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Background: The initial step to interpreting putative biological functions from comparative multi-omics studies usually starts from a differential expressed gene list followed by functional enrichment analysis (FEA). However, most FEA packages are designed exclusively for humans and model organisms. Although parasitic protozoan is the most important pathogen in the tropics, no FEA package is available for protozoan functional (ProFun) enrichment analysis. To speed up comparative multi-omics research on parasitic protozoans, we constructed ProFun, a web-based, user-friendly platform for the research community. Methods: ProFun utilizes the Docker container, ShinyProxy, and R Shiny to construct a scalable web service with load-balancing infrastructure. We have integrated a series of visual analytic functions, in-house scripts, and custom-made annotation packages to create three analytical modules for 40 protozoan species: (1) Gene Overlaps; (2) Over-representation Analysis (ORA); (3) Gene Set Enrichment Analysis (GSEA). Results: We have established ProFun, a web server for functional enrichment analysis of differentially expressed genes. FEA becomes as simple as pasting a list of gene IDs into the textbox of our website. Users can customize enrichment parameters and results with just one click. The intuitive web interface and publication-ready charts enable users to reveal meaningful biological events and pinpoint potential targets for further studies. Conclusion: ProFun is the first web application that enables gene functional enrichment analysis of parasitic protozoans. In addition to supporting FEA analysis, ProFun also allows the comparison of FEA results across complicated experimental designs. ProFun is freely available at http://dalek.cgu.edu.tw:8080/app/profun.

Abstract Background Cytomegalovirus (CMV) colitis significantly complicates the course of inflammatory bowel disease (IBD), frequently leading to severe flare-ups and poor outcomes. The role of antiviral therapy in hospitalized IBD patients with CMV colitis is currently under debate. This retrospective analysis seeks to clarify the influence of antiviral treatment on these patients. Methods We retrospectively reviewed IBD patients diagnosed with CMV colitis via immunohistochemistry staining from colonic biopsies at a major tertiary center from January 2000 to May 2021. The study focused on patient demographics, clinical features, risk factors, prognostic indicators, and antiviral treatment outcomes. Results Among 118 inpatients, 42 had CMV colitis. Risk factors included hypoalbuminemia and antibiotic use. IBD patients with CMV colitis receiving

Background: Trichomonas vaginalis is parasitic protozoan that causes human urogenital infections. Accumulated reports indicated that exosomes released by this parasite play a crucial role in transmitting information and substances between cells during host-parasite interactions. Current knowledge on the protein contents in T. vaginalis exosome is mainly generated from three previous studies that used different T. vaginalis isolates as an experimental model. Whether T. vaginalis exosomes comprise a common set of proteins (core exosome proteome) is still unclear. Methods: To explore the core exosome proteome in T. vaginalis, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the contents of sucrose ultracentrifugation-enriched exosome and supernatant fractions isolated from six isolates. Results: Transmission electron microscopy (TEM) confirmed the presence of exosomes in the enriched fraction. Proteomic analysis identified a total of 1870 proteins from exosomal extracts. There were 1207 exosomal-specific proteins after excluding 436 ‘non-core exosomal proteins’. Among these, 72 common exosomal-specific proteins were expressed in all six isolates. Compared with three published T. vaginalis exosome proteome datasets, we identified 16 core exosomal-specific proteins. These core exosomal-specific proteins included tetraspanin (TvTSP1), the classical exosome marker, and proteins mainly involved in catalytic activity and binding such as ribosomal proteins, ras-associated binding (Rab) proteins, and heterotrimeric G proteins. Conclusions: Our study highlighted the importance of using supernatant fraction from exosomal extract as a control to eliminate ‘non-core exosomal proteins’. We compiled a reference core exosome proteome of T. vaginalis, which is essential for developing a fundamental understanding of exosome-mediated cell communication and host-parasite interaction.

Background: Proper identification of the polymicrobial microorganisms in patients with limb-threatening diabetic foot ulcers (LTDFUs) using conventional culture is insufficient. This prospective study evaluates the potential value of adjuvant molecular testing assisting in identify fastidious micro-organisms in LTDFUs compared to standard treatment alone. Methods: Ninety patients with LTDFUs received interdisciplinary and standard antibiotic treatment in a referral diabetic foot center. A simultaneous 16S amplicon sequencing (16S AS) specimen along with conventional culture collected at admission was used to retrospectively evaluate the microbiological findings and its association with amputation outcomes. Results: The microorganism count revealed by 16S AS overwhelmed that of conventional culturing (17 vs. 3 bacteria/ulcer respectively). The Stenotrophomonas spp. revealed in 29 patients were highly correlated with major (above ankle) amputation (OR: 4.76, 95% CI 1.01–22.56), while only one had been concomitantly identified by conventional culturing. Thus, there were 27 cases without proper antibiotics coverage during treatment. Conclusions: Adjuvant molecular testing assisted identification of fastidious pathogens such as Stenotrophomonas infection and might be associated with major amputation in patients with LTDFUs.

The hepatitis delta virus (HDV) is a unique pathogen with significant global health implications, affecting individuals who are coinfected with the hepatitis B virus (HBV). HDV infection has profound clinical consequences, manifesting either as coinfection with HBV, resulting in acute hepatitis and potential liver failure, or as superinfection in chronic HBV cases, substantially increasing the risk of cirrhosis and hepatocellular carcinoma. Given the complex dynamics of HDV infection and the urgent need for advanced research tools, this article introduces vHDvDB 2.0, a comprehensive HDV full-length sequence database. This innovative platform integrates data preprocessing, secondary structure prediction, and epidemiological research tools. The primary goal of vHDvDB 2.0 is to consolidate HDV sequence data into a user-friendly repository, thereby facilitating access for researchers and enhancing the broader scientific understanding of HDV. The significance of this database lies in its potential to streamline HDV research by providing a centralized resource for analyzing viral sequences and exploring genotype-specific characteristics. It will also enable more in-depth research within the HDV sequence domains.

Abstract Background Iron is an essential element for cellular functions, such as energy metabolism. Trichomonas vaginalis, a human urogenital tract pathogen, is capable of surviving in the environment without sufficient iron supplementation. Pseudocysts (cyst-like structures) are an environmentally tolerated stage of this parasite while encountering undesired conditions, including iron deficiency. We previously demonstrated that iron deficiency induces more active glycolysis but a drastic downregulation of hydrogenosomal energy metabolic enzymes. Therefore, the metabolic direction of the end product of glycolysis is still controversial. Methods In the present work, we conducted an LC‒MS-based metabolomics analysis to obtain accurate insights into the enzymatic events of T. vaginalis under iron-depleted (ID) conditions. Results First, we showed the possible digestion of glycogen, cellulose polymerization, and accumulation of raffinose family oligosaccharides (RFOs). Second, a medium-chain fatty acid (MCFA), capric acid, was elevated, whereas most detected C18 fatty acids were reduced significantly. Third, amino acids were mostly reduced, especially alanine, glutamate, and serine. Thirty-three dipeptides showed significant accumulation in ID cells, which was probably associated with the decrease in amino acids. Our results indicated that glycogen was metabolized as the carbon source, and the structural component cellulose was synthesized at same time. The decrease in C18 fatty acids implied possible incorporation in the membranous compartment for pseudocyst formation. The decrease in amino acids accompanied by an increase in dipeptides implied incomplete proteolysis. These enzymatic reactions (alanine dehydrogenase, glutamate dehydrogenase, and threonine dehydratase) were likely involved in ammonia release. Conclusion These findings highlighted the possible glycogen utilization, cellulose biosynthesis, and fatty acid incorporation in pseudocyst formation as well as NO precursor ammonia production induced by iron-depleted stress. Graphical Abstract

BackgroundCompared to antibiotic treatment, fecal microbiota transplantation (FMT) is a more effective treatment for refractory or recurrent CDI (rCDI). Patients with inflammatory bowel disease (IBD) have a higher incidence of CDI and worse outcomes. There has been no study from Asia to evaluate the cost-effectiveness of FMT for overall rCDI patients and rCDI patients with IBD.MethodsWe applied a Markov model with deterministic and probabilistic sensitivity analyses to evaluate the cost and effectiveness of different treatments for rCDI patients with a time horizon of 1 year from the payer's perspective. We compared the cost and clinical outcomes of FMT through colonoscopy to two antibiotics (vancomycin and fidaxomicin) using data from Chang Gung Memorial Hospital, Taoyuan, Taiwan.ResultsCompared to vancomycin, FMT was cost-effective in overall rCDI patients as well as IBD patients with rCDI [USD 39356 (NT$1,101,971.98)/quality-adjusted life year (QALY) gained in overall patients; USD65490 (NT$1,833,719.14)/QALY gained in IBD patients]. Compared to fidaxomicin, FMT was only cost-effective in overall rCDI patients [USD20255 (NT$567,133.45)/QALY gained] but slightly increased QALY (0.0018 QALY gained) in IBD patients with rCDI.ConclusionFMT is cost-effective, compared to vancomycin or fidaxomicin, for the treatment of rCDI in most scenarios from the payers' perspective in Taiwan.

Abstract Background Pseudomonas aeruginosa intestinal carriage rates are significantly higher in immunosuppressed individuals and hospitalized patients who therefore have increased risk of infections and antibiotic-associated diarrhea. To combat intestinal dysbiosis and decolonize P. aeruginosa from gastrointestinal tract, we investigated the anti-adherence and gut microbiota modulation properties of marine prebiotic fucoidans. Methods Proteomic analysis of culture supernatant was performed by LC–MS/MS. Using lectin-based enzyme-linked immunosorbent assay, hemagglutinin domain interaction and inhibition with biomolecules were studied. We investigated the role of nutritional grade fucoidans in a mouse model and used 16S ribosomal RNA sequencing to examine fecal microbiota composition. Results Analysis of culture supernatant proteins indicated the secretion of two-partner secretion (TPS) family proteins, including TpsA1/CdiA2 and TpsA2/CdiA1. Lectin like activity at the N-terminal of TpsA due to a conserved hemagglutinin domain (Pfam identifier [ID] PF05860) mediates binding to mucins that carry multiple fucosylated glycans. Fucose-rich sulfated polysaccharides (fucoidans) and sulfated dextrans were found to be potent inhibitors of the recombinant N-terminal hemagglutinin domain of TpsA (TpsA-NT-HAD) binding to mucins. In a mouse model, antibiotic-induced dysbiosis was essential for P. aeruginosa gastrointestinal colonization. After prophylactic oral fucoidans supplementation, a higher proportion (60%) of the mice were decolonized over time and resisted re-colonization, this was associated with remarkable expansion of Bacteroides (post-infection day-3 abundance, 29–50%) and consequential reductions in bloom of Enterobacteriaceae and Enterococcaceae populations. In the non-supplemented group, Parabacteroides mediated recovery from dysbiosis but failed to decolonize P. aeruginosa. Conclusions Supplementing diet with marine prebiotic fucoidans can mediate earlier recovery from dysbiosis and decolonization of P. aeruginosa from gut by inhibiting secreted virulence factor (TpsA/CdiA) interaction with mucins and promoting the growth of beneficial Bacteroides population. We suggest the prophylactic use of nutritional grade fucoidans to decolonize P. aeruginosa from gastrointestinal tract of at-risk individuals to prevent infection and transmission of colonizing P. aeruginosa.

Background: Millions of people throughout the world suffer from parasite infections. Traditionally, technicians use manual eye inspection of microscopic specimens to perform a parasite examination. However, manual operations have limitations that hinder the ability to obtain precise egg counts and cause inefficient identification of infected parasites on co-infections. The technician requirements for handling a large number of microscopic examinations in countries that have limited medical resources are substantial. We developed the helminth egg analysis platform (HEAP) as a user-friendly microscopic helminth eggs identification and quantification platform to assist medical technicians during parasite infection examination. Methods: Multiple deep learning strategies including SSD (Single Shot MultiBox Detector), U-net, and Faster R-CNN (Faster Region-based Convolutional Neural Network) are integrated to identify the same specimen allowing users to choose the best predictions. An image binning and egg-in-edge algorithm based on pixel density detection was developed to increase the performance. Computers with different operation systems can be gathered to lower the computation time using our easy-to-deploy software architecture. Results: A user-friendly interface is provided to substantially increase the efficiency of manual validation. To adapt to low-cost computers, we architected a distributed computing structure with high flexibilities. Conclusions: HEAP serves not only as a prediction service provider but also as a parasitic egg database of microscopic helminth egg image collection, labeling data and pretrained models. All images and labeling resources are free and accessible at http://heap.cgu.edu.tw. HEAP can also be an ideal education and training resource for helminth egg examination.

Background: Fecal microbiota transplantation (FMT) has been shown to highly effective in the treatment of recurrent or refractory Clostridioides difficile infection (rCDI) in many countries of the world. Not until 2018, Ministry of Health and Welfare, Taiwan approved the application of FMT for rCDI under a special law. The study reported the first implementation of the technology in the medical center in Taiwan and the preliminary outcome. Methods: FMT was used to treat patients with rCDI in Chang Gung Memorial Hospital. FMT was delivered by gastroenterologists using colonoscope. Strict donor screening was performed according to the guidelines. We followed up the clinical course of patients after FMT. 16S rRNA sequencing of fecal samples for donor, and also recipient before and after FMT was carried out. Results: From September 2018 to June 2020, 39 patients with rCDI received FMT, with a successful rate of 89.7%. Two patients died due to causes unrelated to FMT, and two other cases showed no clinical improvement after the procedure. High school and college students showed the best pass rate during donor screening. The presence of multi-drug resistant pathogen was the most common cause for screening failure. We demonstrated in a case the use of rRNA sequencing as a biomarker indicating for the improvement of dysbiosis in a patient after FMT. Conclusions: FMT was successfully implemented in a medical center in Taiwan and showed a comparable successful rate in treating rCDI, compared to other countries. Safety remains the most important issue when applying FMT in the clinical setting.

The pathogenic influences of uterine bacteria on endometrial carcinogenesis remain unclear. The aim of this pilot study was to compare the microbiota composition of endometrial lavage samples obtained from women with either endometrial hyperplasia (EH) or endometrial cancer (EC) versus those with benign uterine conditions. We hypothesized that specific microbiota signatures would distinguish between the two groups, possibly leading to the identification of bacterial species associated with endometrial tumorigenesis. A total of 35 endometrial lavage specimens (EH, n = 18; EC, n = 7; metastatic EC, n = 2; benign endometrial lesions, n = 8) were collected from 32 women who had undergone office hysteroscopy. Microbiota composition was determined by sequencing the V3−V4 region of 16S rRNA genes and results were validated by real-time qPCR in 46 patients with EC/EH and 13 control women. Surprisingly, we found that Bacillus pseudofirmus and Stenotrophomonas rhizophila – two plastic-degrading bacterial species – were over-represented in endometrial lavage specimens collected from patients with EC/EH. Using computational analysis, we found that the functional profile of endometrial microbiota in EC/EH was associated with fatty acid and amino acid metabolism. In summary, our hypothesis-generating data indicate that the plastic-degrading bacteria Bacillus pseudofirmus and Stenotrophomonas rhizophila are over-represented within the endometrial lavage microbiota of women with EC/EH living in Taiwan. Whether this may be related to plastic pollution deserves further investigation.

Abstract Background Chronic obstructive pulmonary disease (COPD) is a progressive, life-threatening lung disease with increasing prevalence and incidence worldwide. Increasing evidence suggests that lung microbiomes might play a physiological role in acute exacerbations of COPD. The objective of this study was to characterize the association of the microbiota and exacerbation risk or airflow limitation in stable COPD patients. Methods The sputum microbiota from 78 COPD outpatients during periods of clinical stability was investigated using 16S rRNA V3-V4 amplicon sequencing. The microbiome profiles were compared between patients with different risks of exacerbation, i.e., the low risk exacerbator (LRE) or high risk exacerbator (HRE) groups, and with different airflow limitation severity, i.e., mild to moderate (FEV1 ≥ 50; PFT I) or severe to very severe (FEV1

Abstract Background Iron plays essential roles in the pathogenesis and proliferation of Trichomonas vaginalis, the causative agent of the most prevalent non-viral human sexually transmitted infection. We previously demonstrated that under iron deficiency, the endogenous nitric oxide (NO) is accumulated and capable of regulating the survival of T. vaginalis. Herein, we aim to explore the influence of NO on the activity of the pyruvate-reducing enzyme lactate dehydrogenase in T. vaginalis (TvLDH). Methods Levels of lactate and pyruvate were detected for determining glycolysis activity in T. vaginalis under iron deficiency. Quantitative PCR was performed to determine the expression of TvLDH. S-nitrosylated (SNO) proteomics was conducted to identify the NO-modified proteins. The activities of glyceraldehyde-3-phosphate dehydrogenase (TvGAPDH) and TvLDH were measured after sodium nitrate treatment. The effects of protein nitrosylation on the production of cellular reducing power were examined by measuring the amount of nicotinamide adenine dinucleotide (NAD) and the ratio of the NAD redox pair (NAD+/NADH). Results We found that although the glycolytic pathway was activated in cells under iron depletion, the level of pyruvate was decreased due to the increased level of TvLDH. By analyzing the SNO proteome of T. vaginalis upon iron deficiency, we found that TvLDH is one of the glycolytic enzymes modified by SNO. The production of pyruvate was significantly reduced after nitrate treatment, indicating that protein nitrosylation accelerated the consumption of pyruvate by increasing TvLDH activity. Nitrate treatment also induced NAD oxidation, suggesting that protein nitrosylation was the key posttranslational modification controlling cellular redox status. Conclusions We demonstrated that NO-mediated protein nitrosylation plays pivotal roles in the regulation of glycolysis, pyruvate metabolism, and the activity of TvLDH. The recycling of oxidized NAD catalyzed by TvLDH provided the reducing power that allowed T. vaginalis to adapt to the iron-deficient environment.

Gut dysbiosis may precede neonatal sepsis, but the association is still not well-understood. The goal of this study is to investigate the association between gut microbiota and neonatal sepsis, and to seek the evidence of colonization of pathogenic bacteria in the gut before evolving into an invasive infection. A prospective cohort study examined fecal microbiota composition in preterm infants with and without sepsis. Thirty-two very-low-birth-weight (VLBW) preterm infants and 10 healthy term infants as controls were enrolled. The fecal samples collected from the participants at the first, fourth, and seventh weeks of life underwent 16S rRNA amplicon sequencing for measurement of the diversity and composition of the microbiota. The bacterial isolates causing neonatal sepsis were genome sequenced. PCR was performed to confirm the translocation of the bacteria from the gut to the blood. The results showed that VLBW preterm infants with sepsis had lower microbial diversity in the gut at birth compared to preterm infants without sepsis and term infants. The composition of gut microbiome in preterm infants was similar to healthy terms at birth but evolved toward dysbiosis with increasing Proteobacteria and decreasing Firmicutes weeks later. The strain-specific PCR confirmed the presence of causative pathogens in the gut in 4 (40%) out of 10 VLBW preterms with sepsis before or at onset of sepsis, and persistence of the colonization for weeks after antibiotic treatment. The same bacterial strain could horizontally spread to cause infection in other infants. Prolonged antibiotic exposure significantly reduced beneficial Bifidobacterium and Lactobacillus in the gut. In conclusion, preterm infants with gut dysbiosis are at risk for neonatal sepsis, and the causative pathogens may be from the gut and persist to spread horizontally. The association of increased Proteobacteria abundance and decrease in microbiome diversity suggests the need for interventions targeting the gut microbiome to prevent dysbiosis and sepsis in VLBW preterm infants.

Our previous clinical trial showed that a novel concentrated herbal extract formula, YH1 (Rhizoma coptidis and Shen-Ling-Bai-Zhu-San), improved blood glucose and lipid control. This pilot observational study investigated whether YH1 affects microbiota, plasma, and fecal bile acid (BA) compositions in ten untreated male patients with type 2 diabetes (T2D), hyperlipidemia, and a body mass index ≥ 23 kg/m2. Stool and plasma samples were collected for microbiome, BA, and biochemical analyses before and after 4 weeks of YH1 therapy. As previous studies found, the glycated albumin, 2-h postprandial glucose, triglycerides, total cholesterol, and low-density lipoprotein cholesterol levels were significantly improved after YH1 treatment. Gut microbiota revealed an increased abundance of the short-chain fatty acid-producing bacteria Anaerostipes and Escherichia/Shigella. Furthermore, YH1 inhibited specific phylotypes of bile salt hydrolase-expressing bacteria, including Parabacteroides, Bifidobacterium, and Bacteroides caccae. Stool tauro-conjugated BA levels increased after YH1 treatment. Plasma total BAs and 7α-hydroxy-4-cholesten-3-one (C4), a BA synthesis indicator, were elevated. The reduced deconjugation of BAs and increased plasma conjugated BAs, especially tauro-conjugated BAs, led to a decreased glyco- to tauro-conjugated BA ratio and reduced unconjugated secondary BAs. These results suggest that YH1 ameliorates T2D and hyperlipidemia by modulating microbiota constituents that alter fecal and plasma BA compositions and promote liver cholesterol-to-BA conversion and glucose homeostasis.

Graves’ disease (GD) is a systemic autoimmune disease characterized by hyperthyroidism. Evidence suggests that alterations to the gut microbiota may be involved in the development of autoimmune disorders. The aim of this study was to characterize the composition of gut microbiota in GD patients. Fecal samples were collected from 55 GD patients and 48 healthy controls. Using 16S rRNA gene amplification and sequencing, the overall bacterial richness and diversity were found to be similar between GD patients and healthy controls. However, principal coordinate analysis and partial least squares-discriminant analysis showed that the overall gut microbiota composition was significantly different (ANOSIM; p < 0.001). The linear discriminant analysis effect size revealed that Firmicutes phylum decreased in GD patients, with a corresponding increase in Bacteroidetes phylum compared to healthy controls. In addition, the families Prevotellaceae, and Veillonellaceae and the genus Prevotella_9 were closely associated with GD patients, while the families Lachnospiraceae and Ruminococcaceae and the genera Faecalibacterium, Lachnospira, and Lachnospiraceae NK4A136 were associated with healthy controls. Metagenomic profiles analysis yielded 22 statistically significant bacterial taxa: 18 taxa were increased and 4 taxa were decreased. Key bacterial taxa with different abundances between the two groups were strongly correlated with GD-associated clinical parameters using Spearman’s correlation analysis. Importantly, the discriminant model based on predominant microbiota could effectively distinguish GD patients from healthy controls (AUC = 0.825). Thus, the gut microbiota composition between GD patients and healthy controls is significantly difference, indicating that gut microbiota may play a role in the pathogenesis of GD. Further studies are needed to fully elucidate the role of gut microbiota in the development of GD.

Abstract Background CoMut plot is widely used in cancer research publications as a visual summary of mutational landscapes in cancer cohorts. This summary plot can inspect gene mutation rate and sample mutation burden with their relevant clinical details, which is a common first step for analyzing the recurrence and co-occurrence of gene mutations across samples. The cBioPortal and iCoMut are two web-based tools that allow users to create intricate visualizations from pre-loaded TCGA and ICGC data. For custom data analysis, only limited command-line packages are available now, making the production of CoMut plots difficult to achieve, especially for researchers without advanced bioinformatics skills. To address the needs for custom data and TCGA/ICGC data comparison, we have created CoMutPlotter, a web-based tool for the production of publication-quality graphs in an easy-of-use and automatic manner. Results We introduce a web-based tool named CoMutPlotter to lower the barriers between complex cancer genomic data and researchers, providing intuitive access to mutational profiles from TCGA/ICGC projects as well as custom cohort studies. A wide variety of file formats are supported by CoMutPlotter to translate cancer mutation profiles into biological insights and clinical applications, which include Mutation Annotation Format (MAF), Tab-separated values (TSV) and Variant Call Format (VCF) files. Conclusions In summary, CoMutPlotter is the first tool of its kind that supports VCF file, the most widely used file format, as its input material. CoMutPlotter also provides the most-wanted function for comparing mutation patterns between custom cohort and TCGA/ICGC project. Contributions of COSMIC mutational signatures in individual samples are also included in the summary plot, which is a unique feature of our tool. CoMutPlotter is freely available at http://tardis.cgu.edu.tw/comutplotter.

Abstract Background Pathogenic protist membrane transporter proteins play important roles not only in exchanging molecules into and out of cells but also in acquiring nutrients and biosynthetic compounds from their hosts. Currently, there is no centralized protist membrane transporter database published, which makes system-wide comparisons and studies of host-pathogen membranomes difficult to achieve. Results We analyzed over one million protein sequences from 139 protists with full or partial genome sequences. Putative transmembrane proteins were annotated by primary sequence alignments, conserved secondary structural elements, and functional domains. We have constructed the PPTdb (Pathogenic Protist Transmembranome database), a comprehensive membrane transporter protein portal for pathogenic protists and their human hosts. The PPTdb is a web-based database with a user-friendly searching and data querying interface, including hierarchical transporter classification (TC) numbers, protein sequences, functional annotations, conserved functional domains, batch sequence retrieving and downloads. The PPTdb also serves as an analytical platform to provide useful comparison/mining tools, including transmembrane ability evaluation, annotation of unknown proteins, informative visualization charts, and iterative functional mining of host-pathogen transporter proteins. Conclusions The PPTdb collected putative protist transporter proteins and offers a user-friendly data retrieving interface. Moreover, a pairwise functional comparison ability can provide useful information for identifying functional uniqueness of each protist. Finally, the host and non-host protein similarity search can fulfill the needs of comprehensive studies of protists and their hosts. The PPTdb is freely accessible at http://pptdb.cgu.edu.tw.

Congenital short bowel syndrome (CSBS) is a rare condition characterized by an inborn shortening of bowel length with loss of intestinal functions, which often combines malrotation. CXADR-like membrane protein (CLMP) and filamin A (FLNA) gene mutations are the two major causes of this inherited defect. We presented two siblings with the older brother suffering from a laparotomy for bowel obstruction due to malrotation on the 17th day after birth. The younger sister encountered a laparotomy for lactobezoar at 6 months old. CSBS was diagnosed by measurement of the bowel length during the operations. Compound heterozygous CLMP mutations with the paternal allele harboring a long deletion across exon 3–5 and the maternal allele bearing a non-sense mutation of exon 3 (c.235C > T, p.Q79∗) were identified in both cases. They are the first reported familial CSBS caused by novel CLMP mutations in Taiwan.

Accumulated evidence suggests that the endosymbiotic Trichomonasvirus (TVV) may play a role in the pathogenesis and drug susceptibility of Trichomonas vaginalis. Several reports have shown that extracellular vesicles (EVs) released from TVV-positive (TVV+) trichomonads can modulate the immune response in human vaginal epithelial cells and animal models. These results prompted us to examine whether EVs released from TVV+ isolates contained TVV. We isolated small extracellular vesicles (sEVs) from six T. vaginalis isolates that were either TVV free (ATCC 50143), harbored a single (ATCC 30236, ATCC 30238, T1), two (ATCC PRA-98), or three TVV subspecies (ATCC 50148). The presence of TVV subspecies in the six isolates was observed using reverse transcription-polymerase chain reaction (RT-PCR). Transmission electron microscopy (TEM) confirmed the presence of cup-shaped sEVs with a size range from 30–150 nm. Trichomonas vaginalis tetraspanin (TvTSP1; TVAG_019180), the classical exosome marker, was identified in all the sEV preparations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that all the sEVs isolated from TVV+ isolates contain viral capsid proteins derived from the same TVV subspecies in that isolate as demonstrated by RT-PCR. To provide more comprehensive information on the TVV subspecies population in other T. vaginalis isolates, we investigated the distribution of TVV subspecies in twenty-four isolates by mining the New-Generation Sequencing (NGS) RNAseq datasets. Our results should be beneficial for future studies investigating the role of TVV on the pathogenicity of T. vaginalis and the possible transmission of virus subspecies among different isolates via sEVs.

Abstract Background Patient-derived xenograft (PDX) tumor model has become a new approach in identifying druggable tumor mutations, screening and evaluating personalized cancer drugs based on the mutated targets. Methods We established five nasopharyngeal carcinoma (NPC) PDXs in mouse model. Subsequently, whole-exome sequencing (WES) and genomic mutation analyses were performed to search for genetic alterations for new drug targets. Potential drugs were applied in two NPC PDX mice model to assess their anti-cancer activities. RNA sequencing and transcriptomic analysis were performed in one NPC PDX mice to correlate with the efficacy of the anti-cancer drugs. Results A relative high incident rate of copy number variations (CNVs) of cell cycle-associated genes. Among the five NPC-PDXs, three had cyclin D1 (CCND1) amplification while four had cyclin-dependent kinase inhibitor CDKN2A deletion. Furthermore, CCND1 overexpression was observed in > 90% FFPE clinical metastatic NPC tumors (87/91) and was associated with poor outcomes. CNV analysis disclosed that plasma CCND1/CDKN2A ratio is correlated with EBV DNA load in NPC patients’ plasma and could serve as a screening test to select potential CDK4/6 inhibitor treatment candidates. Based on our NPC PDX model and RNA sequencing, Palbociclib, a cyclin-dependent kinase inhibitor, proved to have anti-tumor effects by inducing G1 arrest. One NPC patient with liver metastatic was treated with Palbociclib, had stable disease response and a drop in Epstein Barr virus (EBV) EBV titer. Conclusions Our integrated information of sequencing-based genomic studies and tumor transcriptomes with drug treatment in NPC-PDX models provided guidelines for personalized precision treatments and revealed a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for NPC.

Abstract Background High throughput sequencing technologies have been an increasingly critical aspect of precision medicine owing to a better identification of disease targets, which contributes to improved health care cost and clinical outcomes. In particular, disease-oriented targeted enrichment sequencing is becoming a widely-accepted application for diagnostic purposes, which can interrogate known diagnostic variants as well as identify novel biomarkers from panels of entire human coding exome or disease-associated genes. Results We introduce a workflow named VAReporter to facilitate the management of variant assessment in disease-targeted sequencing, the identification of pathogenic variants, the interpretation of biological effects and the prioritization of clinically actionable targets. State-of-art algorithms that account for mutation phenotypes are used to rank the importance of mutated genes through visual analytic strategies. We established an extensive annotation source by integrating a wide variety of biomedical databases and followed the American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation and reporting of sequence variations. Conclusions In summary, VAReporter is the first web server designed to provide a “one-stop” resource for individual’s diagnosis and large-scale cohort studies, and is freely available at http://rnd.cgu.edu.tw/vareporter.

Trichomonas vaginalis is the causative agent of trichomoniasis, the most prevalent non-viral sexually transmitted infection worldwide. Metronidazole (MTZ) is the mainstay of anti-trichomonal chemotherapy; however, drug resistance has become an increasingly worrying issue. Additionally, the molecular events of MTZ-induced cell death in T. vaginalis remain elusive. To gain insight into the differential expression of genes related to MTZ resistance and cell death, we conducted RNA-sequencing of three paired MTZ-resistant (MTZ-R) and MTZ-sensitive (MTZ-S) T. vaginalis strains treated with or without MTZ. Comparative transcriptomes analysis identified that several putative drug-resistant genes were exclusively upregulated in different MTZ-R strains, such as ATP-binding cassette (ABC) transporters and multidrug resistance pumps. Additionally, several shared upregulated genes among all the MTZ-R transcriptomes were not previously identified in T. vaginalis, such as 5′-nucleotidase surE and Na+-driven multidrug efflux pump, which are a potential stress response protein and a multidrug and toxic compound extrusion (MATE)-like protein, respectively. Functional enrichment analysis revealed that purine and pyrimidine metabolisms were suppressed in MTZ-S parasites upon drug treatment, whereas the endoplasmic reticulum-associated degradation (ERAD) pathway, proteasome, and ubiquitin-mediated proteolysis were strikingly activated, highlighting the novel pathways responsible for drug-induced stress. Our work presents the most detailed analysis of the transcriptional changes and the regulatory networks associated with MTZ resistance and MTZ-induced signaling, providing insights into MTZ resistance and cell death mechanisms in trichomonads.

The three most common sexually transmitted infections (STIs) are Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC) and Trichomonas vaginalis (TV). The prevalence of these STIs in Taiwan remains largely unknown and the risk of STI acquisition affected by the vaginal microbiota is also elusive. In this study, a total of 327 vaginal swabs collected from women with vaginitis were analyzed to determine the presence of STIs and the associated microorganisms by using the BD Max CT/GC/TV molecular assay, microbial cultures, and 16S rRNA sequencing. The prevalence of CT, TV, and GC was 10.8%, 2.2% and 0.6%, respectively. A culture-dependent method identified that Escherichia coli and Streptococcus agalactiae (GBS) were more likely to be associated with CT and TV infections. In CT-positive patients, the vaginal microbiota was dominated by L. iners, and the relative abundance of Gardnerella vaginalis (12.46%) was also higher than that in TV-positive patients and the non-STIs group. However, Lactobacillus spp. was significantly lower in TV-positive patients, while GBS (10.11%), Prevotella bivia (6.19%), Sneathia sanguinegens (12.75%), and Gemella asaccharolytica (5.31%) were significantly enriched. Using an in vitro co-culture assay, we demonstrated that the growth of L. iners was suppressed in the initial interaction with TV, but it may adapt and survive after longer exposure to TV. Additionally, it is noteworthy that TV was able to promote GBS growth. Our study highlights the vaginal microbiota composition associated with the common STIs and the crosstalk between TV and the associated bacteria, paving the way for future development of health interventions targeting the specific vaginal bacterial taxa to reduce the risk of common STIs.

Background: Nasopharyngeal carcinoma (NPC) involves host genetics, environmental and viral factors. In clinical observations, patients of young and old ages were found to have higher recurrence and metastatic rates. Methods: Cytokine array was employed to screen druggable target(s). The candidate target(s) were confirmed through patient-derived xenografts (PDXs) and a new EBV-positive cell line, NPC-B13. Results: Overexpression of epithelial growth factor (EGF) and EGF receptor (EGFR) was detected in young patients than in older patients. The growth of NPC PDX tumors and cell lines was inhibited by EGFR inhibitors (EGFRi) cetuximab and afatinib when used separately or in combination with the cell cycle blocker palbociclib. Western blot analysis of these drug-treated PDXs demonstrated that the blockade of the EGF signaling pathway was associated with a decrease in the p-EGFR level and reduction in PDX tumor size. RNA sequencing results of PDX tumors elucidated that cell cycle-related pathways were suppressed in response to drug treatments. High EGFR expression (IHC score ≥ grade 3) was correlated with poor survival in metastatic patients (p = 0.008). Conclusions: Our results provide encouraging preliminary data related to the combination treatment of EGFRi and palbociclib in patients with NPC.

Oral squamous cell carcinoma is a prevalent malignancy in Taiwan. Here, the authors show that OSCC in Taiwanese show a frequent deletion polymorphism in the cytidine deaminases gene cluster APOBEC3 resulting in increased expression of A3A, which is shown to be of clinical prognostic relevance.

Abstract Background γ-Carboxymuconolactone decarboxylase (CMD) participates in the β-ketoadipate pathway, which catalyzes aromatic compounds to produce acetyl- or succinyl-CoA, in prokaryotes and yeast. Our previous study demonstrated that expression of a CMD homologue that contains two signatures (dualCMD) is negatively regulated by iron in Trichomonas vaginalis. However, we were not able to identify the components of the β-ketoadipate pathway in the parasite’s genome. These observations prompted us to investigate the biological functions of this novel CMD homologue in T. vaginalis. Methods The specific anti-TvCMD1 antibody was generated, and the expression of TvCMD1 in T. vaginalis cultured under iron-rich and iron-deficient were evaluated. Phylogenetic, metabolomic and substrate induction (protocatechuate and benzoate) analysis were conducted to clarify the function of dualCMD in trichomonad cells. Subcellular localization of TvCMD1 was observed by confocal microscopy. The cell cycle-related role of TvCMD1 was assessed by treating cells with G2/M inhibitor nocodazole. Results We confirmed that T. vaginalis is not able to catabolize the aromatic compounds benzoate and protocatechuate, which are known substrates of the β-ketoadipate pathway. Using immunofluorescence microscopy, we found that TvCMD1 is spatially associated with the basal body, a part of the cytoskeletal organizing center in T. vaginalis. TvCMD1 accumulated upon treatment with the G2/M inhibitor nocodazole. Additionally, TvCMD1 was expressed and transported to/from the basal body during cytokinesis, suggesting that TvCMD1 plays a role in cell division. Conclusion We demonstrated that TvCMD1 is unlikely to participate in the β-ketoadipate pathway and demonstrated that it is a novel basal body-localizing (associated) protein. This model sheds light on the importance of genes that are acquired laterally in the coevolution of ancient protists, which surprisingly functions in cell cycle regulation of T. vaginalis.

Emerging evidence has shown the oncogenic roles of leptin in modulating cancer progression in addition to its original roles. Analyses of transcriptomic data and patients’ clinical information have revealed leptin’s prognostic significance in renal cell carcinoma (RCC). However, its biological effects on RCC progression have not yet been explored. Clinical and transcriptomic data of a RCC cohort of 603 patients were retrieved from The Cancer Genome Atlas (TCGA) and analyzed to reveal the correlation of leptin with clinical outcomes and the hierarchical clustering of gene signatures based on leptin levels. In addition, cox univariate and multivariate regression analyses, cell migration upon leptin treatment, identification of putative leptin-regulated canonical pathways via ingenuity pathway analysis (IPA), and the investigation of induction of Wnt5a, ROR2, and Jun N-terminal Kinases (JNK) phosphorylation activation were performed. We first observed a correlation of high leptin levels and poor outcomes in RCC patients. Knowledge-based analysis by IPA indicated the induction of cancer cell migration by leptin, which was manifested via direct leptin treatment in the RCC cell lines. In RCC patients with high leptin levels, the planar cell polarity (PCP)/JNK signaling pathway was shown to be activated, and genes in the axis, including CTHRC1, FZD2, FZD10, ROR2, WNT2, WNT4, WNT10B, WNT5A, WNT5B, and WNT7B, were upregulated. All of these genes were associated with unfavorable clinical outcomes. WNT5A and ROR2 are pivotal upstream regulators of PCP/JNK signaling, and their correlations with leptin expression levels were displayed by a Pearson correlation analysis. The inhibition of signal transduction by SP600125 reversed leptin-mediated cell migration properties in RCC cell lines. The results indicate the prognostic impact of leptin on RCC patients and uncover its ability to promote cell migration via PCP/JNK signaling.

Ghrelin is a peptide hormone, originally identified from the stomach, that functions as an endogenous ligand of the growth hormone secretagogue receptor (GHSR) and promotes growth hormone (GH) release and food intake. Increasing reports point out ghrelin’s role in cancer progression. We previously characterized ghrelin’s prognostic significance in the clear cell subtype of renal cell carcinoma (ccRCC), and its pro-metastatic ability via Snail-dependent cell migration. However, ghrelin’s activity in promoting cell invasion remains obscure. In this study, an Ingenuity Pathway Analysis (IPA)-based investigation of differentially expressed genes in Cancer Cell Line Encyclopedia (CCLE) dataset indicated the potential association of Aurora A with ghrelin in ccRCC metastasis. In addition, a significant correlation between ghrelin and Aurora A expression level in 15 ccRCC cell line was confirmed by variant probes. ccRCC patients with high ghrelin and Aurora A status were clinically associated with poor outcome. We further observed that ghrelin upregulated Aurora A at the protein and RNA levels and that ghrelin-induced ccRCC in vitro invasion and in vivo metastasis occurred in an Aurora A-dependent manner. Furthermore, MMP1, 2, 9 and 10 expressions are associated with poor outcome. In particular, MMP10 is significantly upregulated and required for the ghrelin-Aurora A axis to promote ccRCC invasion. The results of this study indicated a novel signaling mechanism in ccRCC metastasis.

The pathogenic influences of uterine bacteria on endometrial carcinogenesis remain unclear. The aim of this pilot study was to compare the microbiota composition of endometrial lavage samples obtained from women with either endometrial hyperplasia (EH) or endometrial cancer (EC) versus those with benign uterine conditions. We hypothesized that specific microbiota signatures would distinguish between the two groups, possibly leading to the identification of bacterial species associated with endometrial tumorigenesis. A total of 35 endometrial lavage specimens (EH, n = 18; EC, n = 7; metastatic EC, n = 2; benign endometrial lesions, n = 8) were collected from 32 women who had undergone office hysteroscopy. Microbiota composition was determined by sequencing the V3-V4 region of 16S rRNA genes and results were validated by real-time qPCR in 46 patients with EC/EH and 13 control women. Surprisingly, we found that Bacillus pseudofirmus and Stenotrophomonas rhizophila - two plastic-degrading bacterial species - were over-represented in endometrial lavage specimens collected from patients with EC/EH. Using computational analysis, we found that the functional profile of endometrial microbiota in EC/EH was associated with fatty acid and amino acid metabolism. In summary, our hypothesis-generating data indicate that the plastic-degrading bacteria Bacillus pseudofirmus and Stenotrophomonas rhizophila are over-represented within the endometrial lavage microbiota of women with EC/EH living in Taiwan. Whether this may be related to plastic pollution deserves further investigation. [ABSTRACT FROM AUTHOR]

Background: Next-generation sequencing promises the de novo genomic and transcriptomic analysis of samples of interests. However, there are only a few organisms having reference genomic sequences and even fewer having well-defined or curated annotations. For transcriptome studies focusing on organisms lacking proper reference genomes, the common strategy is de novo assembly followed by functional annotation. However, things become even more complicated when multiple transcriptomes are compared. Results: Here, we propose a new analysis strategy and quantification methods for quantifying expression level which not only generate a virtual reference from sequencing data, but also provide comparisons between transcriptomes. First, all reads from the transcriptome datasets are pooled together for de novo assembly. The assembled contigs are searched against NCBI NR databases to find potential homolog sequences. Based on the searched result, a set of virtual transcripts are generated and served as a reference transcriptome. By using the same reference, normalized quantification values including RC (read counts), eRPKM (estimated RPKM) and eTPM (estimated TPM) can be obtained that are comparable across transcriptome datasets. In order to demonstrate the feasibility of our strategy, we implement it in the web service PARRoT. PARRoT stands for Pipeline for Analyzing RNA Reads of Transcriptomes. It analyzes gene expression profiles for two transcriptome sequencing datasets. For better understanding of the biological meaning from the comparison among transcriptomes, PARRoT further provides linkage between these virtual transcripts and their potential function through showing best hits in SwissProt, NR database, assigning GO terms. Our demo datasets showed that PARRoT can analyze two paired-end transcriptomic datasets of approximately 100 million reads within just three hours. Conclusions: In this study, we proposed and implemented a strategy to analyze transcriptomes from non-reference organisms which offers the opportunity to quantify and compare transcriptome profiles through a homolog based virtual transcriptome reference. By using the homolog based reference, our strategy effectively avoids the problems that may cause from inconsistencies among transcriptomes. This strategy will shed lights on the field of comparative genomics for non-model organism. We have implemented PARRoT as a web service which is freely available at http://parrot.cgu.edu.tw. [ABSTRACT FROM AUTHOR]