated with a near-complete restoration of a normal effector T cell\u2013to-Treg ratio. Using coculture and mixed bone marrow chimeric mouse experiments, we demonstrated a cell-intrinsic contribution of IFNGR1 to SAVI mouse T cell survival. Thus, our results demonstrate that STING gain of function amplifies contributions of IFNGR1 in macrophages and T cells, including Tregs. Furthermore, our results indicate that a STING gain-of-function mutation causes a type II interferonopathy in mice.https://doi.org/10.1172/jci.insight.155250DS1). We observed that deletion of Ifngr1 protects against autoinflammatory lung disease. In contrast, deletion of Ifnar1 or Ifnlr1 IFN receptors had no significant effect on lung disease in SAVI mice. However, certain features of disease were not corrected by deletion of any IFN receptor. For example, we observed that splenomegaly persists in SAVI mice, regardless of which IFN receptor is deleted  and type I (IFN-\u03b1) pathways  after treatment with types I, II, or III IFN. We found that IFN-\u03b3 caused SAVI mouse BMDMs to upregulate Cxcl9 more robustly than in WT BMDMs. In contrast, IFN-\u03b2 equivalently induced Cxcl9 expression in WT and SAVI mouse macrophages, and IFN-\u03bb had no effect on Cxcl9 expression  animals. We found that SAVI mouse CD4+ and CD8\u03b1+ T cells exhibited significant upregulation of the IFN-\u03b3 reporter  reporter  have been targeted therapeutically in SAVI , 14, 34,\u2013/\u2013Ifnar1 STING N153S (a/bPtprc (CD45.1/2) STING N153S mice were also previously generated (\u2013/\u2013Ifngr1 (\u2013/\u2013Ifnlr1 (eYFPIfng mice) . \u2013/\u2013Ifnlr1 mice were gifts from Megan Baldridge (Washington University). For antigen-presentation assays, OTI mice were purchased from The Jackson Laboratory (003831). For mixed bone marrow chimera studies, 6-week-old syngeneic aThy1 (Thy1.1) C57BL/6J mice were purchased from The Jackson Laboratory (000406). In all experiments, heterozygous STING N153S animals were compared with cohoused WT STING littermate control animals. For histological assessment of lungs, FACS analysis, and ISG expression, 13- to 16-week-old mice were euthanized, and their organs were harvested and processed for downstream applications. For T cell proliferation studies or bone marrow isolation, mice aged 4\u20136 weeks were studied.All mice were bred and cohoused in specific pathogen\u2013free facilities at the University of Pennsylvania Perelman School of Medicine and at Washington University. Heterozygous STING N153S (SAVI) mice were generated as previously described  . Ifngr1\u20132O, and glacial acetic acid), rinsed in 70% ethanol, and encased in SeaPlaque low-melting temperature agarose  to preserve orientation prior to paraffin embedding. Tissue sections (3 \u03bcm thick) were stained with H&E, and slides were imaged on a Nikon Eclipse Ci-L microscope equipped with a DS-Fi3 camera, using NIS Elements Basic Research software (Nikon). Quantitation of lung inflammation was performed with ImageJ (NIH) software by a histologist blinded to the identity of specimens as previously described  and washed in 70% ethanol before paraffin embedding and sectioning. To facilitate serial sectioning of inguinal fat pads through the inguinal node, resected fat pads were pinned flat and stained overnight in Davidson\u2019s fixative  (https://artyomovlab.wustl.edu/phantasus/) (2(1 + x) transformation. The top 1.5 \u00d7 104 genes rank ordered by mean expression were analyzed for differential expression using the limma package  (Gene expression from the publicly available WT and E100411)  was analntasus/) . Transcr package . Pathwaynrichr/) . Statistnrichr/)  as descrnrichr/) .3 rpm. Homogenates were centrifuged at 1.5 \u00d7 104g for 5 minutes at 4\u00b0C, and RNA from 50 \u03bcL of supernatant was extracted using the RNeasy Mini Kit (Qiagen) per the manufacturer\u2019s instructions. The TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems) was used to measure mRNA expression. Primers and probes were purchased from Integrated DNA Technologies. Ct values for all target genes were normalized to Ct values of the housekeeping gene Gapdh. Fold change in target gene expression was reported as 2\u2013\u0394\u0394Ct, normalized to the average expression observed in WT mice.Lung tissue was frozen on dry ice and stored at \u201380\u00b0C until tissue disruption in 250 \u03bcL Dulbecco\u2019s phosphate-buffered saline (DPBS) using a MagNA Lyser (Roche) for 60 seconds at 6 \u00d7 10For Evans blue staining, WT and STING N153S mice were anesthetized, and 25 \u03bcL of 5% Evans blue dye in PBS was injected into 1 forefoot and 1 hindfoot. Mice were euthanized 15 minutes following dye injection and dissected for quantitation of retroperitoneal and inguinal lymph nodes. Any mesenteric lymph nodes were counted as 1 node.Spleens and thymi were removed from animals and kept on ice in FACS buffer  supplemented with 5% FBS . To obtain single-cell suspensions, organs were mechanically dissociated and passed through 70 \u03bcm cell strainers and rinsed with ice-cold 20 mL PBS. Red blood cells were lysed in 2 mL ACK lysis buffer  for 3 minutes, and the cell suspensions were washed once in FACS buffer, counted, and prepared for downstream applications. To isolate bone marrow cells for mixed chimera studies or BMDM differentiation, femurs and tibias were dissected from animals, and the marrow was flushed from bones, followed by debris removal by filtration through 70 \u03bcm cell strainers. For T cell isolation, bulk splenic T cells were isolated using the EasySep Mouse T Cell Isolation Kit (StemCell 19751), and purity (routinely ~90%\u201399%) assessed by flow cytometric analysis of CD3\u03b5 expression.6 marrow cells were cultured for 8 days at 37\u00b0C in 5% CO2 in 10 mm petri dishes containing 40 ng/mL recombinant mouse macrophage colony-stimulating factor  in 10 mL complete DMEM  supplemented with 10% FBS, 2 mM L-glutaMAX , 1\u00d7 nonessential amino acids , 1 mM sodium pyruvate , 10 mM HEPES , and 100 U/mL penicillin and 100 mg/mL streptomycin . For T cell coculture and costimulation assays, isolated T cells or splenocytes were incubated at 37\u00b0C in 5% CO2 in complete RPMI 1640 Medium , containing 20% FBS, all supplements (see above), and 55 \u03bcM \u03b2-mercaptoethanol .To generate BMDMs, 5 \u00d7 10Escherichia coli , 10 ng/mL recombinant murine IFN-\u03b3 , or 100 ng/mL recombinant murine IFN-\u03bb3 . Gene expression was quantitated 4 hours later by qRT-PCR. Cell surface marker expression was analyzed 24 hours later by flow cytometry.BMDMs were treated with 100 U/mL murine IFN-\u03b2 from To confirm the neutralizing capacity of the anti\u2013IFN-\u03b3 antibody, 20 \u03bcg/mL anti-mouse IFN-\u03b3 antibodies  or isotype control antibody  were incubated with recombinant murine IFN-\u03b3 (10 ng/mL) for 30 minutes at room temperature and then added to BMDMs and incubated for 4 hours, followed by RNA isolation and ISG expression analysis by qRT-PCR. For anti-mouse antibodies used in flow cytometry and T cell proliferation assays, refer to 257\u2013264)  in complete media for 1 hour at 37\u00b0C, followed by 3 washes in PBS. OVA-loaded BMDMs were then plated at 25,000 cells per well in T cell media  in 96-well round-bottom plates. CD8\u03b1+ T cells were isolated from OT-I mouse spleens by magnetic bead separation  and stained with 1 \u03bcM CFSE  in PBS for 20 minutes at room temperature, followed by 2 washes in T cell media. Next, 100,000 CD8\u03b1+ T cells were added to the OVA-loaded BMDMs in the 96-well plates and cultured for 3 days. Proliferation of T cells was measured using an LSR II flow cytometer (BD Biosciences) and analyzed by FlowJo v10 software.Antigen-presentation assays were performed as previously described . BMDMs wFc-mediated interactions were blocked by incubating cell suspensions in 1 \u03bcg TruStain FcX anti-mouse CD16/32 in 50 \u03bcL FACS buffer for 20 minutes on ice. To stain surface antigens, cell suspensions were incubated with fluorescence-conjugated antibodies in 50 \u03bcL FACS buffer for 30 minutes on ice and washed 3 times. To assess cellular viability, cell suspensions were washed once in DPBS to remove residual FBS and then stained as indicated with 1:1000 Zombie NIR , LIVE/DEAD Fixable Aqua , or LIVE/DEAD Fixable Violet  in 200 \u03bcL DPBS for 15 minutes on ice. For intracellular FoxP3 expression, cells were fixed in FoxP3 Fixation/Permeabilization Buffer  and stained with FoxP3 antibody per the manufacturer\u2019s instructions. For evaluation of apoptosis, surface-antibody labeled cells were washed twice in 1\u00d7 Annexin V Binding Buffer and stained with Annexin V  per the manufacturer\u2019s instructions. In all experiments, stained cells were acquired on an Attune NxT Flow Cytometer (Thermo Fisher) or LSR II (BD Biosciences), and data analysis was conducted using FlowJo v10 software.7 splenocytes were washed twice in DPBS to remove residual FBS prior to a 15-minute incubation at room temperature in 1 \u03bcM carboxyfluorescein succinimidyl ester  in 1.25 mL DPBS. The CFSE-labeling reaction was quenched with 12.5 mL RPMI supplemented with 20% FBS. Antibody-coated wells were washed twice with DPBS and plated with 1 \u00d7 106 CFSE-labeled cells and resuspended in a total volume of 1 mL complete RPMI, before a 72-hour incubation at 37\u00b0C in 5% CO2. Cells were stained with Zombie NIR viability dye and antibodies against CD45, CD3\u03b5, CD4, and CD8\u03b1 in order to assess cellular division in CD4+ and CD8\u03b1+ T cells. The division index, defined as the average number of cell divisions that a cell in the original population underwent, was determined using the Proliferation Modeling tool in FlowJo. For T cell coculture assays, 1 \u00d7 105 isolated CD45.1 WT T cells were incubated with 1 \u00d7 105 CD45.2 WT or STING N153S T cells in 200 \u03bcL complete RPMI.One day prior to splenocyte isolation, 24-well tissue culture plates were coated with 1 \u03bcg/mL hamster anti-mouse CD3\u03b5 and CD28 antibodies in 1 mL DPBS and incubated overnight at 4\u00b0C. Following red blood cell lysis, 2 \u00d7 10\u2013/\u2013Ifngr1 STING N153S mice were mixed at a 1:1 ratio in DPBS without FBS. Seven-week-old Thy1.1 mice were administered a single lethal dose of irradiation (10 Gy) . Next, 5 hours after irradiation, 5 \u00d7 106 cells were transplanted via intravenous retro-orbital injection. Donor and recipient animals were all male to mitigate graft-versus-host disease. At 13 weeks of age, spleens were harvested from Thy1.1 mixed bone marrow recipients for FACS analysis of circulating immune cells. Experiments were similarly conducted with bone marrow isolated from CD45.1/2 WT and CD45.2 \u2013/\u2013Ifngr1 animals.Isolated bone marrow cells from CD45.1/2 STING N153S and CD45.2 P value is reported as a Q value in Enrichr . The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance no. A3381-01) and the University of Pennsylvania Perelman School of Medicine (assurance no. A3079-01). All efforts were made to minimize animal suffering.This study was performed in accordance with the recommendations in the WAS, CAM, AJL, and FRZ performed experiments and analyzed data. WAS performed tissue harvests and performed many of the T cell studies, including the mixed bone marrow chimeric mouse experiments. CAM performed all of the revision experiments, including coculture studies and BMDM experiments, analyzed data, and carried out manuscript revision. FRZ performed thymocyte analysis. AJL revised the final version of the manuscript and performed qRT-PCR analysis at the revision stage. JJM guided experiments. WAS wrote the initial draft and edited subsequent versions. JJM, CAM, AJL, SP, and FRZ wrote and edited the final version of the manuscript. All authors reviewed the final version of the manuscript. JJM conceived the project and guided all experiments. All authors have critically reviewed the manuscript."}
+{"text": "However, these are limited by solely depicting\u00a0a single healthy or diseased state with no ability to show variations. This leaves tertiary educators limited in their capability to offer \u2018hands\u2010on\u2019 examples of important disorder presentations. In recent years, high schools have undergone exponential growth in their employment of technology, and many now host engineering societies, information technology groups and STEM\u2010based activities. Linking up with local secondary schools presents an ideal opportunity to engage school students in the co\u2010creation of high\u2010quality, accurate and hands\u2010on resources that can be used within medical programme teaching. If structured correctly, this endeavour can be performed in a way that benefits both high school and university students.2We assessed limitations between the teaching requirements and available models in our medical programme's laboratories. For example, it would be helpful to show the natural progression of a fatty heart, as well as a brain lesion in multiple sclerosis, neither of which were available from the current resources. This would enable an educator to hold up the model in class, point to important features and then pass it around for group examination, something not possible when using more modern teaching interventions such as virtual reality.3In many cases, tertiary institutions assist local schools through guest presentations, awarding prizes, and offering campus visits. Inversely, through this project, school students assisted our medical programme. Many high schools have a wide range of resources, such as 3D printers and computer expertise, that can add great value to the creation of teaching resources. Although in some cases virtual 3D models using a computer or tablet may be able to showcase diseases,"}
+{"text": "BRCA1/2 are responsible for 16\u201320% of the risk for hereditary BC. Other susceptibility genes have been identified; Fanconi Anemia Complementation Group M (FANCM) being one of these. Two variants in FANCM, rs144567652 and rs147021911, are associated with BC risk. These variants have been described in Finland, Italy, France, Spain, Germany, Australia, the United States, Sweden, Finnish, and the Netherlands, but not in the South American populations. Our study evaluated the association of the SNPs rs144567652 and rs147021911 with BC risk in non-carriers of BRCA1/2 mutations from a South American population. The SNPs were genotyped in 492 BRCA1/2-negative BC cases and 673 controls. Our data do not support an association between FANCM rs147021911 and rs144567652 SNPs and BC risk. Nevertheless, two BC cases, one with a family history of BC and the other with sporadic early-onset BC, were C/T heterozygotes for rs144567652. In conclusion, this is the first study related contribution of FANCM mutations and BC risk in a South American population. Nevertheless, more studies are necessary to evaluate if rs144567652 could be responsible for familial BC in BRCA1/2-negatives and for early-onset non-familial BC in Chilean BC cases.Breast cancer (BC) is the most common cancer among women worldwide.  BRCA1/2 genes are responsible for only 16\u201320% of the risk for hereditary BC on average  and an especially reduced survival in patients who had not received radiotherapy , suggesting that rs147021911 carriers have a reduced BC survival, but postoperative radiotherapy may diminish this survival disadvantage [FANCM rs147021911 in 492 BRCA1/2-negative BC cases and 673 controls. One hundred percent of the cases and controls were C/C. Considering the small sample set and the fact that this is a rare variant in the majority of the populations, this result could be expected. According to the Ensembl database, this variant is monomorphic in controls from the five super populations in the 1000 Genomes Project . With respect to BC cases, according to the ExAC database [FANCM rs147021911 mutations and BC in South America.The 4 domain . This mucontrols . The mut = 0.01) . In 2016 = 0.01) . The autdvantage . In thisdatabase , this mudatabase ,28,29. Tdatabase ,28,29. IFANCM variant rs144567652 generates a premature stop codon, resulting in the loss of 118 amino acids from the C-terminal. The functional characterization of the mRNA transcript derived from the rs144567652 allele shows that the variant causes the skipping of exon 22, introducing a premature stop codon. In addition, genetic complementation assays revealed the mutated protein lacks DNA repair activity. These findings support the idea that the FANCM rs144567652 mutation is pathogenic. Gracia-Aznarez et al. (2013) [BRCA1/2-negative familial BC cases and 3896 controls from Italy, the Netherlands, Australia, and Spain, and the variant was detected in 10 cases (0.3%) and 5 controls (0.1%), with an estimated OR of 2.29 . These authors detected a low frequency of this variant (0.0011 in cases and 0.00077 in controls), with a variable prevalence in the available populations . Given the important role of FANCM in DNA repair, Gracia-Aznarez et al. (2013) suggested that it would be interesting to analyze this gene in a greater number of samples to better understand its contribution to BC [BRCA1/2-negative familial BC cases and 6625 controls from Italy, France, Spain, Germany, Australia, the United States, Sweden, and the Netherlands, confirming an association between truncating FANCM mutations and BC risk, with a carrier frequency of 0.21% in cases and 0.06% in controls, and an OR of 3.93 . When the Peterlongo et al. data were combined with those of Gracia-Aznarez et al., overall, the variant was detected in 28 of 12,044 (0.23%) familial cases and 9 of 10,521 (0.09%) controls, corresponding to an OR of 2.83  [BRCA1/2 mutations. Proband F188P1 was an early-onset familial BC case, and F579P1 presented with bilateral BC. Ultimately, no significant association was found between the rs144567652 mutation and familial BC risk, likely due to the sample sizes used. Therefore, more studies are necessary to evaluate if rs144567652 could be responsible for familial BC in BRCA1/2-negatives and for early-onset non-familial BC in Chilean BC cases.The . (2013)  studied on to BC . Peterlo= 0.007) . It mustBRCA1/2-negative BC patients from Chilean families was enrolled from Corporaci\u00f3n Nacional del Cancer (CONAC) files. None of the families fulfilled the criteria for other BC-related syndromes, including ataxia-telangiectasia, Li-Fraumeni, or Cowden syndrome. A sample of 492 n = 673) was recruited from CONAC files. DNA samples were taken from unrelated individuals with no personal or familial history of cancer who provided consent for anonymous testing. These individuals were interviewed and informed as to the aims of the study. DNA samples were obtained according to all ethical and legal requirements. The control sample was matched to the case population for age and socioeconomic strata.A healthy Chilean control sample  (forward: 5\u2032-AGTTTGCTCAATGGTGTAGATCT-3\u2032 and reverse: 3\u2032-TGTTAGCCATCCTTTTCACAGAT-5\u2032). A 402-pb PCR fragment containing rs144567652 was generated by standard polymerase chain reaction (PCR). Sanger sequencing was performed in an ABI 3730xl automated fluorescence-based sequencer and BigDye v.3.1 terminator system .https://www.r-project.org/ accessed on 29 June 2022). Fisher\u2019s exact test was used to test the association between genotypes/alleles and case/control status. Odds ratios (OR) with 95% confidence intervals (CI) were calculated to estimate the strength of the associations . A two-tailed p-value < 0.05 was used as the criterion of significance.The Hardy-Weinberg equilibrium assumption was assessed in the control sample using a goodness-of-fit chi-square test (HW Chisq function included in the \u2018Hardy Weinberg\u2019 package v1.4.1 for R, Foundation for Statistical Computing, Vienna, Austria, URL:"}
+{"text": "There are well-established guidelines for treating hypertension (HTN), yet only half of patients with HTN meet the defined target of\u2009<\u2009140/90. Team-based care (TBC) is an evidence-based strategy for improving blood pressure (BP) management and control. TBC is defined as the provision of health services by at least two health professionals \u201cwho work collaboratively with patients and their caregivers to accomplish shared goals to achieve coordinated, high-quality care\u201d. However, primary care practices experience challenges to implementing TBC principles and care processes; these are more pronounced in small independent practice settings (SIPs). Practice facilitation (PF) is an implementation strategy that may overcome barriers to adopting evidence-based TBC to improve HTN management in SIPs.Using a stepped wedge randomized controlled trial design, we will test the effect of PF on the adoption of TBC to improve HTN management in small practices (<\u20095 FTE clinicians) in New York City, and the impact on BP control compared with usual care. We will enroll 90 SIPs and randomize them into one of three 12-month intervention waves. Practice facilitators will support SIPs to adopt TBC principles to improve implementation of five HTN management strategies . The primary outcome is the adoption of TBC for HTN management measured at baseline and 12\u00a0months. Secondary outcomes include the rate of BP control and sustainability of TBC and BP outcomes at 18\u00a0months. Aggregated data on BP measures are collected every 6\u00a0months in all clusters so that each cluster provides data points in both the control and intervention conditions. Using a mixed methods approach, we will also explore factors that influence the effectiveness of PF at the organization and team level.This study will provide much-needed guidance on how to optimize adoption and sustainability of TBC in independent primary care settings to reduce the burden of disease related to suboptimal BP control and advance understanding of how facilitation works to improve implementation of evidence-based interventions.NCT05413252.ClinicalTrials.gov; The online version contains supplementary material available at 10.1186/s12913-023-09533-1. Hypertension (HTN) accounts for nearly 400,000 preventable CVD-related deaths per year in the U.S. , 2. TeamDespite the effectiveness of TBC, primary care practices experience significant barriers to implementing TBC care processes \u201311. For Barriers to implementing TBC are more pronounced in small independent primary care practices (SIPs) . SIPs  to scale the adoption of evidence-based practice models like TBC , 22\u201326. All enrolled practices are members of the New York City (NYC) Department of Health and Mental Hygiene (DOHMH)\u2019s NYC Regional Electronic Adoption Center for Health (REACH) Network. The Network is managed by the DOHMH Bureau of Equitable Health Systems (BEHS). NYC REACH supports over 2,000 small- and medium-sized independent practices with a range of services to enhance infrastructure  and optimize care processes to improve health outcomes. Overwhelmingly, participating practices are in socioeconomically disadvantaged, racially diverse neighborhoods with evidence of significant disparities in HTN control and CVD-related outcomes more generally \u201338.Using a stepped wedge randomized controlled trial (RCT) design, we randomly assign 90 practices to one of three waves Table . Waves aThe study design and assessments are informed by principles of TBC, and Armenakis\u2019s theory of organizational readiness . ArmenakWe hypothesize that additional organizational features  will moderate the effectiveness of PF Fig.\u00a0. We furtEligible practices must have fewer than 5 full-time equivalent (FTE) primary care providers \u2013 MD, DO, NP, PA \u2013 providing primary care), a minimum of two staff , minimum of 200 patients in the practice that have a diagnosis of HTN, and less than 75% of patient with HTN have a BP\u2009<\u2009140/90 in the past 6\u00a0months.The study recruits eligible practices from the NYC REACH network. First, NYC REACH recruitment staff uses their existing database to pre-screen practices based on the eligibility criteria. Next, recruitment staff reach out by phone to the lead clinicians to describe the study and assess interest. If interested, they are asked to respond to survey questions that confirm eligibility. If eligible, lead clinicians are asked to sign a memorandum of understanding (MOU) which outlines expectations and requirements for participation. BEHS uses several approaches to retain practices once all sites are recruited and randomized. For practices waiting for their wave to begin the intervention, BEHS\u2019 primary strategy is to stay in regular contact through a series of activities that were not related to the project. This includes their usual modes of communication to keep practices up-to-date on relevant changes in health care policies and to provide trainings and staff orientations, again, on topics that are not related to the project\u2019s main aim. Practice facilitators made contact by email, phone, and/or in person 1\u00a0month prior to the start of the subsequent study waves.During the usual care period, patients at the sites receive standard HTN care delivered by their primary care providers.Facilitators collaborate with practice leadership and staff to implement TBC principles ) to facilitate the adoption of five evidence-based HTN management strategies , 42, 43.In visit 1, facilitators use an adapted version of the Primary Care Team Guide Assessment (PCTGA)  to estabPractice facilitators attend 15\u00a0h of didactic and experiential training over three days. BEHS facilitators are all certified facilitators and have received additional training on motivational interviewing, and implementing quality improvement strategies and systems . Therefore, the training focuses on increasing capacity to support practices to apply principles of TBC in the context of improving the implementation of the five HTN management strategies. Specifically, the training reviews TBC principles, HTN management strategies, the activities that they are expected to complete during each visit, including conducting workflow and RCA, and uses case examples to engage facilitators in role-playing exercises. Practice facilitators are also trained to complete the assessment tools. Prior to attending the trainings, facilitators are expected to review the PF toolkit, complete required readings, and view required videos. The required readings focus on teams, team functioning, and the principles of TBC, while the videos focus on leadership, communication, and fostering mutual trust and support in primary care practices.Facilitators participate in weekly one-on-one meetings with a PF supervisor. Supervision meetings review site visits and facilitators/barriers to implementing TBC and HTN management strategies. Supervisors use the PCTGA and other tools that the PFs completed, as well as PF visit data from Salesforce, a cloud-based customer relations management system ,  to 5 (Strongly agree) to statements related to TBC. Participants also complete the 5-item monitoring progress toward goals subscale of the team process measure. Respondents rate the extent to which their team participates in activities related to goal monitoring on a scale of 1  to 5 (To a very great extent). Practice staff in each wave complete the TBC Assessment Tool at baseline and 12- and 18-month period (end of intervention period). Both surveys were adapted to include language specifically related to HTN management. This allows us to measure the degree to which practice teams adopt TBC strategies for HTN management.To measure our primary outcome Table , adoptioWe assess the percentage of patients with BP\u2009<\u2009140/90 . Data onFacilitators are expected to complete activities outlined in the toolkit before, during, and after each visit in the 12-month intervention and document these activities using Salesforce. Practice facilitators complete a visit checklist that reflects items outlined in Table Sustainability of the primary  and secondary outcomes  will be assessed at 18\u00a0months, six months after the intervention period ends.Practice staff are asked to complete a survey at baseline which assess practice and staff level characteristics, and organizational readiness for change. The study collects staff-level sociodemographic data including role, race, ethnicity, sex, age, country of birth, and spoken languages. Practice-level characteristics include practice location, ownership, number of staff, and NCQA Patient-Centered Medical Home recognition.Strongly disagree) to 5 (Strongly agree) [Poor) to 5 (Excellent) [All staff are asked to answer the statement \u201cOverall, I am satisfied with my current job\u201d and rate their satisfaction from 1 (y agree) . Additioy agree) . Finallycellent) .Strongly disagree) to 5 (Strongly agree) [To measure organizational readiness, we use the 24-item Organizational Change Recipients\u2019 Beliefs Scale (OCRBS). Response options ranged from 1 (y agree) .Qualitative interviews explore the implementation process and the potential for sustainability guided by CFIR. In a purposive sample of eight sites per wave, two staff members per site are selected to participate in an interview six months after the end of the intervention. The interviews explore specific elements of the transformation processes to achieve TBC and HTN management and outcome goals, team member experiences with TBC, how various challenges were identified and addressed, satisfaction with the PF process, and strategies for sustaining gains.In addition, qualitative interviews will be conducted with the practice facilitators at 12\u00a0months to further assess the implementation process, including contextual facilitation barriers, and facilitators that influenced TBC implementation, review adaptations to components and of TBC, what worked/did not work, and recommendations for enhancing sustainability and scale up. Informed consent will be obtained from all subjects. All study participants will receive an email describing the study with a link to the survey; the landing page will include the consent form, and clicking the link will indicate consent. Lead clinicians and other staff members invited to participate in qualitative interviews will be asked to provide verbal consent. Those who complete the interview will receive a $50 gift card as compensation. The study protocol was reviewed and approved by the New York University Institutional Review Board.The primary outcome is the overall average score on the TBC assessment tool. The secondary outcome is patient BP control. The analysis of the effect of PF on the TBC adoption outcome measure will be based on a linear mixed model. Specifically, to assess the PF intervention effect compared to usual care, we will use a model with a random site effect, and fixed effects for time and study condition. Condition will be dummy coded with the control condition as a reference category. The fixed-effects coefficient for the contrast of the PF condition with the control condition will indicate the direction and magnitude of the difference on the overall average TBC score. Inclusion of a fixed effect for time accounts for any general time trends. We also will explore whether the time coefficient should have a random effect to capture differences in time trends across practice sites. Confidence intervals (95%) will be reported to convey precision. In addition to the effects of time and condition, covariates  will be considered for inclusion in the mixed-effects model to reduce the within-group variance, if the potential covariate is predictive of the outcome.rate of BP control in each practice site and adjusts for differences in the number of HTN patients across sites. Comparisons of conditions  can be converted to incidence rate ratios (IRRs) by exponentiating the appropriate fixed-effects coefficient or contrast.For the BP secondary outcome, a generalized linear mixed model will be used. The count of HTN patients with BP control (<\u2009140/90) will be regressed on fixed effects for time and condition, the natural logarithm of the total number of HTN patients as an offset, and a random site effect. For the count outcome, a Poisson model will be used, but we also will consider a negative binomial mixed model if overdispersion is found. The offset term allows us to model the glmmTMB package using R software [Models will be fit using the software , 52. EacPotential moderator effects will be examined by including separately each organizational characteristic as a main effect and in interaction with the treatment condition indicator (Control vs. PF vs. Sustainability). A significant interaction effect would indicate the effect of the condition depends on the moderator, and contrasts of the control and PF periods at different levels of the moderator would be undertaken to understand the pattern of the interaction. We will conduct further analysis to explore the possibility that changes in organizational readiness mediate the effect of PF on primary and secondary outcomes. We will conduct preliminary analyses to assess whether PF (versus Control) is associated with changes at the site level. If there is a detectable effect of the PF intervention on these measures, we will assess whether the changes in these measures are associated with changes in overall average TBC and HTN management scores and changes in the rate of BP control using the Baron and Kenny \u201cproduct method\u201d as a starting point of the analysis . If necer\u2009=\u20090.35; ICC\u2009=\u20090.32) [d\u2009=\u20090.5 . Power was 0.92 to detect this effect with the planned number of sites. To allow for withdrawal of a small number of sites after baseline, we plan to enroll 90 practice sites (30 in each wave). Power for the secondary blood pressure outcome was also calculated by simulating ten thousand datasets. In all conditions, we assume an average of 150 patients with HTN per site and time period . The intraclass correlation for BP control was specified as ICC\u2009=\u20090.85 based on an estimate from our previous EvidenceNOW study [With the pre-specified number of sites (90) and design (Table \u2009=\u20090.32) , and a sOW study . For theWe will calculate adherence to the PF protocol as the number of components fully or partially implemented divided by the total number of possible components . We willInterviews are transcribed verbatim and imported into Atlas.ti for analysis. The study uses an abductive content analysis that systematically integrates practices from inductive analysis   and deduThe analysis integrates the qualitative and quantitative data in a nested, parallel mixed-methods design \u201364. EachPF is a promising strategy to support the effective implementation of TBC in SIPs, but is understudied in small practices that serve immigrant and minority populations that are experiencing significant disparities in CVD-related health outcomes \u201367. ThisAn additional strength of the study is the partnership between NYU investigators and the NYC Department of Health and Mental Health\u2019s practice network, a group of practices that serve primarily, low-income, minority, and immigrant populations. Embedding research in their PF program, BEHS increases the potential for sustainable changes.Potential limitations may include the potential for practices dropping out of the study, lack of fidelity due to intervention adaptions, and non-response bias. Additionally, we anticipate that facilitators may experience challenges in meeting with staff due to high patient volume or staffing turnover. However, the BEHS has long-standing relationships with their network practices and providers have noted the advantage of the PF process which facilitates engagement , 68. If Despite the potential limitations, this study provides much-needed guidance on how to optimize the adoption and sustainability of TBC in independent primary care settings to reduce the burden of disease related to suboptimal BP control and address current gaps in prior research .Additional file 1."}
+{"text": "E)-2-nonenal were newly generated after stir-frying with OAV > 1, and contributed mushroom, fatty, and green odors to SF-CSC. Diallyl trisulfide, nonanal, (E)-\u03b2-ionone, \u03b2-sesquiphellandrene, and (E)-2-decenal were considered as the potential key aroma compounds (KACs) to distinguish the CSCs after different heat treatment. After cooking, the total titratable acidity of CSC increased and the sensory properties changed significantly. This study provides valuable information and guidance on the sensory and flavor changes of thermal processing fermented vegetables.Chinese spicy cabbage (CSC) is a common traditional fermented vegetable mainly made of Chinese cabbage. In addition to eating raw, boiling and stir-frying are the most common cooking methods for CSC. To identify the impacts of boiling or stir-frying on the quality of CSC, the physicochemical properties, flavor compounds, and sensory properties of CSC were analyzed. A total of 47 volatile flavor compounds (VFCs) were detected by gas chromatography\u2013mass spectrometry. Sulfide was determined as the main flavor compound of CSC, mainly contributed by cabbage, garlic, and onion odors. The content of sulfide decreased significantly after cooking. Nonanal, geranyl acetate, and linalool were newly generated after boiling with odor activity value (OAV) > 1, and contributed fatty, sweet, fruity, and floral odors to BL-CSC. 1-Octen-3-one, 1-octen-3-ol, octanal, nonanal, and ( Chinese spicy cabbage  is a traditional fermented vegetable in northeast China and Korea. CSC is usually made from cabbage, scallion, ginger, garlic, red pepper, salt, and other ingredients, and fermented by various microorganisms present in the raw materials . Among tBoiling or stir-frying are the most common processing methods for daily consumption of CSC besides direct eating. After heat treatment, the number of microorganisms in CSC decrease significantly, delaying the changes in pH, acidity, organic acid, microbial population, and carbon dioxide, which could stabilize the quality of CSC and effectively maintain its shelf life ,6. ThereIn this study, gas chromatography\u2013mass spectrometry (GC-MS), principal component analysis (PCA), odor activity value (OAV), and fold change (FC > 2 or <0.5) were used to investigate the key aroma compounds (KACs) causing flavor differences in CSC after boiling or stir-frying. And the possible generation pathways of KACs were analyzed. In addition, the effects of boiling or stir-frying on the physicochemical and sensory properties of CSC were evaluated. This study provides valuable insights into the impact of cooking on the flavor of CSC.After cooking, the total titratable acidity (TTA) concentration increased significantly from 1.57 \u00b1 0.01 g/100 g to 1.63 \u00b1 0.01 g/100 g, which might be due to the increase in the organic acids escaping from the cells after heat treatment . There wThe composition and concentration of VFCs in fermented vegetables had an important influence on sensory characteristics and consumer preference . As showTo further investigate the discriminatory VFCs contributing to the CSC after cooking, the PCA was applied. As shown in The odor profiles of CSC not only depend on the composition and concentration of VFCs but are also affected by odor threshold (OT), which determines the OAV value of flavor compounds. In general, a compound with OAV greater than 1 was considered to be the main contributor of aroma, and the contribution and importance of the compound to the aroma increased with the increase in OAV . A totalSulfides were the most abundant VFCs in the CSC. In total, 15, 11, and 13 sulfides were detected in CK-CSC, BL-CSC, and SF-CSC, respectively. Sulfides were considered to be the most significant volatiles to determine the overall aroma of CSC because of the low threshold . They orCompared with CK-CSC, the sulfide content in BL-CSC and SF-CSC significantly decreased by 62.58% and 42.43%, respectively b. Most sE,E)-2,4-Heptadienal, (E)-2-decenal, and -2,4-decadienal were identified as the KACs of CK-CSC with OAVs > 1, which contributed significantly to the nutty, waxy, and fatty flavor. -2,4-Heptadienal  and nonanal  contributed greatly to the aroma of BL-CSC. Nonanal and (E)-2-heptenal  were newly generated in the BL-CSC. -2,4-Heptadienal, octanal , nonanal, (E)-2-nonenal , (E)-2-decenal, and -2,4-decadienal were the KACs of SF-CSC. -2,4-Heptadienal was the KAC in all the three CSC groups with no significant difference. (E)-2-Decenal increased significantly in SF-CSC, while octanal, (E)-2-nonenal, and benzaldehyde  were newly generated after the CSC stir-frying.Aldehydes are important characteristic flavor compounds in vegetables, which are generally associated with amino acid catabolism, ketoacid decarboxylation, and unsaturated fatty acid oxidation . As showCompared with CK-CSC, the aldehyde content in BL-CSC decreased by 46.51% but increased by 91.18% in SF-CSC b. AldehyE)-\u03b2-ionone was the key aroma compound in CK-CSC and BL-CSC, and imparted the seaweed, violet, and fruity odor. As a terpene derivative, (E)-\u03b2-ionone occurs in red peppers and is generated by the degradation of carotenoids by LAB [E)-\u03b2-ionone was significantly decreased in BL-CSC and disappeared in SF-CSC. Similar results showed that the content of (E)-\u03b2-ionone in watermelon juice decreased significantly after heat treatment [Ketones are generated through amino acid catabolism and fat degradation, resulting in sweet, creamy, or buttery odors with low odor thresholds ,45. Comps by LAB . After creatment . After sreatment . Adding reatment .Acids are known to impart a sour odor to fermented vegetables. Compared with CK-CSC, the acid content in BL-CSC increased by 8.48%, and in SF-CSC increased by 157.31% b. AceticEsters provide fruity, sweet, and floral flavor to fermented vegetables, generally originating from the esterification of short-chain acids with alcohols ,46. As sE)-2-octen-1-ol in CK-CSC, linalool and geraniol in BL-CSC, and 1-octen-3-ol in SF-CSC, as shown in E)-2-Octen-1-ol had little effect on the flavor of CK-CSC due to a high threshold. (E)-2-Octen-1-ol was not detected after boiling, which was consistent with a previous study indicating that the content of (E)-2-octen-1-ol decreased sharply in the heating of watermelon juice [Alcohols exhibit a sweet and floral flavor with a high threshold, and are essential for the production of esters and aldehydes . Four alon juice . Linalooon juice ,50. 1-Ocon juice .In total, five terpenes, \u03b2-sesquiphellandrene, zingiberene, \u03b2-bisabolene, farnesene, and curcumene, were detected in CSC samples, most of which were decreased significantly after cooking . Most teNitriles are important flavor compounds in fermented cruciferous plants and generated by glucosinolate degradation under acidic conditions . BenzeneTo further distinguish the main flavor compounds and their conveyed odors between different groups, the KAC differences among the three CSC samples were further screened based on the analysis of OAV (OAV > 1) and fold change (FC > 2 or <0.5). The histograms show the VFCs with greater than two-fold change, and the newly generated or disappeared a,b, indiE)-2-decenal, diallyl sulfide, -2,4-decadienal, and dimethyl disulfide disappeared after boiling, resulting in the lack of waxy, sweet, and onion odor compared with CK-CSC. And seven KACs including cis-propenyl propyl disulfide, diallyl disulfide, \u03b2-sesquiphellandrene, diallyl trisulfide, dimethyl trisulfide, (E)-\u03b2-ionone, and allyl methyl disulfide were significantly reduced (FC < 0.5) in BL-CSC, which reduced the pungent, herbal, garlic, cabbage, and seaweed odor to a certain extent. Comparing SF-CSC with CK-CSC (E)-2-nonanal, octanal, 1-octen-3-ol, nonanal, and 1-octen-3-one after stir-frying gave SF-CSC green, fatty, and mushroom odors. Acetic acid and (E)-2-decenal with FC > 2 increased the SF-CSC acidic and orange odor contrasted with CK-CSC. Meanwhile, (E)-\u03b2-ionone and \u03b2-sesquiphellandrene disappeared resulting in reduced fruity odor of SF-CSC. And five KACs including dimethyl trisulfide, dipropyl disulfide, cis-propenyl propyl disulfide, 2-phenethyl isothiocyanate, and diallyl trisulfide that decreased by greater than two-fold were all sulfides, which would reduce the cabbage, burnt green onion, radish, pungent, and garlic odor in SF-CSC. The KACs increased and newly generated in SF-CSC were more than those in BL-CSC, with more abundant odor changes, which was due to higher cooking temperature, and the peanut oil added in the stir-frying process that was related to the increase of aldehydes, acids, and alcohols [Comparing BL-CSC with CK-CSC a, three h CK-CSC b, the nealcohols ,51,53.E)-\u03b2-ionone, \u03b2-sesquiphellandrene, and (E)-2-decenal.The potential markers causing aroma differences among the three CSC samples were screened by analyzing VFCs with OAV > 1, and FC > 2 or <0.5, newly generated or disappeared as shown in E)-2-decenal, octanal, and (E)-2-nonenal increases in SF-CSC were generated from the oxidation of oleic acid and linoleic acid in peanut oil [E,E)-2,4-decadienal was generated from linoleic acid and arachidonic acid in peanut oil after stir-frying, and transferred to cooking fumes, resulting in a decrease in CSC after cooking [The possible formation pathways of KACs after cooking are shown in anut oil . However cooking .E)-\u03b2-Ionone was the main KAC contributing a fruity and seaweed odor in CK-CSC with OAV = 105.49, and the odor intensity decreased in BL-CSC with OAV = 32.94, disappearing in SF-CSC. Aldehydes including -2,4-decadienal (OAV > 300), nonanal (OAV > 60), octanal (OAV > 50), and -2,4-heptadienal (OAV > 40) mainly presented fatty odors in SF-CSC. 1-Octen-3-one  and 1-octen-3-ol (OAV = 51.28) presented a mushroom odor in SF-CSC, which was the unique flavor of CSC after stir-frying.In order to distinguish the odor profiles among different CSC groups, a total of 20 KACs with OAV > 1 and definite odor description were selected to form an odor wheel as shown in The overall flavors of CSCs were weakened after cooking, as the total OAV values decreased significantly in BL-CSC  and SF-CSC , compared with CK-CSC . As shown in Glossiness is an indicator of CSC appearance, which reflects the organoleptic property that affects consumers\u2019 appetite. The CSCs after cooking were characterized by a lower perception of glossiness than CK-CSC. The browning of BL-CSC and SF-CSC were caused by thermal processes, which might be due to the sugar\u2013sugar reactions at high temperatures, or the Maillard reactions between reducing sugars and ammonia compounds, such as proteins, amino acids, and amides .In addition to appearance, aroma was an important sensory attribute in determining the consumer acceptance of CSC. Five taste qualities  of CSCs were evaluated as shown in Brassica rapa ssp. pekinensis) and other materials were purchased from a local supermarket in Yantai, Shandong Province, China. CSC was produced according to Xing, et al. [Chinese cabbage  according to Choi, et al.  with modm/z. The linear retention index (LRI) of volatile compounds was calculated based on the series of n-alkanes (C10\u2013C40). The mass spectra of the volatile compounds were compared to those in the NIST 17 mass spectral library or in previously published articles. By comparing the values of the LRI with those of published values, identities were confirmed whenever possible. The relative concentration of each volatile compound was quantified as equivalent to the internal standard by the peak area.For volatile compound analysis, 2 g of CSC sample was put into a headspace vial with 15 \u03bcL (0.01 mg/mL) 3-nonanone , and stirred (300 rpm) at 70 \u00b0C for 10 min. Volatiles were adsorbed using divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber with a thickness of 50/30 \u00b5m for 30 min. After extraction, the fiber was introduced into the chromatograph for desorption at 250 \u00b0C for 5 min in split mode. GC-MS system  with SH stabilwax column (60 m \u00d7 0.25 mm \u00d7 0.25 \u03bcm) was used for analysis. Helium (1 mL/min) was used as the carrier gas. The GC was programmed as follows: 40 \u00b0C holding for 3 min, increasing the temperature to 115 \u00b0C at 5 \u00b0C/min, increasing to 150 \u00b0C at 2 \u00b0C/min, increasing to 230 \u00b0C at 7 \u00b0C/min, and holding for 10 min. The mass spectrometry was performed at 70 eV in electron impact ionization mode with a scan range of 50~550 n = 3). The differences among groups were determined by one-way analysis of variance (ANOVA) with Duncan\u2019s multiple range test (p < 0.05) using SPSS statistics version 24 . Principal component analysis (PCA) and the heatmap were obtained using TBtools software . The OAVs of flavor compounds were calculated according to Yang, et al. [All experimental data are displayed as mean \u00b1 SD -\u03b2-ionone, \u03b2-sesquiphellandrene, and (E)-2-decenal changed greatly among the three CSC groups, so these five KACs were identified as the characteristic potential markers of aroma differences.Two cooking methods of boiling and stir-frying had a great influence on the physicochemical properties, flavor components, and sensory qualities of CSC. Compared with CK-CSC, the TTA of CSCs after cooking significantly increased; however, the total sugar contents significantly decreased after stir-frying. A total of 47 VFCs were detected in CSC by GC-MS, and 21 VFCs with OAV > 1 and definite odor description were selected as the KACs including 8 sulfides, 6 aldehydes, 2 ketones, 1 acid, 1 ester, 2 alcohols, and 1 terpene. Compared with CK-CSC, seven KACs decreased significantly, four KACs disappeared, and three KACs were newly generated in BL-CSC, resulting in the disappearance of onion and waxy odors, more prominent acidic and pungent odors, and reduced fatty odor. However, in the SF-CSC group, two KACs increased and five KACs decreased significantly, two KACs disappeared, and five KACs were newly generated, which mainly resulted in the decrease in cabbage, garlic, pungent, fruity, floral, and burnt green onion odors, and increase in mushroom, acidic, green, and waxy odors. Although the total OAV of KACs exhibiting fatty odor was reduced in SF-CSC, the fat flavor in SF-CSC was stronger by sensory evaluation than that of CK-CSC, which might be due to the abundance of aldehydes detected in SF-CSC, leading to mutual enhancement of fat flavors between different aldehydes. The contents of diallyl trisulfide, nonanal, (Through different cooking methods, some beneficial flavor compounds were newly generated or increased, contributing abundant flavors to CSC. However, cooking caused browning of CSC and a reduction in characteristic sulfides. Therefore, on the basis of traditional cooking, it is necessary to combine cold processing methods such as high-pressure and microwave to inactivate microorganisms and enzymes to retain more flavors of CSC. In addition, the parameters in the cooking process could be further studied to develop more diverse and flavorful CSC products. This study also provides a reference for consumers in choosing different CSC products, and contributes to the industrial production of higher quality and more convenient CSC products."}
+{"text": "Change is a very complex and multifaceted phenomenon that is intertwined with the understanding of nursing practice, so, resistance to change in nursing can be considered as an important challenge. Knowing the reasons for this resistance can help in solving it in nursing. Therefore, the present study was conducted with the aim of investigating the reasons for resistance to change in nursing as an integrated review.This integrative review was conducted using the Whittemore & Knafl method in 5 stages, including problem identification, searching the literature, evaluating primary sources, analyzing data, and presenting the results. Databases like SID, Irandoc, Magiran, Google Scholar, Web of Science, PubMed, CINAHL, and Scopus were searched using the keywords; \u201cResistance\u201d, \u201cChange\u201d, \u201cNursing\u201d, \u201cResistance to Change\u201d and their Persian equivalents in the time range of 2000 to January 2023. After applying inclusion criteria and assessing the articles using Bowling\u2019s Quality Assessment Tool, finally, 15 papers were included from 2964.After reviewing and critically appraisal of the qualified articles, the findings were placed in three main categories including; (1) individual factors, (2) interpersonal factors, and (3) organizational factors and six subcategories.Undoubtedly, change is an integral component in nursing care, and resistance to it is the result of a set of individual, interpersonal and organizational factors that change managers should pay special attention to in order to make changes due to the reasons of this resistance, and the development process of developing changes in the clinical field is easily possible. Change is a very complex and multifaceted phenomenon that is intertwined with the understanding of nursing practice , 2, and Generally defining the concept of \u201cresistance to change\u201d is not easy , but basOrganizational resistance can be caused by power and conflict, or be the result of differences in functional orientation, structure, and organizational culture . Some ofImplementing change in the healthcare system is difficult, challenging, and often has short-term results , especiaStudies show that; Nurses are inherently resistant to clinical change , and theResistance has historically been viewed with negative consequences due to its potential impact on organizational success . HoweverAccepting change in the core of nursing and health care is considered a challenge, and some of these challenges are related to the movement of information and knowledge from research to the implementation of evidence-based best practices . It is bWhat is obvious is that resistance to change in nursing care can be an important challenge, although various studies have addressed the concept of change, however, very few and scattered studies have focused on the reasons for resistance in nursing. Therefore, this study was conducted with the aim of an integrated overview of the reasons for resistance to change in nurses. An integrative review is a specialized review method that summarizes empirical or theoretical studies that have already been conducted to provide a more comprehensive understanding of a specific phenomenon or healthcare problem . In factThis study is an integrated review based on articles related to the reasons for resistance to change in nursing which was conducted to collect data from various studies. This integrative review was conducted using the Whittemore & Knafl method in 5 stages of review, including (a) problem identification, (b) searching the literature, (c) evaluating data from primary sources, (d) analyzing data, and (e) presenting the results, using of this method also increases the rigor of this study \u201332.Based on the Whittemore & Knafl method, 1) in the first stage, the following question was set to answer the study\u2019s aim: What are \u201cthe reasons for resistance to change in nursing\u201d?2) In the second stage, searching for articles was conducted by two researchers in the time range from 2000 to January 2023. We searched databases such as; Persian database, Google Scholar, Web of Science, PubMed, CINAHL, and Scopus by using the keywords; \u201cResistance\u201d, \u201cChange\u201d, \u201cNursing\u201d, and \u201cResistance to Change\u201d in English and Persian separately or combined by using the Boolean operators(AND and OR). In this stage; the results of the comprehensive search included 2964 articles after reviewing them based on the inclusion criteria such as: accessing the full text of the article, including the keywords in the title and abstract of the article, and writing in Persian and English language, finally 2949 were removed, and the 15 articles were included.3) In this stage, two researchers evaluated the data and the content of selected studies for their quality by using \u201cBowling\u2019s Quality Assessment Tool\u201c , which cThe data extraction was based on the main data from the 15 articles included: the publication\u2019s year, the language of the study, the main keywords and the methodology of the study, and also the results.4) In the fourth stage, data were evaluated independently by two researchers and updated in a continuous process, after that, the data were analyzed and interpreted. This process was verified by three authors including; documenting the required data through five methodological stages, and analyzing separately by researchers.All of the extracted data were read by researchers and determined significant items and similar and different data were assessed and examined throughout the data analysis process.Eventually, confirmation and verification were performed by all authors to ensure that all 15 reviews were thoroughly evaluated in all of the methodological stages and the results were matched by the research questions of the study.Whittemore and Knafl (2005) state that assessing the quality of the included evidence is not essential in a supplementary review . All stuFor our review, studies were deemed to be of relatively high quality, and studies that were in the moderate or low-quality range were omitted.5) In the last step, the results were obtained according to the framework of 15 articles that were selected. It is necessary to mention that was found no paper in Persian, and all articles included in this study were in English  individual factors, 2) interpersonal factors, and (3) organizational factors and six subcategories , six sub-categories, and thirty-seven codes were identified.What is clear to us is that change in improving patient outcomes is common and important in the current healthcare systems . The proindividual factors; It is one of the factors that can be based on the individual attitude and understanding and personality characteristics of nurses. Attitudes toward an impending change may be positive, negative, or neutral. Resistance to change in nursing is probably based on negative and defensive feelings toward change such as fear, uncertainty, doubt, disappointment, mistrust, confusion, and anger [Based on the findings of the present study, nd anger . The finnd anger . Tendencnd anger . In the nd anger .All personnel in an organization does not react equally to ongoing changes in their organization . A feeliinterpersonal factors of employees. Studies show that communication barriers in the organization ultimately affect the implementation, quality, and sustainability of change [Among other effective factors that can cause RtC in nursing is f change . Employef change , and thef change . The quaf change . This faf change , 49.organizational factors which are in three sub-categories; management factors, organizational values, and structural factors are placed. As mentioned, accepting change at the core of nursing and health care is a challenge because nurses are not only inflexible but also adept at strengthening the existing [The third factor of resistance to change in nursing, is the existing . Therefoexisting .The effect of RtC can strengthen the negative effect on organizational effectiveness and organizational commitment, and the lack of leadership support will amplify with time. In this regard, it seems that managers supporting change in the organization can play an important role in improving resistance . The resOrganizational culture is characterized as a set of anticipated behaviors that are for the most part supported inside the group , can plaOrganizational values including organizational culture, negative organizational perception, and conflict with organizational identity also play a fundamental role to cause nurses\u2019 resistance to change. Understanding organizational orientations may hinder the adoption of new evidence-based programs and practices . Also, cStructural factors such as organizational characteristics, resources and budget, job characteristics, and environmental changes, along with other organizational factors, are effective factors in creating resistance to change in health care workers. Higgs and Rowland (2010) emphasize factors such as environmental changes, organizational characteristics, resources, and budgets as broad factors affecting the change process , 59. OrgOrganizational change can be called a stressful factor in the work environment , but altFinally, based on the results obtained from selected studies, due to the nature of the nursing profession on the one hand and the occurrence of rapid and large changes in clinical environments and care organizations on other hand, several factors can cause resistance to these changes and affect the care and safety of patients. This resistance is influenced by three important factors, individual, interpersonal, and organizational factors. The effects of these factors, can directly and indirectly, affect the proper care of patients. Therefore, paying attention to these factors to improve them with education, improving communication, efficient and collaborative management, understanding organizational values, and developing organizational structure can reduce resistance to changes in patient care environments.Changes in the nursing environment are an integral part of nursing practice. The findings of this integrated review confirm the complexity and multifaceted nature of these resistances. A set of individual, interpersonal, and organizational factors in nurses leads to resistance to change and is considered an important challenge in nursing care. Knowing these factors can help reduce resistance and improve the quality of nursing care. So nursing managers and decision makers should pay special attention to this in order to make changes. So that, nurses can provide safe and qualified care for their clients and improve the level of health and satisfaction of patients.The limitations of this study include: not searching for articles in languages other than English and Persian, so our search strategies may have under-represented studies in other languages, such as Spanish and Portuguese."}
+{"text": "Our results can be used to evaluate the effect of different sampling protocols on abundances of clones within an individual as well as the commonality of clones across individuals. Using synthetic and empirical TCR amino acid sequence data, we perform simulations to study expected clonal commonalities across multiple individuals. Based on our formulae, we compare these simulated results with the analytically predicted mean and variances of the repertoire overlap. Complementing the results on simulated repertoires, we derive explicit expressions for the richness and its uncertainty for specific, single-parameter truncated power-law probability distributions. Finally, the information loss associated with grouping together certain receptor sequences, as is done in spectratyping, is also evaluated. Our approach can be, in principle, applied under more general and mechanistically realistic clone generation models.Diverse T and B cell repertoires play an important role in mounting effective immune responses against a wide range of pathogens and malignant cells. The number of unique T and B cell clones is characterized by T and B cell receptors (TCRs and BCRs), respectively. Although receptor sequences are generated probabilistically by recombination processes, clinical studies found a high degree of sharing of TCRs and BCRs among different individuals. In this work, we use a general probabilistic model for T/B cell receptor clone abundances to define \u201cpublicness\u201d or \u201cprivateness\u201d and information-theoretic measures for comparing the frequency of sampled sequences observed across different individuals. We derive mathematical formulae to quantify the mean  A major component of the adaptive immune system in most jawed vertebrates is the repertoire of B and T lymphocytes. A diverse immune repertoire allows the adaptive immune system to recognize a wide range of pathogens\u00a0 that are presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs). T cells that each carry a type of TCR mature in the thymus and undergo V(D)J recombination, where variable (V), diversity (D), and joining (J) gene segments are randomly recombined\u00a0 1, 2, and 3, located within the V region, among which the CDR3B cells can also respond to different antigens via different B cell receptors (BCRs) that are comprised of heavy and light chains. As with TCRs, the mechanism underlying the generation of a diverse pool of BCRs is VDJ recombination in heavy chains and VJ recombination in light chains. Positive and negative selection processes sort out about 90% of all BCRs that react too weakly or strongly with certain molecules\u00a0, all expressed in terms of the general set of clone generation probabilities or clone populations. One of our metrics is the expected \u201cM-overlap\u201d or \u201cM-publicness,\u201d defined as the expected number of clones that appear in samples drawn from M separate individuals. This quantity is a clinically interpretable limit of the expected \u201csharing number\u201d defined in Elhanati et\u00a0al.  has been used to characterize the number of T cell receptor sequences possible within a continuous range of generation probabilities  be the probability density associated with the distribution of traits, as depicted in Fig.\u00a0p(x). If we discretize the values of probabilities, the number of clones with a certain relative frequency clone countp(x). If p(x) is not available from data or a model, an alternative representative starts with the number density n(x), which can be estimated by sampling a process which follows p(x). The probability that a continuous trait x is drawn twice from a continuous distribution p(x) is almost surely zero. Hence, the corresponding number counts n(x) are either 1 if X is sampled) or 0 otherwise, as shown in Fig.\u00a0X is of clonotypei if i. Then, if i is thus p(x) from Let mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimUsing the mathematical quantities defined above, we develop a simple statistical model for BCR and TCR sequences distributed among individuals. Although our model is applicable to both BCR and TCR sequences, we will primarily focus on the characterization of TCRs for simplicity. B cells undergo an additional process of somatic hypermutation and class switching leading to a more dynamic evolution of the more diverse B cell repertoire , we can describe the probability of a T-cell population m by a multinomial distribution over individual probabilities m is a cell of clone i. Repeated draws (with replacement) would provide the samples for the estimator i T cells from the thymus, the proliferation and apoptosis rates of clone i cells, and interactions manifested as, e.g., carrying capacity as model parameters. Probability distributions for At any specific time, individual s et\u00a0al.  and can k). Other diversity/entropy measures such as Simpson\u2019s indices, Gini indices, etc. Rempala and Seweryn  can be computed using the marginalized probabilityR [defined by Eq.\u00a0 (Eq.\u00a0) allows M individuals is defined asM-individual probabilityi appears in at least one of the M individualsi cell occurs at all in the total population, while i cell appears in each of the M individuals.Here, we consider the distribution i and j both appear in at least one of the M individuals We will also need the joint probability that clones ing Eqs.\u00a0, we findM-population richness can also be found in terms of M randomly selected individuals, the \u201cM-overlap\u201d or \u201cM-publicness\u201dUsing the above definitions, we can express the mean total-population richness as and Eq.\u00a0,18\\documM-population probability M individuals, M-overlap. Clearly, M-publicness, we can identify the expected M-privateness as M individuals, i.e., that occur in at most M-overlap associated with a certain cell-type distribution are captured by the variance As with Eqs.\u00a0 and 10)10), we cting Eq.\u00a0 by the M(see Eq.\u00a0) to findM tested individuals. To find the probability that i, we use the Poisson binomial distribution describing independent Bernoulli trials on individuals with different success probabilities A. Equation\u00a0N replaced by S. Uniform random sampling from a multinomial results in another multinomial. Thus, if we use the full multi-individual probabilityi appears in the sample from individual m ism becomesThe results above are described in terms of the entire cell populations entclass1pt{minimawith Eq.\u00a0 and 16)\\document in Eqs.\u00a0, 17), , a, a\\docum in Eqs.\u00a0, except k clones are shared by all M samples isk integers that can be selected from the set A. Equation\u00a0\\documentThe sampling theory derived in the previous sections is useful for understanding the effect of different sampling distributions on measurable quantities such as the proportion of shared TCRs and BCRs among different individuals. Figures Using d by Eq.\u00a0. In Fig.SONIA package\u00a0\\document use Eq.\u00a0 and \\docfrom Eq.\u00a0 to find To examine the variance associated with a given expected number of shared empirical TRB CDR3 sequences, we show the Fano factor \u00ae Ryzen Threadripper 3970 using Numba to parallelize the calculation of Eqs.\u00a0 38), we cWe end with a brief discussion of information loss upon coarse-graining which arises when analyzing lower-dimensional experimental/biochemical classifications of clones that are commonly used. Such lower-dimensional representations can be obtained through spectratyping\u00a0 differential entropy o Jaynes  using thAs an example, we compute the information loss associated with discretizing the truncated power lawtion Eq.\u00a0. AnalytiEquation\u00a0 is plottWhile the connection between continuous probability distributions and their discretized counterparts has important consequences for sampling, information loss also occurs in spectratyping when an already discrete random variable is coarse grained. In this scenario, the information loss can be quantified uniquely  by the entropy difference of the distributions\u00a0  to quantify the expected overlap between cell-sequence samples\u00a0\\documentFinally, in the context of coarse-graining, or spectratyping (Ciupe et\u00a0al."}
+{"text": "Multicomponent solid forms of low molecular weight drugs, such as co-crystals, salts, and co-amorphous systems, are a result of the combination of an active pharmaceutical ingredient (API) with a pharmaceutically acceptable co-former. These solid forms can enhance the physicochemical and pharmacokinetic properties of APIs, making them increasingly interesting and important in recent decades. Nevertheless, predicting the formation of API multicomponent solid forms in the early stages of formulation development can be challenging, as it often requires significant time and resources. To address this, empirical and computational methods have been developed to help screen for potential co-formers more efficiently and accurately, thus reducing the number of laboratory experiments needed. This review provides a comprehensive overview of current screening and prediction methods for the formation of API multicomponent solid forms, covering both crystalline states  and amorphous forms (co-amorphous). Furthermore, it discusses recent advances and emerging trends in prediction methods, with a particular focus on artificial intelligence. Drug absorption after oral administration of active pharmaceutical ingredients (APIs) inter alia depends on their physicochemical properties , their dose, and the local environment within the gastrointestinal tract. It has been reported that up to 90% of the currently developed APIs and about 40% of the approved drugs have poor biopharmaceutical properties as a consequence of their insufficient solubility in water ,2. The pIn 1989, Desiraju defined the term crystal engineering as \u201cthe comprehension of intermolecular interactions within crystal packing, and the use of this knowledge to engineer novel solid materials possessing specific physical and chemical characteristics\u201d . CrystalIn 2018, the U.S. Food and Drug Administration (FDA) defined pharmaceutical co-crystals as crystalline materials composed of a neutral API and a second neutral molecule generally defined as a co-former . Co-crysAmorphous solid dispersion technology has evolved since the 1960s, with polymers used as carriers. However, polymers have limitations, such as hygroscopicity and low drug loading. In 2009, Chieng et al.  introducSuitable co-formers are crucial to designing stable co-crystal, salts, and co-amorphous systems with desirable properties, such as high solubility, fast dissolution rate, good diffusion permeability, high physicochemical stability and a possible synergistic pharmacological effect . Over thKa based models [In recent years, advanced prediction methods, especially computer-assisted methods, have been developed to improve the specificity, sensitivity and accuracy of co-former screening and the formation prediction of API multicomponent solid forms. d models ,26,27,28d models ,31,32,33d models ,36,37,38d models ,40,41,42d models ,44; moled models ,46,47; td models ,48,49,50d models ,53,54,55d models ,58,59,60d models ,61,62. Ed models ,64,65. TKa Rule is a simple approach to predict the formation of co-crystals or salts [Ka (\u0394pKa) between acidic/basic APIs and basic/acidic co-formers is calculated as follows:The \u0394por salts ,68. The Ka can be used to estimate the tendency for proton transfer between a given API and co-former. When \u0394pKa is higher than 3, a significant difference in the acidity or basicity of the API and co-former is obtained, indicating a preferable formation of a salt. When \u0394pKa is lower than 0, the acidity or basicity of the API and co-former are similar and thus the formation of a co-crystal is expected where non-charge-assisted hydrogen bond interactions occur between the API and co-former. However, when \u0394pKa is between 0 and 3, the difference in acidity or basicity is not large enough to clearly favor the formation of salt over a co-crystal or vice versa. In such cases, the \u0394pKa rule is not the most reliable approach to predict the formation of a salt or co-crystal [\u0394p-crystal ,69. A co-crystal .Ka rule was validated by Cruz-Cabeza using 6465 crystalline structures from the CSD [Ka is greater than 4 or less than -1, respectively. When \u0394pKa is between -1 and 4, \u0394pKa and the likelihood of proton transfer showed a linear relationship (Ka between 0 and 3), which is between the two extremes of salt and co-crystal [Ka rule is not applicable for predicting the formation of salts or co-crystals. Childs et al. [Ka lower than 3. They prepared a total of 20 multicomponent solid forms of theophylline and guest molecules, which included 13 co-crystals, five salts, and two within the salt\u2013co-crystal continuum. It is important to note that the formation of salts or co-crystals is influenced by several factors, including the solvent used (and its polarity), temperature, API or co-former concentration, and crystal packing interactions. Therefore, the values of \u0394pKa alone cannot predict with certainty the formation of salts or co-crystals, but they can provide useful information about the likelihood of their formation.The \u0394p the CSD . Multicotionship . A \u201csalt-crystal . In suchs et al. , investiarom and OH\u00b7\u00b7\u00b7Narom) are preferred over the corresponding supramolecular homosynthons (COOH\u00b7\u00b7\u00b7COOH and OH\u00b7\u00b7\u00b7OH). Supramolecular synthons enable the prediction of the possible interactions between different molecules and the assessment of their propensity to form stable co-crystals. Through the analysis of complementary synthons in the molecular components, it becomes possible to narrow down the potential co-formers for a given target molecule.Recognizing supramolecular interactions is crucial for designing crystals. After introducing the term \u201ccrystal engineering\u201d in 1989, Desiraju  introducThe use of supramolecular synthons has been explored only in multicomponent crystal forms ,32,70. S\u03b1) and acceptor (\u03b2) are generated from the maxima and minima of the MEP and calculated using Equations (2) and (3), respectively.min and MEPmax are the local minima and maxima on the MEPs (unit: kJ\u00b7mol\u22121). The prediction of multicomponent crystal forms based on MEPs considers that all hydrogen bond sites on the surface of a molecule are not restricted by the internal molecular structure and are free to interact with other molecules in the solid-state environment. The molecular arrangement, steric constraints and packing effects are not taken into account [E is the sum of all contacts across the surface of every molecule in the crystal, which can be calculated using Equation (4) [\u03b1i are hydrogen bond donor sites, and \u03b2j are hydrogen bond acceptor sites. Positive or negative unpaired sites locate low electrostatic potential gaps or regions, making no contribution to the overall electrostatic interaction energy [The calculated MEPs generated using the density functional theory in the gas phase are used to identify surface site interaction points and predict electrostatic interactions at the surface of the molecules . The str account . The inttion (4) .(4)E=\u2212\u2211n energy .\u03b1i interacts with the first most negative \u03b2j, the second most positive \u03b1i with the second most negative \u03b2j, and so forth until all of the interaction sites are covered [E) between the total E of pure components and the E of the multicomponent crystal forms, using the following Equation (5).Ecrys, E1 and E2 are the interaction site pairing energies of a multicomponent crystal of stoichiometry 1n2m, and the pure component solids, 1 and 2, respectively. The interaction site pairing energy of a multicomponent crystal is calculated in the same way as for a pure component solid. A high value of \u0394E indicates a stronger interaction between two different components and a higher probability of forming a multicomponent crystal form. The minimum value of \u0394E is 0 kJ\u00b7mol\u22121, indicating that the formation of multicomponent crystal forms must increase the interaction energy [According to Etter, the first most positive  covered . Based o covered  proposedn energy . The speE was over 11 kJ\u00b7mol\u22121, the possibility of co-crystal formation is larger than 50%. Grecu et al. [E values and the ones presenting a larger value of \u0394E were consistent with the experimental results in most cases. Furthermore, the MEPs method showed little difference when compared to the COSMO-based methods (see below). The MEPs method can also be an effective tool to (i) investigate the driving force of co-crystals/salts formation and explain the ratio of APIs and co-formers in multicomponent crystal forms [n-pyridinealdazines , the I\u00b7\u00b7\u00b7N halogen bond interaction is the primary interaction [n-pyridinealdazines was 4 > 3 > 2 according to the Vs,min  values located near the pyridine-N atom for n = 2, 3 and 4  synthons. Overall, the validated approach provides a promising framework for virtual multicomponent crystal forms screening and could potentially be applied in drug discovery.The prediction reliability of MEPs has until now only been demonstrated for multicomponent crystal forms. For example, Pagliari et al.  applied u et al.  successfal forms ,70, and al forms . Apart fal forms  and chalal forms  formatioeraction . The \u03c3-  3 and 4 . WzgardaHBP was first developed by Galek et al.  as a logAPI-co-former is larger than HBPAPI-API and HBPco-former-co-former, co-crystallization is likely to occur [propensity value is calculated as the difference of largest heteromeric interactions and largest homomeric interactions as shown in Equation (6).HBP was developed by the Cambridge Crystallographic Data Centre and has been integrated into the Mercury program  to identto occur . In Figu2 of nicotinamide group and the carbonyl group of lenalidomide were found to have a high propensity for heteromeric interactions (0.94), while homomeric interactions between lenalidomide\u2013lenalidomide have a propensity of 0.84 for -NH- with carbonyl group, and 0.82 between nicotinamide\u2013nicotinamide for -NH2 with carbonyl group  is the lattice energy of multicomponent crystal forms AmBn consisting of molecules A and B in a stoichiometric ratio m:n, and Elatt(A) and Elatt(B) are the lattice energies of the pure components A and B, respectively. The probability of forming a multicomponent crystal form is higher when the \u0394Elatt is more negative. It is unlikely that multicomponent crystal forms are formed if \u0394Elatt has a positive value. Distinct from other methods, this prediction technique does not make any assumptions about hydrogen bonds and relies solely on the lattice energy. Factors such as temperature, pressure, solvent effects, and kinetics are not considered in the calculations [Lattice energy is another tool to predict multicomponent crystal forms. The energy difference between multicomponent crystal forms and pure individual components can be used as an indication of whether co-crystallization is expected to occur spontaneously, i.e., if it is thermodynamically favored compared to the crystallization of each starting material separately . Consideulations .Elatt of the crystal can be calculated by three methods [The  methods : the fir methods , to calc methods , and the methods . Chan et methods  performe methods  used the methods  proposed methods  compared methods  found thMolecular complementarity (MC) was first introduced by F\u00e1bi\u00e1n in 2009  to invesMC is mostly used as a preliminary screening tool instead of being used solely to determine the formation of multicomponent crystal forms. For example, Li et al.  used bot\u03b4, unit: MPa0.5) is shown in the following equation:Ev is the energy of vaporization, and Vm is the molar volume. The mutual solubility of two components is determined by their closeness in \u03b4 values. Desai and Patravale [The solubility parameter theory was proposed by Hildebrand and Scott in 1950 . Accordiatravale  applied \u03b4t), or Hildebrand solubility parameter, into three partial solubility parameters, as follows [\u03b4d are dispersion forces (atomic dispersion), \u03b4p are \u2018polar\u2019 interactions, and \u03b4h are hydrogen bonds. These partial solubility parameters are most commonly calculated based on the Hoftyzer\u2013Van Krevelen and Fedors group contribution methods, as follows: [i is the structural group within the molecule, Fdi, Fpi, Fhi and Vi are the group contributions to the dispersion forces, polar forces, hydrogen bonding energy and molar volume, respectively.The Hildebrand and Scott approach is based on regular solution theory and works best for non-polar molecules interacting via weak dispersion forces . In orde follows :(9)\u03b4t2=\u03b4follows: (10)\u03b4d=\u2211\u03b4t) (Equation (11)) to predict the miscibility between the drug and carriers, and Mohammad et al. [\u03b4t < 7 MPa0.5.In 1999, Greenhalgh et al.  proposedd et al.  proposed\u03b4d and \u03b4p have thermodynamically similar effects, while \u03b4h has a different effect in nature [\u03b4d and \u03b4p parameters were combined as a volume-dependent solubility parameter, and \u03b4v, and Ra(v) factor were introduced as follows:According to Bagley et al. , \u03b4d and n nature , because\u03b4v against \u03b4h, i.e., the Bagley diagram, has been applied in different aspects, such as predicting the miscibility of two materials and the duration of drug intestinal absorption [Ra(v) is not higher than 5.6 Mpa0.5.The two-dimensional plot of sorption ,116. Albsorption  found thVan Krevelen and Hoftyzer  introduc0.5. For the purpose of convenient visualization of the spherical, instead of using the ellipsoidal solubility plot, a modified radius (Ra) equation (Equation (15)) was proposed [1  and another point HSP2  in the Hansen space Ra=Ra(v), \u03b4t criteria. Since there were large deviations from the experimental results for Ra(v) and \u03b4t criterion was chosen in further discussion in both MIN and MOP cases. The overall success rates of co-former prediction by the \u0394\u03b4t criterion were 65.5% and 62.5% for MIN and MOP, respectively. In co-amorphous systems prediction, the \u0394\u03b4t value between florfenicol and oxymatrine was determined to be 3.87 MPa0.5, indicating good miscibility between the API and co-former, contributing to the successful formation of a co-amorphous system [\u03b4t, as it could offer more accurate information about the forces present between the molecules  [The HSP method was first used in co-crystal formation prediction in 2011 , and lats system . The HSPactions) .The COSMO-RS theory was developed by Klamt and co-workers  to prediKa [Hex (Equation (16)) of the API and co-former with a given stoichiometry as compared with the pure materials [HAB is the enthalpy of the supercooled stoichiometric mixture of components A and B. HA and HB are the enthalpy of the pure components A and B, respectively, and x is the mole fraction of each component. The probability of forming a multicomponent crystal form is higher when the \u0394Hex is more negative.COSMO-RS theory has been used in several research fields, including the prediction of solubility  and pKa Ka , identifKa , solvateKa , and theKa . It is aKa ,70. EvenKa , the relKa ,70. On tKa . Therefoaterials .(16)\u0394HeHex < \u22121 kcal/mol. Surprisingly, the COSMO-RS method showed up to 100% overall success rate in predicting the formation of multicomponent crystal forms of pymetrozine [Hex < \u22123.0 kcal\u00b7mol\u22121 and \u0394Hex < \u22122.00 kcal\u00b7mol\u22121, respectively. Alhadid et al. [Although the COSMO-RS method ignores the solid-state order, it could successfully predict the formation of 21 new multicomponent crystal forms of 2-amino-4,6-dimethoxypyrimidine  with 63 etrozine  and minoetrozine  when \u0394Hed et al.  calculatd et al. . When exd et al. . The COSd et al.  used mixIn recent years, AI methods, especially machine learning (ML), have emerged as promising and effective strategies to predict both multicomponent crystal forms and co-amorphous systems . This meThe main limitation in applying deep learning (DL) approaches in crystal engineering lies in the critical bottleneck of data availability, while the model performance also heavily relies on the key factor of data quality. The CSD comprises an extensive repository of high-quality positive samples , providing valuable support for DL applications . In data0.5), and a larger \u0394pKa. Wang et al. [Chambers et al.  later bug et al.  found thter Horst et al.  proposedEnon-bonded) between the API and co-former. A higher absolute value of \u0394Enon-bonded was indicative of a greater probability of co-amorphous formation. This model was developed based on 105 solvent-based cases involving 13 different drugs, and successfully predicted five new co-amorphous combinations of gefitinib and four new co-amorphous combinations of erlotinib.In recent years, computational methods have also been developed to screen co-formers more accurately and efficiently. A modified, non-bonded interaction energy model was introduced by Deng and co-workers  to prediCo-crystals, salts and co-amorphous systems are multicomponent solid forms containing one or more types of molecules. They are able to offer significant benefits to the bioavailability, solubility, dissolution, stability, permeability, and other physicochemical and pharmacokinetic properties of APIs. Choosing appropriate co-formers for a specific API is essential to develop solid formulations with ideal properties, which generally requires a significant amount of time and resources. Over the past few years, several knowledge-based methodologies, as well as computer-assisted prediction methods, have emerged exhibiting efficacy and accuracy in co-former selection and prediction of the formation of multicomponent solid forms. More strategies have been developed for multicomponent crystalline forms than amorphous forms. Some methodologies, such as HSP, and other new approaches, such as artificial intelligence, are applicable for both solid-state forms. Though none of the single approaches can completely and accurately predict the formation of multicomponent solid forms, each approach can offer valuable guidance in selecting the most suitable co-formers. The implementation of multiple screening/prediction approaches can result in considerable enhancements in the effectiveness and accuracy of predicting the formation of co-crystals, salts and co-amorphous systems, as confirmed by previous studies.It can be expected that in the future, more investigations will continue to promote screening/prediction efficiency by using multiple approaches as well as developing novel models. One direction could be the integration and combination of various approaches mentioned in this review. For instance, combining machine learning algorithms with molecular docking methods could hold potential to improve the precision of multicomponent solid-state forms  formations. The combination of quantum mechanical simulations with virtual screening techniques offers opportunities to delve deeper into the intricate interplay of thermodynamic stability and intermolecular interactions of multicomponent systems. High-throughput virtual screening methods taking into account advancements in computing power coupled with experimental screening to validate the predictions will speed up the identification of potential co-crystals and co-amorphous forms.Collaborations between computational scientists and experimentalists are required to arrive at synergistic approaches to co-crystal and co-amorphous discovery. As researchers explore novel combinations of approaches, significant advancements in the field are foreseeable. These collective endeavors are poised to pave the way for the expedited discovery and development of innovative drug formulations, which could have a transformative impact on the pharmaceutical industry."
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