Vancomycin-resistant enterococci (VRE) are nosocomial pathogens with genetic plasticity and widespread antimicrobial resistance (AMR). To prevent the spread of VRE in the hospital setting, molecular epidemiological approaches such as pulsed-field gel electrophoresis and multilocus sequence typing have been implemented for pathogen outbreak surveillance. However, due to the insufficient discriminatory power of these methods, whole-genome sequencing (WGS), which enables high-resolution analysis of entire genomic sequences, is being used increasingly. Herein, we performed WGS of VRE using both short-read next-generation sequencing (SR-NGS) and long-read next-generation sequencing (LR-NGS). Since standardized workflows and pipelines for WGS-based bacterial epidemiology are lacking, we established three-step pipelines for SR- and LR-NGS, as a standardized WGS-based approach for strain typing and AMR profiling. For strain typing, we analyzed single-nucleotide polymorphisms (SNPs) of VRE isolates and constructed SNP-based maximum-likelihood phylogenies. The phylogenetic trees constructed using short and long reads showed good correspondence. Still, SR-NGS exhibited higher sensitivity for detecting nucleotide substitutions of bacterial sequences. During AMR profiling, we examined AMR genes and resistance-conferring mutations. We also assessed the concordance between genotypic and phenotypic resistance, which was generally better for LR-NGS than SR-NGS. Further validation of our pipelines based on outbreak cases is necessary to ensure the overall performance of pipelines. Enterococcus faecium and Enterococcus faecalis, are frequently associated with human disease outbreaks characterized by high morbidity rates in the context of both hospital- and community-acquired infections , among which vanA and vanB are the most frequently implicated in VRE outbreaks. VanA is responsible for high levels of resistance to vancomycin and teicoplanin, while vanB and vanC are only associated with vancomycin resistance  through the transfer of mobile genetic elements or the development of mutations, which constitute advantages over nosocomial pathogens . Glycopecus spp. . Howevercus spp. . Vancomysistance . The traliflavus . Unfortuliflavus .The widespread AMR of enterococci necessitates strategies for clinical management of infections, including active surveillance and rapid recognition of outbreaks. To elucidate the mechanisms underlying AMR and prevent the dissemination of bacteria, molecular epidemiological analysis of infections is needed. Conventional molecular diagnostic approaches to the analysis of the genetic relatedness of strains include pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PFGE was among the techniques ushering in the era of molecular epidemiology, and has long been considered the gold standard for bacterial strain typing . HoweverStaphylococcus aureus and carbapenem-resistant Enterobacterales  sequencing platforms are designed to yield much longer reads (tens of kilobases) without the need for polymerase chain reaction (PCR) amplification and are currently represented by Single-Molecule Real-time sequencing (SMRT) of Pacific Biosciences  and Nanopore sequencing   for investigating outbreaks. WGS has high discriminatory power for clonal isolates and can reveal the transmission route of hospital infections caused by various species, including methicillin-resistant cterales . Next-geord, UK) . Howeverord, UK) . Nanoporord, UK) .In this study, we performed bacterial WGS using both short-read and long-read next-generation sequencing  for epidemiological analysis of VRE. We developed pipelines involving three-step analyses of SR- and LR-NGS data for strain typing and AMR profiling, respectively. Moreover, noting the differences between SR- and LR-NGS, the strain typing and AMR detection methods were evaluated using clinical VRE isolates.E. faecalis; the remainder were E. faecium. The AMR of each isolate to vancomycin, teicoplanin, gentamicin, streptomycin, tetracycline, and daptomycin was detected based on the minimal inhibitory concentration (MIC), in accordance with Clinical & Laboratory Standards Institute (CLSI) guidelines .A total of 23 samples, including 13 clinical isolates and ten College of American Pathologist samples were analyzed in this study. Clinical isolates were collected from Seoul National University Bundang Hospital. For each isolate, species identification was performed and AMR was detected using the MicroScan WalkAway system . Three species were identified as idelines . For isoPrior to WGS of the VRE isolates, each isolate was cultured on a blood agar plate and subjected to DNA extraction. Genomic DNA extraction of colonies was performed using InstaGene Matrix  according to the manufacturer’s instructions and the concentration was measured using a Quantus fluorometer .For SR-NGS, WGS of bacterial DNA was performed using the iSeq 100 (Illumina). DNA libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina) according to the manufacturer’s instructions. After adapter ligation, barcoding was performed using the Nextera Index primers (Illumina); 23 isolates per flow cell were run in multiplex. The completed sample libraries were quantified using the Quantus fluorometer to achieve a standardized input of 100 ng per sample. Libraries were pooled and diluted to the final loading concentration of 200 pM with 1% PhiX (Illumina) spike-in, and sequenced on iSeq 100 i1 REAGENT cartridge (Illumina) using 150-bp paired-end reads.MinION (Oxford Nanopore Technologies) was chosen as the LR-NGS platform and WGS was performed on 20 isolates (DNA samples of three isolates were not available). For library preparation, DNA was end-repairelid and A-tailed with NEBNext FFPE DNA Repair Mix and the NEBNext Ultra II End Repair/dA-Tailing Module . Ligation of the sequencing adapters was performed using the SQK-LSK109 kit (Oxford Nanopore Technologies) and each sample was barcoded using the EXP-NBD104 kit (Oxford Nanopore Technologies). Each step was followed by purification with Agencourt AMPure XP beads (Beckman Coulter). Libraries were pooled in equimolar amounts and adjusted to a final concentration of 1 pM. Twenty isolates were sequenced per flow cell  using MinKNOW software .Demultiplexed FASTQ files were exported from iSeq 100 and subjected to quality control (QC) using FastQC . The QC indices were chosen regarding manufacturer’s recommendations and observed run data. Specifically, we evaluated the iSeq data according to the number and mean length of the reads, percentage of reads with an average quality (Phred) score > 30, and the GC content. Then, the reads were trimmed and filtered using CLC Genomics Workbench . The quality threshold was 0.05 and the maximum read length was 150 bp.Raw fast5 files were basecalled, sorted according to their barcodes, and converted into FASTQ format using Guppy v.4.2.2 (Oxford Nanopore Technologies). Trimmed reads with an average quality score > 7 were retained. EPI2ME, a real-time bioinformatic tool specifically designed for Nanopore sequencing, was used to conduct QC of reads in conjunction with the What’s In My Pot (WIMP) workflow. The QC statistics of the MinION reads included quality scores, sequence lengths, and overall yields. Length filtering was performed using CLC Genomics Workbench; reads shorter than 500 bp were discarded.E. faecium strain  using the “map reads to reference” function of CLC Genomics Workbench. On the other hand, the trimmed MinION data were subjected to reference-based assembly using the read mapping function of the Long Read Support plugin, which is specifically used for the assembly of error-prone long reads. Variant calling of mapped reads was performed using the “basic variant detection” function, and genetic alterations including single-nucleotide variants (SNVs) and small insertions and deletions (INDELs) were detected. Variants with coverage below 10 and frequency lower than 35% were discarded. The variant calls of each isolate were combined and the nucleotide positions were determined. The consensus sequence of each isolate was aligned to determine substitutions at specific positions and single-nucleotide polymorphisms (SNPs) were identified. The trees were constructed using maximum-likelihood method under the Jukes–Cantor  algorithms using the Long Read Support plugin . The Wilcoxon signed rank test was used to compare two groups of non-parametric distribution. Maximum-likelihood phylogenies of SR- and LR-NGS, generated using CLC Genomics Workbench, were exported into R software in Newick format for further analysis. The ic trees . For com package . The tan package .VRE isolates were subjected to SR-NGS and QC was performed using FastQC. The FastQC results indicated that a total of 5,149,064 reads were generated per run, and were of high quality . The mean read length of each isolate ranged from a minimum of 136 bases to a maximum of 147 bases Table 1.E. faecium, and five isolates had a noticeably lower depth of coverage than the others. Two of those five isolates were E. faecalis, consistent with the results obtained by MicroScan WalkAway prior to DNA extraction. However, for the other three isolates, discordance was observed between the sequencing results and MicroScan WalkAway identifications. While all three isolates were confirmed to be E. faecium by biochemical tests, E. faecalis and E. casseliflavus were present in two of them according to the WIMP analysis performed using EPI2ME. The remaining species was identified as Bacillus spp. and thus excluded from the downstream analysis.Following quality filtering and length trimming, the median ratio of trimmed short reads to raw reads was 99.72, compared to 79.8 for the long reads (data not shown). The preprocessed reads of 20 isolates were assembled using the reference sequence of E. faecium (VREfm) isolates mapped to the reference genome were subjected to variant calling using CLC Genomics. For comparative analysis of the SNVs generated by the short- and long-read sequencing platforms, a nonparametric test was performed to assess differences in the number of SNVs; the results are plotted in 10−6; data not shown).Fifteen vancomycin-resistant The SNPs between the two VREfm isolates were identified and visualized using a heatmap. As shown in Various types of AMR  were evaluated based on the presence of AMR genes and resistance-inducing point mutations in the assembled contigs of short reads and corrected long reads. No point mutation leading to AMR was found, but several AMR genes were detected. For the representative antibiotics, the phenotypic resistance determined by MIC and genotypic resistance predicted by SR- and LR-NGS were compared. The representative antibiotics refer to vancomycin, teicoplanin, aminoglycoside, tetracycline, and daptomycin.van gene clusters; vanA, vanB, and vanC were the three most prevalent van genes in VRE isolates. A vanA cluster consisting of seven genes (vanRSHAXYZ) was detected in 13 isolates using SR-NGS, and in 15 isolates using LR-NGS. A vanB gene cluster consisting of seven genes (vanRSYWHBX) and vanC cluster consisting of five genes (vanC(XY)TRS) were detected in a single sample by both SR- and LR-NGS. The type of vancomycin resistance was confirmed using the MicroScan WalkAway system followed by PCR. The results were compared to those of the NGS vancomycin resistance profiling. Although the vancomycin resistance revealed by LR-NGS accorded with the PCR genotyping results, two vanA isolates were classified as vancomycin susceptible by SR-NGS -Ii, which belongs to the aminoglycoside acetyltransferase (AAC) family of AMEs, in ten and 13 isolates, respectively; AAC(6’)-Ii was found in nine of the isolates by both methods. The aad(6) aminoglycoside nucleotidyltransferase (ANT) gene, which confers resistance to high-level streptomycin, was found in two isolates by both SR- and LR-NGS, while the ANT(9)-Ia gene was found in four and six isolates, respectively. According to both SR- and LR-NGS, six isolates harbored the APH(3’)-IIIa gene, which encodes an aminoglycoside phosphotransferase (APH); five of those isolates were common to both methods. Finally, the AAC(6’)-Ie-APH(2’’)-Ia gene, which encodes a bifunctional enzyme acting on all aminoglycosides except streptomycin, was detected by SR- and LR-NGS in five and six isolates, respectively; four of those isolates were common to both methods. Each isolate was considered to be aminoglycoside resistant if genes encoding aminoglycoside-modifying enzymes (AMEs) were detected. SR- and LR-NGS AMR profiling revealed Phenotypic resistance of high-level gentamicin and streptomycin was determined by MIC breakpoints of > 500 and > 1,000 μg/mL, respectively. All nine isolates that were phenotypically resistant to gentamicin and streptomycin harbored at least one AME gene according to LR-NGS. However, no AME gene was detected by SR-NGS in one gentamicin-resistant isolate, or in an isolate resistant to both gentamicin and streptomycin. Concordance between phenotypic and genotypic resistance was observed for seven of the nine VRE isolates. Every isolate that showed the phenotypic resistance to gentamicin, streptomycin, or both were also identified as aminoglycoside-resistant strains by LR-NGS  gene was found in one sample by LR-NGS. The msrC gene, which encodes an ABC-F ribosomal protection protein that confers resistance to macrolide and streptogramin B, was detected in four and three isolates by SR- and LR-NGS, respectively; one sample was common to both methods. The lsaA gene, which belongs to the ABC-F subfamily and confers resistance to lincosamide and streptogramin A (Sa), and the vatD gene  were detected in one sample by LR-NGS. In total, 17 and 18 isolates were shown to be MLS resistant by SR- and LR-NGS, respectively .The E. faecium) ampicillin, as well as clindamycin and low-level aminoglycosides. They also have the capacity to acquire and disseminate genetic determinants of AMR such as vancomycin, erythromycin, and high-level aminoglycosides , variant calling , and strain typing and AMR detection . In this study, the steps were evaluated using clinical VRE isolates, and the results were compared between SR- and LR-NGS. For strain typing, we performed reference-based assemblies to analyze the SNPs of VRE isolates and constructed SNP-based phylogenies using a maximum-likelihood algorithm. SR-NGS detected significantly more SNPs than LR-NGS Figure 2sistance , but thesistance . Moreovehttps://www.ncbi.nlm.nih.gov/, PRJNA798443.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: SO and KP conceived and planned the experiments. HC and SN performed DNA extraction and next-generation sequencing. SO, KP, and HC analyzed the data and interpreted the results. SO performed the statistical analysis and wrote the manuscript. SO, KP, HC, and SN contributed to the final version of the manuscript and approved the submitted version.This work was supported by the research fund No. 2019-ER5402-01 from the Korea Disease Control and Prevention Agency.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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The US Food and Drug Administration (FDA) recently restricted characterizing flavors in tobacco products. As a result, ice hybrid–flavored e-cigarettes, which combine a cooling flavor with fruit or other flavors , emerged on the market. Like menthol, ice-flavored e-cigarettes produce a cooling sensory experience. It is unclear if ice hybrid–flavored e-cigarettes should be considered characterizing flavors or menthol, limiting regulatory action. Monitoring the public’s conversations about ice-flavored e-cigarettes on Twitter may help inform the tobacco control community about these products and contribute to the US FDA policy targets in the future.This study documented the themes pertaining to vaping and ice flavor–related conversations on Twitter. Our goal was to identify key conversation trends and ascertain users’ recent experiences with ice-flavored e-cigarette products.Posts containing vaping-related  and ice-related  terms were collected from Twitter’s streaming application programming interface from January 1 to July 21, 2021. After removing retweets, a random sample of posts (N=2001) was selected, with 590 posts included in the content analysis. Themes were developed through an inductive approach. Theme co-occurrence was also examined.Many of the 590 posts were marked as (or consisted of) marketing material , contained positive personal testimonials , and mentioned disposable pods . Other themes had relatively low prevalence in the sample: neutral personal testimonials , cannabidiol products , negative personal testimonials , “official” flavor description , ice-flavored JUUL , information seeking , and comparison to combustible tobacco . The most common co-occurring themes in a single tweet were related to marketing and disposable pods .Our findings offer insight into the public’s experience with and understanding of ice-flavored e-cigarette products. Ice-flavored e-cigarette products are actively marketed on Twitter, and the messages about them are positive. Public health education campaigns on the harms of flavored e-cigarettes may help to reduce positive social norms about ice-flavored products. Future studies should evaluate the relationship between exposure to personal testimonials of ice-flavored vaping products and curiosity, harm perceptions, and experimentation with these products among priority populations. The use of e-cigarettes, or vaping, among adolescents and young adults is a public health concern ,2. One fIce hybrid–flavored products may contain menthol. Menthol is a flavor additive that produces pleasant cooling sensations and analgesic effects in the throat and mouth, which reduces the harshness of combustible smoke and nicotine’s irritating effects on the airways ,12. ReseOne approach that may be helpful in informing regulatory efforts is the tracking and analyzing of real-world conversations and depictions of novel tobacco products on Twitter . MonitorThis study used Twitter data to document the themes in posts pertaining to ice flavors. Examining and monitoring the public’s conversations about ice-flavored e-cigarettes may help inform the tobacco control community about these products and uncover trends, knowledge, and attitudes toward e-cigarette flavors. Findings from this study may contribute to the US FDA policy targets and tobacco control campaigns in the future.Twitter posts containing both vaping-related  tweets that were included in the content analysis.personal testimonials , marketing, official  flavor description, disposable pod devices, JUUL, cannabidiol (CBD) products, and other. A tweet could be classified into multiple themes. To establish interrater reliability, the same subsample of posts  were double coded, with percent agreement ranging from 99.2% to 100% and Cohen κ ranging from 0.90 to 1.00. JPA served as an arbitrator resolving discrepancies between the coders.The codebook consists of the following themes: personal testimonials consisted of positive, negative, and neutral testimonies, personal testimonials theme, and only includes its components: positive, negative, and neutral testimonies. The other category was also excluded since it had few co-occurrences with other themes.Descriptive analyses were conducted first to show the prevalence of each theme. Further, pairwise co-occurrence analyses were used to evaluate connections across themes, since tweets often included more than one theme. R base package  was used to construct the co-occurrence matrix, and the igraph package (version 1.2.6) was used to visualize the results. Since the theme related to All Twitter posts in this data set were publicly available and anonymized, and all analyses adhered to the terms and conditions, terms of use, and privacy policies of Twitter. To further protect privacy, posts exemplifying themes were paraphrased; no tweets are reported verbatim.The protocol was approved by the university’s institutional review board (protocol HS-18-00697).marketing  of ice-flavored products and personal testimonials . Mentions of using ice-flavored disposable pod devices  was also a common theme. Other themes had relatively low prevalence in the sample: “ice-flavored” CBD products , official flavor description , and ice-flavored JUUL . Positive sentiment  was the most prevalent theme among personal testimonials, while the other testimonial themes were less common: neutral sentiment , negative sentiment , information seeking , and comparison to combustible tobacco .Descriptive characteristics of the 590 tweets from this corpus and example paraphrased posts are presented in marketing, positive sentiment, and disposable pod devices. The marketing of ice-flavored disposable pod devices was mentioned in 73 (12.4%) of the 590 tweets; the marketing of ice-flavored CBD products was mentioned in 40 (6.8%) of the tweets; the marketing of ice-flavored products and official flavor descriptions was mentioned in 37 (6.3%) of the tweets. Positive testimonies about disposable devices were observed in 31 (5.3%) of the tweets, and conversations about disposable devices containing CBD were observed in 26 (4.4%) of the tweets. Pairwise co-occurrences of the remaining themes were found in 2% of the tweets  and refillable  e-cigarette products . These dPersonal testimonials was another common theme in this study, while positive sentiment was the most common category among testimonials. These findings are consistent with previous studies showing that Twitter users are exposed to positive messages about vaping and tobacco [ tobacco ,26. Posi tobacco . Given t tobacco ,26. For This study was limited to the analysis of discussions on ice-flavored e-cigarette products and may not pertain to other e-cigarette flavors. In addition, relatively few  tweets among all identified ice tweets were analyzed , which limits the generalizability of our results. Moreover, the word “cool” was one of the four terms used to identify ice-flavored e-cigarette posts. “Cool” is a colloquialism and is commonly used to express the enjoyment of someone or something. The inclusion of this search term resulted in a collection of many posts that were unrelated to e-cigarette flavors; nonetheless, we found this limitation unavoidable and worked diligently to analyze pertinent tweets. This study focused on the text of the Twitter posts but did not code website links or accompanying images. Previous studies demonstrated that there is value in examining both text and image. In other words, it is possible that additional themes would have emerged had we coded images. Findings may not generalize to other time periods or other social media platforms. Our findings may not extend to all Twitter users or to the population of the United States.Our findings may offer valuable insights into the public’s experience with and understanding of ice-flavored e-cigarette products. In this study, we found that Twitter discussions about ice-flavored e-cigarette products focused on marketing, personal testimonials, and ice-flavored disposable pod devices. Future studies should evaluate the relationship between exposure to personal testimonials of ice-flavored vaping products and curiosity, harm perceptions, and experimentation with these products among priority populations. Public health education campaigns informing the public on the harms of flavored e-cigarettes may be helpful in reducing positive social norms about ice-flavored products, while flavor regulatory actions may include ice flavors to prevent new products from circumventing current regulations. For instance, banning tobacco marketing posts on Twitter may reduce the exposure to ice-flavored e-cigarette product promotions. In addition, social media might be more influential than traditional marketing because participants can be actively engaged in the content they are viewing ,22. Clos


Thus, we investigated the role of gut microbial dysbiosis in CMI-induced depression-like behavior. Hierarchical cluster analysis of sucrose preference test (SPT) results was adopted to classify the CMI rats into depression-like behavior (CMI + Dep) or non-depression-like behavior (CMI + Non-Dep) phenotypes. First, 16S ribosomal RNA sequencing analysis showed both β-diversity and relative abundance of several gut bacteria significantly differed between the CMI + Dep and CMI + Non-Dep rats. Next, transplantation of fecal microbiota from CMI + Dep rats visibly altered the relative abundance of gut microbiota and also induced depression-like behavior in the antibiotics-treated pseudo-germ-free rats. In conclusion, these findings suggested that dysbiosis in gut microbial composition contributed to the onset of CMI-induced depression-like behavior and that exogenous regulation of gut microbiota composition could be a potential therapeutic strategy for CMI and related depression-like behavior.Depression is common among patients who have chronic myocardial infarction (CMI). Despite their frequency, depression and CMI are bidirectional related conditions, each is a risk for the other, and they often co-exist, suggesting shared or interacting pathomechanisms. Accumulating data revealed the effects of gut microbiota in terms of regulating depression  With the optimization of evidence-based treatments in modern society, the mortality of myocardial infarction significantly decreased . HoweverRecently, gut microbiota is confirmed to strongly affect brain function and the cardiovascular system . BidirecIn the classical model of depression, behavioral changes of rats included anhedonia, behavioral despair, and psychomotor retardation . Anhedonad libitum access to food and water. Animals were allowed to acclimate for 7 days before experiments. All experimental protocols and animal handling procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health . This study was ethically approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University .One hundred male SD rats  were purchased from Hunan SJA Laboratory Animal Co., Ltd., . Animals were housed under controlled temperature (22 ± 2°C), controlled relative humidity (60 ± 5%), and a 12-h/12-h light/dark cycle with This study was divided into two parts. The first part of the study was designed to compare the difference of the gut microbiota between the Sham group, CMI + Dep group, and CMI + Non-Dep group. Fifty male SD rats were randomly used to establish the CMI models and 10 to the sham models. In the second part, 30 male SD rats were randomly used to establish pseudo-germ-free rat models and 10 to the control group. Then fecal microbiota transplantation was performed on the 30 pseudo-germ-free rats. The fecal microbiota for transplantation was collected from the CMI + Dep group and CMI + Non-Dep group rats. Detailed experimental schedule is shown in Left anterior descending coronary artery ligation was performed to produce a CMI rat model . The ratCardiac function was evaluated by using echocardiography . Under lAfter all the experiments were done, rats were sacrificed, and their hearts were repeatedly rinsed with phosphate-buffered saline (PBS) buffer solution to remove the redundant tissues such as right ventricle, right atrium, and great vessels. Ventricular muscle tissues were taken and fixed with 4% paraformaldehyde solution. Samples were alcohol-dehydrated and paraffin-embedded, sections were made, and Masson staining was performed. Masson stain blue was used for collagen fibers and red for cardiomyocytes. The experimental process is shown in Depression-like behavior was evaluated by SPT and FST. All tests were conducted individually, in the morning (between 9 and 11 a.m.). The detailed schedule is shown in This test consisted of a two-bottle procedure in which rats were given the choice between water and 1% sucrose solution. Rats were habituated to drink water from two bottles during days 1 and 2. At day 3, rats were deprived of water and food. At day 4, rats were exposed to two identical bottles again: one was water, and another was 1% sucrose solution. The bottles containing water and sucrose were weighed before and at the end of this period. The sucrose preference [sucrose consumption/(sucrose consumption + water consumption)] was used as a measure for depression-like behavior . The liqIn FST, rats were forced to swim under inescapable conditions. Initially, rats tried to escape but eventually adopted a static posture in which they make only the movements necessary to maintain their head above water. The immobility time was used as a measure for depression-like behavior. Specifically, all rats were placed individually in a clear plastic cylinder  containing 20 cm of water, maintained at 23 ± 1°C. Rats were forced to swim for five consecutive minutes, and immobility time was recorded .ad libitum to the pseudo-germ-free group rats for 14 consecutive days. At the same time, the control group rats were administered to drinking water ad libitum. All drinking water was sterile and renewed every day.Forty male SD rats were acclimated to a new environment for 7 days. Thirty rats were randomly chosen to perform the pseudo-germ-free rat models and 10 to the control group. Broad-spectrum antibiotics  were dissolved in drinking water and administered After finishing the behavioral tests, feces from each group of rats were collected individually. All rats were separately placed in a clean cage containing sterilized filter paper. Fecal samples were collected immediately after defecation in a sterilized centrifuge tube. The filter paper was replaced for each rat. Feces were stored in a −80°C freezer until analysis or transplantation .Fecal microbiota was prepared by diluting 1 g of fecal sample obtained from CMI + Dep group rats in 10 ml of sterile saline. The fecal material was suspended, and 0.2 ml of the suspension was administered by gavage to each recipient pseudo-germ-free rats for 14 consecutive days . Fecal s16S rRNA gene is mainly composed of defensive zone and variable zone. The conserved region represents the affinity between the microbiome, and the variable region represents the difference. Among them, V3–V4 had good specificity and complete database information . In viewReceiver operating characteristic (ROC) curves illustrated the diagnostic ability of a binary classifier system with the true positive rate (sensitivity) as the ordinate and the false positive rate (1-specificity) as the abscissa. The ROC curves were used to distinguish rats with CMI-induced depression-like behavior from all other rats. The value of the area under the curve (AUC) represents the accuracy of the diagnosis.post hoc Tukey’s tests for pair-wise comparisons. A p < 0.05 (two-tailed) was considered significant for all tests.Data was presented as the mean ± standard error of the mean (SEM). All statistical analyses were performed using GraphPad Prism 8 . For the hierarchical cluster analysis of SPT results to define CMI + Dep and CMI + Non-Dep groups, the data were first standardized as Z scores. Hierarchical cluster analysis was performed using Ward’s method and applying squared Euclidean distance as the distance measure. Correlation analysis was conducted using Pearson’s product-moment coefficient. The diagnostic cutoff value, sensitivity, specificity, and accuracy of each bacterium were determined using ROC curve analysis. Group means were compared by one-way analysis of variance (ANOVA), followed by p < 0.05; p < 0.05; p < 0.05; Echocardiography revealed that LVEF and %FS were both significantly greater in Sham rats than in CMI rats  significantly differed among the Sham group, the CMI + Dep group, and the CMI + Non-Dep group . At the Alphaproteobacteria, order Rhodospirillales, family Rs-E47_termite_group, and genus Roseburia negatively influenced depression-like behavior in rats (Sucrose preference test and FST were adopted as measures to evaluate the effects of gut microbiota composition on depression-like behaviors. Correlations between the sucrose preference percentage of the CMI + Dep group and the CMI + Non-Dep group and the relative abundance of eight bacteria were analyzed. Correlations between the immobility time and the relative abundance of eight bacteria were also analyzed. Although the results were not statistically significant, they showed clear trends in the influence of gut microbiota on depression-like behaviors. The relative abundance of class  in rats .Receiver operating characteristic curves were constructed to evaluate the diagnostic ability of the gut bacteria in depression . The besp < 0.05, Pseudo-germ-free models were established by administering antibiotics for 14 days. Gut microbiota from CMI + Dep group and CMI + Non-Dep group rats were transplanted into the gastrointestinal tract of pseudo-germ-free rats through feces for another 14 consecutive days . FST andp < 0.05, 16S rRNA gene sequencing result showed that the number of OTUs ranged from 688 to 1,135. A flower plot showed that gut microbiota in the four groups were distinct . There wGemmatimonadetes, the other orders, the family Rikenellaceae, the family Bacteroidales RF16 group, the other families, the genus Rikenellaceae_RC9_gut_group, and the genus Ruminococcus_1 significantly increased in Dep group (Streptococcaceae and the species Lactococcus_garvieae_subsp._garvieae decreased in Dep group (Fecal transplantation significantly altered the relative abundances of gut microbiota in Dep and Non-Dep rats. At six levels, a total of 25 gut bacteria in the fecal samples had substantial differences among control rats and rats receiving vehicle, Dep rats fecal bacteria, or Non-Dep rats fecal bacteria . Compareep group , whereasep group .Left anterior descending coronary artery ligation has been commonly used to create myocardial infarction models . In the Gut microbiota diversity has been strongly associated with mood-relating behaviors, such as depression . AccumulIn the present results, gut microbiota was significantly altered in the CMI + Dep or CMI + Non-Dep rats compared to sham-operated rats, suggesting a role of gut microbiota in the individual differences of depression-like phenotype. Furthermore, transplantation of fecal microbiota from CMI + Dep rats induced depression-like behavior in the antibiotics-treated pseudo-germ-free rats. However, transplantation of fecal microbiota from Non-Dep rats showed no tendency to depression-like behavior in the antibiotic-treated pseudo-germ-free rats, compared to the control group. Distinct gut microbial compositions were identified following fecal microbiota transplant. Taken all together, these results suggest that change in gut microbiota played an important role in the depression-like phenotypes in rats with CMI.Streptococcus accounted for a large amount of deaths worldwide through diverse disease manifestations. Cardiac injury and life-threatening acute cardiac complications are more common in Streptococcus infection, compared to other bacterial infections (Streptococcus was recently reported to be higher in depression patients compared to healthy people (Streptococcus and the genus Streptococcus were enriched in the fecal samples of Dep rats. Therefore, Streptococcus infection was possibly associated with the onset and symptom of depression-like behavior in CMI rats.fections . Intriguy people . This reRikenellaceae, Bacteroidales_RF16_group, Ruminococcus_1, and Lactococcus_garvieae_subsp._garvieae. High abundance in Rikenellaceae, Bacteroidales_RF16_group, and Ruminococcus_1 was observed in antibiotics-treated pseudo-germ-free rats treated with fecal microbiota from CMI + Dep rats. In contrast, Lactococcus_garvieae_subsp._garvieae observably decreased in antibiotics-treated pseudo-germ-free rats treated with CMI + Dep rats’ fecal microbiota, compared to the other groups. Although no study has reported on the role of these gut microbiota in depression, there were similar trends in Bacteroidales and Lactococcus in depression patients. Ma et al. found that the alterations of Ruminococcus were closely related to abnormalities of depression symptoms and inflammatory cytokines. Rikenellaceae correlated with butyric and valeric acids (Rikenellaceae, Bacteroidales, Lactococcus, and/or Ruminococcus in depressed patients with CMI. Collectively, gut microbiota may play a role in these behavioral abnormalities in CMI rats.Our study revealed significant inconsistencies in the abundance of ic acids , and shoic acids . NonetheThere were some limitations in our study. First, we did not investigate the influence of sex difference in gut microbiota composition in CMI-related depression, although the previous studies showed a gender-dependent difference in the gut microbiota composition . SecondlIn conclusion, the CMI-induced depression-like behavior was notably associated with dysbiosis in gut microbiota, and change in specific gut bacteria may be involved in the pathogenesis of this comorbidity. In light of the links between depression and CMI, the regulation of gut microbiota may be a potential therapeutic target for depression. However, further detailed studies are clearly needed to validate the role of gut microbiota in the pathogenesis and therapeutic mechanisms of the depression-like behavior in CMI patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Renmin Hospital of Wuhan University.All authors critically reviewed and approved the final version for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this Research Topic, the multiple aspects of this dilemma are examined from different angles by researchers across a range of disciplines.Nanoparticles (NPs) are on the verge of being established as a mainstay of modern medicine. Currently, numerous synthetic nanomaterials derived from various organic or inorganic materials such as lipids, proteins, synthetic/natural polymer, and metals are available for therapeutic and diagnostic purposes. Due to their structural versatility, storage stability, and ease of functionalization, NPs have extensive fields of applications spanning from environmental science to medicine. In medicine, NPs are incessantly being improved for drug delivery, tissue engineering, and diseases diagnosis, just to name a few. However, Stanisic et al. present a cost-effective, bio-based synthesis of antiviral silver NPs and 3D flexible nanostructure composite materials, which effectively suppressed infection of fibroblasts with a murine coronavirus as a proxy for SARS-CoV-2. Furthermore, the authors present an innovative approach to tracing coronavirus infection in vitro utilizing high-resolution magic-angle spinning NMR spectroscopy.The global outbreak of SARS-CoV2 has held us hostage for the past 2 years. A novel class of NP-based mRNA vaccines has emerged as a highly effective weapon in the fight against the virus. . Howevere.g. for the isolation of novel active compounds or the production of therapeutic extracts  sativa, an annual blooming herb rich in thymoquinone (It is believed that plants were used for therapeutic purposes for at least 60.000 years, based on fossil records . In modeextracts . Medicaloquinone . NanosusJeitler et al. aimed at optimizing the composition of lipid-structured NPs in order to enhance their efficiency safety and uptake by buccal epithelial cells. By careful evaluation of NP-to-cell interactions, they identified caveolin-mediated endocytosis as a major uptake route. These findings might guide prospective biopharmaceutical studies.NPs can specifically be designed to overcome several limitations of free therapeutics. In terms of targeted delivery, they offer immense potential to bypass biological barriers . The oraNoubissi Nzeteu et al. recapitulates in depth NP-based immunotherapy for pancreatic cancer and provides an outlook on future treatment possibilities.Nanomaterials have been used extensively for the development of novel cancer therapeutics . Particuin vivo studies, accelerating the introduction of nanomedicine into clinical practice.In conclusion, this collection of related articles highlights novel strategies to understand the physiological and pathological properties of NPs. A comprehensive understanding of NP behavior in clinical settings will eventually be obtained from cell-based and


Necroptosis is a regulated form of cell necroptotic process, playing a pivotal role in tumors. In renal cell cancer (RCC), inhibiting necroptosis could promote the proliferation of tumor cells. However, the molecular mechanisms and prognosis prediction of necroptotic-process-related genes in RCC are still unclear. In this study, we first identified the necroptotic process prognosis-related genes (NPRGss) by analyzing the kidney renal clear cell carcinoma (KIRC) data in The Cancer Genome Atlas . We systematically analyzed the expression alteration, clinical relevance, and molecular mechanisms of NPRGss in renal clear cell carcinoma. We constructed an NPRGs risk signature utilizing the least absolute shrinkage and selection operator (LASSO) Cox regression analysis on the basis of the expression of seven NPRGss. We discovered that the overall survival (OS) of KIRC patients differed significantly in high- or low-NPRGs-risk groups. The univariate/multivariate Cox regression revealed that the NPRGs risk signature was an independent prognosis factor in RCC. The gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were used to explore the molecular mechanisms of NPRGss. Immune-/metabolism-related pathways showed differential enrichment in high-/low-NPRGs-risk groups. The E-MTAB-1980, TCGA-KIRP, GSE78220, the cohort of Alexandra et al., and IMvigor210 cohort datasets were respectively used as independent validation cohorts of NPRGs risk signature. The patients in high- or low-NPRGs-risk groups showed different drug sensitivity, immune checkpoint expression, and immune therapy response. Finally, we established a nomogram based on the NPRGs risk signature, stage, grade, and age for eventual clinical translation; the nomogram possesses an accurate and stable prediction effect. The signature could predict patients’ prognosis and therapy response, which provides the foundation for further clinical therapeutic strategies for RCC patients. As the third most commonly diagnosed urological cancer, renal cell carcinoma (RCC) causes over 179,000 deaths every year worldwide . Severalvia a series of compounds, drugs, and agents inducing or manipulating the necroptosis-related pathway has also emerged as a novel approach for bypassing apoptosis resistance and supporting antitumor immunity in cancer therapy. Necroptosis is considered to play an important role in renal carcinoma cells. Necroptosis resistance has been reported to promote the proliferation of renal carcinoma cells (Necroptosis is a tightly regulated cell death (RCD) mimicking the morphological features of necrosis , which ima cells . High exma cells . ExploriThis study identified necroptotic process prognosis-related genes (NPRGss) and explored the molecular mechanism and clinical relevance of these genes in KIRC. Although previous research has constructed the necroptosis-related genes risk signature in KIRC, these models still have limitations in predicting prognosis and guiding clinical treatment . Herein,https://www.ebi.ac.uk/QuickGO/) (https://xena.ucsc.edu/) and Array Express database (https://www.ebi.ac.uk/arrayexpress). The TCGA-KIRC includes 535 tumors and 72 normal samples. E-MTAB-1980 dataset includes 101 RCC samples with exactly clinical survival information. The TCGA-KIRP includes 285 tumor samples. The TCGA-KIRC and TCGA-KIRP mRNA expression value was normalized and transformed to a CPM unit utilizing the edgeR packages. Furthermore, patients’ clinical information from the TCGA-KIRC and E-MTAB-1980 were respectively collected for subsequent analysis.To identify necroptotic-process-related genes, we referred to high-quality articles  and a puuickGO/) . Thirty-Then, we downloaded three external cohorts with immune therapy, including GSE78220 , IMvigorhttps://www.gencodegenes.org/human/). The highly conserved microRNA family data were downloaded from the miRcode database to conduct microRNA and lncRNA target prediction (http://www.mircode.org/).The lncRNA annotation data were obtained from the Gencode database (limma R package downloaded from the Bioconductor (https://www.bioconductor.org/). We have adjusted the p-value by the Benjamini–Hochberg method to control the false discovery rate (FDR). The gene with an adjusted p-value (FDR)<0.05 and a |log2(fold change)| > 0.5 was regarded as DEG. To determine the NPRGss, we matched the TCGA-KIRC clinical information and mRNA expression data of genes. Then, we conducted the univariate Cox progression analysis by utilizing the coin R-package. The prediction performance was evaluated using the hazard regression model. We further used the VennDiagram R-package to depict a Venn diagram to represent the overlapping genes between the DEGs and prognosis-related genes. These overlapping genes were defined as NPRGss. A p-value<0.05 was considered statistically significant difference. To validate the expression alteration of NPRGs, we downloaded the immunohistochemistry data from the Human Protein Atlas (HPA) database (https://www.proteinatlas.org/) for further validation. This database currently contains 44 human tissue protein data, and the protein data cover 15,323 genes for which there are available antibodies. In the selection of protein immunohistochemistry, we selected data from the same antibody and tried to select the same patient on this basis.The differential expression genes (DEGs) between two groups were screened using the survminer and survival R-package. The ideal cutoff point of NPRGs expression was determined by X-tile software , and then, the protein–protein interaction (PPI) network was visualized by the Cytoscape software.The Pearson correlation coefficient (PCC) among genes was calculated by applying the https://www.gsea-msigdb.org/gsea/index.jsp). The TCGA-KIRC expression dataset was subjected to the gene set variation analysis (GSVA) to calculate the activity score of different terms. The relationship between the NPRGss expression and activity scores was estimated by calculating the Pearson correlation coefficient (PCC). The |PCC| > 0.3 and p-value< 0.05 were considered moderately correlated. The |PCC| > 0.6 and p-value< 0.05 were considered as highly correlated.The dataset of the cancer-related hallmark pathways, cell components, biological process, molecular functions, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were downloaded from the GSEA website  regression analysis was carried out to rule out genes with an overfitting tendency and construct a prognosis-related signature with the glmnet R-package. In the TCGA training cohort, the characteristic gene signatures were established by the LASSO-Cox regression analysis. The optimal penalty parameter l that correlated with the minimum 10-fold cross-validation was selected to screen the signatures. The lambda value with the minimum mean square error was used to reduce the prediction error of the model. Then, we used the coefficients obtained from the LASSO-Cox regression algorithm and gene expression level to yield a risk score. The risk-score equation was shown as follows:The patients were randomly divided into the training and testing cohorts with a ratio of 1:1 using the “Meanwhile, we also use the TCGA-KIRC testing cohort, E-MTAB-1980 cohort, and TCGA-KIRP cohort to verify the risk model. Receiver operating characteristic (ROC) curve analyses were performed to estimate the accuracy of models by employing the survivalROC R-package. Kaplan–Meier survival curve demonstrated the prognosis difference between high- and low-risk cohorts. The univariate and multivariate Cox regression analyses were carried out for risk scores and clinical variables. The variables with p<0.05 in both univariate and multivariate Cox regressions were considered independent risk factors.clusterProfiler R-package to conduct a pre-ranked gene set enrichment analysis (GSEA). The top 10 significant enrichment terms of hallmark pathways, KEGG pathways, and Gene Ontology (GO)  were demonstrated.The TCGA-KIRC cohort was divided into high- or low-NPRGs-risk groups based on the median NPRGs risk score. We first calculated the DEGs between the high- and the low-risk group. The gene with |logFC|>0.5 and adjusted p-value was regarded as expressed differently. Then, all DEGs were ranked by logFC and subjected to estimate R-package. The gene sets representing 24 immune cell types across tumors were obtained from the published research  (The DEGs between the high- and the low-NPRGs-risk group were subjected to public drug prediction (y/query) . A rankey/query) . Then, wy/query) . ThroughThe variables  that correlated with the prognosis of patients estimated by univariate Cox regression analysis in TCGA-KIRC, E-MTAB-1980, and TCGA-KIRP cohorts were subjected to multivariate Cox regression analysis. Due to the lack of grade data in TCGA-KIRP, the variables  were used to construct the analysis. The stepwise regression method based on the minimum of Akaike information criterion was utilized to screen variables. Then, we utilized the rms R-package to construct a nomogram to evaluate the probability of 3-/5-/7-year overall survival (OS). The area under the time-dependent receiver operating characteristic curve (time-dependent AUC), concordance index (C-index), and calibration plots were utilized to estimate the discriminative accuracy. The AUC values and C-index >0.7 were regarded as reasonable estimations. Then, we depicted the decision curve analysis (DCA) to estimate the clinical benefits of the nomogram.The unpaired t-test analysis was performed to compare the differences between the two groups. The log-rank test was utilized to evaluate prognosis differences. The chi-square test was used to test the constituent ratio differences between different groups. The univariate/multivariate Cox regression analysis was employed to estimate the relationship between variables and patients’ prognoses. The Pearson method was calculated to evaluate the correlation between the two groups.The specific workflow of this study is shown in To further detect the contribution of NPRGss for KIRC patients, we established an NPRGs signature using the LASSO Cox regression analysis based on OS. The TCGA-KIRC cohort was divided into the TCGA training and testing cohorts at 1:1. Then, a prognostic model was established applying LASSO Cox regression analysis based on the expression values of 12 NPRGss in the TCGA training cohort  among the mRNA expression of these genes was calculated , 24. HenTo further explore the predictive ability of NPRGs risk signature in patients’ prognosis, we stratified the KIRC training cohort into a high- or low-NPRGs-risk groups according to the median risk score. In the training cohort, the number of death increased, accompanied by the increasing risk score  was the independent prognostic factor for TCGA-KIRC patients  is considered an immunogenic tumor . The intPrevious research has indicated the importance of necroptosis-related genes in KIRC and constructed the risk models to predict the prognosis of patients . HoweverAlthough the NPRGs risk signature is more robust for predicting prognosis and immune therapy response, there are several limitations, including the various cohorts with different cancer types or sample numbers, data format, and gene detecting technology. The retrospective collection of clinical data and lack of clinical information of the therapy cohort could cause different prediction discrepancies and instability of the model. Validating our models as an independent predictor of prognosis and therapy response required prospective studies. In addition, the molecular mechanism of NPRGss in KIRC is still unclear, and individual bioinformatic analyses still need further experimental validation.In conclusion, our study highlighted the importance of NPRGss in KIRC and established a novel NPRGs risk signature that was verified to be an independent prognostic factor for OS in KIRC. The signature could be extended to the prognosis prediction of KIRP patients. The NPRGs risk signature can predict immunotherapeutic response, which provides a foundation for developing therapeutic strategies based on NPRGss in KIRC.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Conception and design: JL, XL, ED, ZZ, YL. Development of methodology: JL, XL, YQ. Acquisition of data: JL, XL, YQ, YL. Analysis and interpretation of data : JL, XL, YQ. Writing, review, and/or revision of the manuscript: JL, XL, YQ, ED, ZZ. Administrative, technical, or material support: JL, XL, ED, ZZ, YL. Study supervision: ED, ZZ. All authors contributed to the article and approved the submitted version.This work was supported by The National Natural Science Foundation of China (NO. 22076138) and Tianjin Science and Technology planning project (NO. 21JCYBJC01750).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Actinic keratoses (AKs) are the most common neoplastic lesions and are recognized as a precursor to squamous cell skin cancer. Photodynamic therapy (PDT) is a therapeutic option for multiple AKs in line with field cancerization. The aim of this study was to assess the effectiveness of PDT on patients with AKs using a meta-analysis, in order to evaluate the possible superiority of one treatment over the others. For this purpose, the PubMed, MEDLINE, Scopus, OVID, Science Direct, British Journal of Dermatology, Research Gate, and Embase databases were searched in March 2022. The search terms used were 'photodynamic therapy' and 'actinic keratosis'. We utilized the random-effects meta-analysis model to compare methyl aminolevulinate PDT (MAL-PDT) and the combination of a nanoscale-lipid vesicle formulation with the prodrug 5-aminolevulinic acid (BF-200 ALA) on a complete response (CR) of the lesions. Our meta-analysis indicated that the comparison of BF-200 ALA versus MAL-PDT showed marginally higher CRs than MAL-PDT. According to scientific literature, actinic keratoses (AKs) are clinically observed lesions where dysplastic keratinocytes appear and which precancerous lesions of the skin have the potential to develop into squamous cell carcinomas (SCCs) in the future -2. The rPDT is a modern and effective technique for AK and field cancerization -10. The The scope of this study was to compare the effectiveness of BF-200 ALA with MAL-PDT on patients with AK. More specifically, we compared both therapies using meta-analysis based on scientific literature in order to find out if these therapies show a significant statistical difference.Materials and methodsLiterature Search and Data ExtractionWe searched the following databases: PubMed, MEDLINE, CINAHL, Scopus, OVID, Embase, Science Direct, Research Gate, and British Journal of Dermatology. Studies were included from the last two decades. The search terms were "photodynamic therapy AND actinic keratosis." We included randomized clinical trials, randomized prospective studies, intraindividual randomized trials, and randomized comparison studies involving patients with actinic keratosis and photodynamic therapy such as MAL-PDT therapy, BF-200 ALA, C-PDT, DL-PDT, ablative fractional laser resurfacing (AFXL)-PDT, and MAL-DL-PDT.Data extraction was undertaken separately for each intervention. All relevant information was extracted for each study: first author/year of publication; type of study; type of therapy/number of patients; age; skin phototype, number of lesions treated; and results. The search was also limited to English-language articles. The reference lists of the included articles have also been searched for additional studies.Data Synthesis and AnalysisThe data evaluating the primary and secondary outcomes for each trial was expressed as a relative risk ratio (RR) with a 95% confidence interval (CI). More than two studies were available for the same treatment comparison on lesion complete remission (CR), we summarized RRs and 95% CIs using fixed-effects and random-effects meta-analysis models . The CocResultsThe study has been designed and the results have been reported based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement Figure  21]..21].Our literature search yielded 759 studies. After excluding case reports, systematic reviews, duplicate studies, studies that did not present any relevant data or did not conform to the inclusion or exclusion criteria, and studies that referred to diseases other than actinic keratosis and treatment other than photodynamic therapy, we ended up with 20 studies.We performed one meta-analysis of randomized controlled clinical trials (RCTs) based on comparisons on CR Figure . OverallDiscussionPDT is widely used in clinical practice to cure AK and many other diseases. Between the two prodrugs, BF-200 ALA and MAL, the only licensed form is MAL-PDT . SeveralPost-marketing surveillance trials with a total sample size of n = 4109 observed that the lesion-specific clearance rate for PDT was 74%  56-87%) compared to other treatments such as ingenol mebutate gel, diclofenac sodium, and imiquimod cream . A studyC-PDT is known worldwide as an effective treatment for multiple AKs and in large areas with high response rates and excellent cosmetics -34. LimiThe general characteristics of the studies are summarized in Table This review has some limitations. The types of studies weren’t similar in the meta-analysis we performed. Because AK is a chronic disease, the follow-up period could be considered relatively short. There weren’t many studies using the same treatment protocol for the AKs. Common PDT treatment variations include topical sensitizer, incubation time, light source, exposure time, and lesion preparation. Moreover, some of the studies analyzed were blinded, and thus the other non-blinded studies were susceptible to bias. Another limitation is that all studies had PDT as a common treatment, but not all patients continued the therapy.Studies have shown that all treatments  are effective in patients with AK and can be clinically applied. In this systematic review, our meta-analysis showed that the comparison of BF-200 ALA versus MAL-PDT yielded a marginally higher CR than MAL-PDT. More clinical trials are needed in order to strengthen our results.


Syphilis self-testing (SST) may help expand syphilis test uptake among female sex workers. However, there has been no studies on examining SST among female sex workers. We aim to examine SST experience and its determinants among female sex workers in China.A venue-based, cross-sectional study of female sex workers was conducted in eight Chinese cities in 2019. Participants completed a survey including socio-demographic characteristics, sexual behaviors, and SST history. Multivariable logistic regression was conducted to evaluate the associated factors with SST.p < 0.001), used drugs before or during sex , tested for other sexually transmitted infections (STIs) in the past 6 months , ever tested in the hospital , and ever tested in the community  were more likely to perform syphilis self-testing.Among 1,287 Chinese female sex workers, 72.1%  had ever tested for syphilis, and 5.9%  had ever used syphilis self-testing. Among syphilis self-testers, more than half  reported that the self-test was their first syphilis test, around one-fifth  reported that syphilis self-testing results influenced the price of commercial sex. After adjusting for covariates, female sex workers who received anal sex in the past month (adjusted odds ratio [aOR]: 2.6, 95%CI: 1.5–4.3, Syphilis self-testing has the potential to expand testing coverage, and increase testing frequency with limited potential harms among FSW. Further evaluation on the intervention effects based on syphilis self-testing among FSW are needed. Syphilis continues to be a major public health concern globally, with an estimation of around 6 million new cases of syphilis occurring every year among adults aged between 15 and 49 years in the world , around Currently, syphilis testing among female sex workers mainly depends on facility-based services such as voluntary counseling and testing (VCT) clinics, but existing facility-based syphilis testing and management resources are limited and it highly dependent on active participation . ObviousSyphilis self-testing may help improve test uptake among female sex workers. Syphilis self-testing is the process whereby a person collects his/her own blood, performs the test, and interprets the result themselves . An immuThe purpose of this study was to examine syphilis self-testing experience and its determinants among female sex workers in China.We conducted a venue-based, cross-sectional study in eight cities  in Chinese seven provinces between August 17 and October 17, 2019 . These eWe partnered with eight local community-based organizations (CBO) that have years of experience in sexually transmitted diseases (STD) intervention and outreach for female sex workers, including condom promotion, sexual health education, HIV and syphilis rapid testing and counseling, and linkage to care . Prior to this study, the sampling frame was generated based on a map of commercial sex venues created by local CBO in each study site according to geographic area and type of venues. A convenience sampling method was used to recruit eligible participants.Female sex workers were categorized into two tiers (high- and low-tier) based on the clientele's socioeconomic status . Low-tieAll eligible participants were required to meet the following criteria: born biologically female; aged 18 or above; earn money or goods through commercial sex  at least once in the past 3 months; volunteered to participate in the survey and signed an informed consent.An online survey link was created by using the Wenjuanxing, an e-questionnaire platform , which allowed participants to complete the survey with smartphone or computer tablets. The survey questionnaire and procedures were designed based on discussions with the local CBO stakeholders, policy makers and international HIV/STI experts. A pilot survey among 50 female sex workers was conducted to simplify the survey administration and improve the comprehensibility. The pilot data was not included in the final analysis.In the formal survey, interested participants who clicked on the survey link were required to sign an online informed consent by clicking on a “button” indicating that the participant has read the consent form and agrees to participate. Eligible participants then self-administered the survey, with the assistance of outreach workers if needed. To avoid repeated participation, each participant's mobile phone number was verified with a code. The surveys completed within 5 min were deemed invalid according to our pilot study results. Participants were provided with $2 USD for their time for completing the survey.Syphilis self-testing kits can be accessed on e-commence platforms or through existing HIV self-testing programs in China. More than 10 brands of syphilis self-testing kits are available on the two largest e-commerce platforms in China (Taobao and Jingdong). All the kits used an immune colloidal gold technology to detect Treponema pallidum specific antibodies in the blood, which cannot distinguish between current and past infection. All the kits were approved by the China Food and Drug Administration with an excellent sensitivity and specificity. The cost per test kit ranges from 2·5 to 15 US dollars .We asked about the syphilis self-testing history through the following questions: location where the kit was obtained, whether the individual performed self-testing alone or with a sexual partner, self-test results, post-test healthcare seeking behaviors, subsequent changes in syphilis testing frequency after initial use of a self-test kit, potential harms of self-testing, influences of performing self-testing on sex exchange, and experiences of being pressured to take a syphilis self-test.The survey also asked about: 1) social-demographic characteristics, including: age, marital status, place of residence, annual income, and education level. 2) Sexual behaviors: time of providing commercial sex in the current location, number of cities worked for selling sex, number of clients served in the past month, charge for vaginal sex, whether condoms were used consistently when engaged in commercial sex in the past month, illicit substance use, STI testing history. Consistent condom use was defined as always using a condom during commercial sex.P < 0.05. All analyses were performed using SAS .We conducted a descriptive analysis on sociodemographic information, sexual behavior, and syphilis self-testing history. Univariable and Multivariable logistic regression was conducted to explore associated factors with syphilis self-testing. The variables of age, legal marital status, educational attainment, and annual income were adjusted in the multivariable model. In addition, a sub-analysis was performed to explore the correlates of syphilis self-testing among participants who had ever used HIV self-testing. Statistical significance was defined as This study was approved by the Ethics Committee of the Dermatology Hospital of Southern Medical University (2019017).A total of 1,287 female sex workers completed the survey and were included in the final analyses. Among them, 928 women  had ever tested for syphilis while 76 participants  had ever used syphilis self-testing.Most participants were between 18 and 35 years old , being married , had a junior high school degree , had an annual income between USD $5,001 and $14,000 , being from low tier venues , being employed by a boss , being a resident in the sampling province , and working in the current province for more than 1 year . The average amount of payment received for vaginal sex was USD $20 (IQR: 15–45). A minority of female sex workers used condoms consistently when engaged in vaginal sex , oral sex  and anal sex  with clients in the past month, respectively. Most women reported having never used drugs before or during sex . Over half reported bulk purchasing of condoms , and quite few individuals reported bulk purchasing of syphilis self-testing kits . Almost all women reported having ever received HIV/STD related services in the past year . A total of 1,072 women  had ever tested for HIV, and 103  had ever used HIV self-testing. Around half of individuals reported ever testing for other STDs  in the past 6 months  reported that the self-test was their first ever syphilis testing, and most  reported that the self-testing was undertaken in conjunction with HIV self-testing. About one-third of women  reported an increase in the frequency of syphilis testing after initial use of a self-test kit. A minority of women reported even giving  or selling  self-test kits to their clients. The most common site to obtain a syphilis self-test kits was from a community-based organization , followed by online drug stores  or uncertain  result in their most recent syphilis self-test, and most of them  sought care following syphilis self-testing. The majority  sought care within 2 weeks of self-testing, and three-quarters  sought care either at a specialty sexually transmitted infection (STI) clinic or in a Center for Disease Control and Prevention.Among the 76 syphilis self-testers, a small proportion  reported that syphilis self-testing results influenced the price of sex, and 9 women  reported ever experiencing pressure to undertake syphilis self-testing. The most common types of pressure were physical violence , psychological stress , and threatening to end a relationship  reported some difficulties in performing self-testing, including collecting blood with a tube  or pricking fingers . The most commonly reported motivations for syphilis self-testing were that they wanted to know their infectious status , and they recently had high risk contact . The most common reason for not using self-testing was that they have never heard of syphilis self-testing before , using drugs before or during sex , tested for other sexually transmitted infections in the past 6 months , ever tested in the hospital , and ever tested in the community , ever tested in the hospital , and ever tested in the community . The following two factors were negatively associated with syphilis self-testing: receiving HIV/STD related services , being diagnosed with other STIs . In the formal survey, interested participants who clicked on the survey link were required to sign an informed consent and complete eligibility screening.CW developed the initial concept for the manuscript and drafted an initial manuscript. YW conducted the statistical analysis. XL and BY edited and contributed content to the final draft. All authors have read and approved the final manuscript.This work was supported by Guangdong Medical Research Foundation (A2019402).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.