The influence of male HBV infection on sperm quality, embryonic development, and assisted reproductive outcomes.

STUDY QUESTION: What is the impact of male hepatitis B virus (HBV) infection on sperm quality, embryonic development, and assisted reproductive outcomes? SUMMARY ANSWER: Male HBV infection did not affect assisted reproductive outcomes, but HBV is capable of impairing human sperm and embryo formation in the early stages following fertilization. WHAT IS KNOWN ALREADY: HBV is found in germ cells and early embryos of patients with HBV. HBV may impair human sperm function via increasing reactive oxygen species. STUDY DESIGN, SIZE, DURATION: We conducted a retrospective cohort study of 1581 infertile couples, including 496 male patients clinically confirmed to have hepatitis B infection, and a laboratory study of effects of HBV proteins on early embryos, using human embryonic stem cells (hESCs), human sperm, and golden hamster oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 1581 infertile couples (24-40 years of age) who were admitted to a reproductive medicine center to undergo ART for the first time from January 2019 to November 2021 were selected as the study subjects. The case group was composed of 469 couples with hepatitis B surface antigen (HBsAg)-seropositive men and seronegative women (368 for IVF and 101 for ICSI treatment). The negative control group was composed of 1112 couples where both men and women were seronegative for hepatitis B antigen. We divided these couples into three comparison groups (IVF/ICSI, IVF, and ICSI). IVF of human sperm and hamster oocytes was used to evaluate the influence of the HBV HBs protein on formation of 2-cell embryos. Mitochondrial membrane potential (MMP) of hESCs was assayed via a fluorescence intensity system. Immunofluorescence staining of the phosphorylated histone H2A.X was applied to identify DNA damage to hESCs caused by the HBV X (HBx) protein. MAIN RESULTS AND THE ROLE OF CHANCE: Sperm concentration, total sperm number, and sperm with normal morphology were decreased in the couples with HBV-infected males in couples who were undergoing IVF/ICSI (male HBV(+) vs control: 469 vs 1112 individuals; sperm number, P < 0.01; normal sperm morphology, P < 0.01), IVF (368 vs 792; sperm number, P < 0.01; normal sperm morphology, P ≤ 0.05), and ICSI (101 vs 306; sperm number, P < 0.01; normal sperm morphology, P < 0.001). There was no significant difference in the number of embryo cleavages, blastocyst formation, biochemical pregnancy rate, clinical pregnancy rate, and live-birth rate between case and control groups. The 2PN fertilization rate in IVF/ICSI (P < 0.01) and ICSI (P < 0.05) couples, and the number of 2PN-fertilized oocytes in IVF (P < 0.001) couples were lower in couples with male HBV infection compared to control couples. HBV HBs protein reduced the MMP of human sperm and decreased 2-cell embryo formation in IVF of human sperm and zona-free-hamster oocyte. A reduction in fluorescence intensity and immunofluorescence staining of phosphorylated histone H2A.X indicated that HBx caused MMP impairment and DNA damage in human early embryonic cells, respectively. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: HBV can be examined in samples of sperm or discarded IVF early embryos from HBsAg-seropositive men and seronegative women. The hESC model in vitro may not fully mimic the natural embryos in vivo. WIDER IMPLICATIONS OF THE FINDINGS: This study furthers our understanding of the influence of male HBV infection on embryonic development. Our results suggest that a semen-washing process may be necessary for male patients with HBV undergoing ART to minimize the potential negative effects of HBV infection on the early embryo. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by grants from the National Natural Science Foundation of China, grant numbers 81870432 and 81570567 to X.Z., 81571994 to P.S., and 81950410640, the Natural Science Foundation of Guangdong Province, China (No. 2023A1515010660 to X.Z.), and the Li Ka Shing Shantou University Foundation (Grant No. L11112008). The authors have no conflicts of interest.

Metagenomic evidence for antibiotics-driven co-evolution of microbial community, resistome and mobilome in hospital sewage.

Overconsumption of antibiotics is an immediate cause for the emergence of antimicrobial resistance (AMR) and antibiotic resistant bacteria (ARB), though its environmental impact remains inadequately clarified. There is an urgent need to dissect the complex links underpinning the dynamic co-evolution of ARB and their resistome and mobilome in hospital sewage. Metagenomic and bioinformatic methods were employed to analyze the microbial community, resistome and mobilome in hospital sewage, in relation to data on clinical antibiotic use collected from a tertiary-care hospital. In this study, resistome (1,568 antibiotic resistance genes, ARGs, corresponding to 29 antibiotic types/subtypes) and mobilome (247 types of mobile genetic elements, MGEs) were identified. Networks connecting co-occurring ARGs with MGEs encompass 176 nodes and 578 edges, in which over 19 types of ARGs had significant correlations with MGEs. Prescribed dosage and time-dependent antibiotic consumption were associated with the abundance and distributions of ARGs, and conjugative transfer of ARGs via MGEs. Variation partitioning analyses show that effects of conjugative transfer were most likely the main contributors to transient propagation and persistence of AMR. We have presented the first evidence supporting idea that use of clinical antibiotics is a potent driving force for the development of co-evolving resistome and mobilome, which in turn supports the growth and evolution of ARB in hospital sewage. The use of clinical antibiotics calls for greater attention in antibiotic stewardship and management.

Machine Learning Techniques for Antimicrobial Resistance Prediction of Pseudomonas Aeruginosa from Whole Genome Sequence Data.

Aim: Due to the growing availability of genomic datasets, machine learning models have shown impressive diagnostic potential in identifying emerging and reemerging pathogens. This study aims to use machine learning techniques to develop and compare a model for predicting bacterial resistance to a panel of 12 classes of antibiotics using whole genome sequence (WGS) data of Pseudomonas aeruginosa. Method: A machine learning technique called Random Forest (RF) and BioWeka was used for classification accuracy assessment and logistic regression (LR) for statistical analysis. Results: Our results show 44.66% of isolates were resistant to twelve antimicrobial agents and 55.33% were sensitive. The mean classification accuracy was obtained ≥98% for BioWeka and ≥96 for RF on these families of antimicrobials. Where ampicillin was 99.31% and 94.00%, amoxicillin was 99.02% and 95.21%, meropenem was 98.27% and 96.63%, cefepime was 99.73% and 98.34%, fosfomycin was 96.44% and 99.23%, ceftazidime was 98.63% and 94.31%, chloramphenicol was 98.71% and 96.00%, erythromycin was 95.76% and 97.63%, tetracycline was 99.27% and 98.25%, gentamycin was 98.00% and 97.30%, butirosin was 99.57% and 98.03%, and ciprofloxacin was 96.17% and 98.97% with 10-fold-cross validation. In addition, out of twelve, eight drugs have found no false-positive and false-negative bacterial strains. Conclusion: The ability to accurately detect antibiotic resistance could help clinicians make educated decisions about empiric therapy based on the local antibiotic resistance pattern. Moreover, infection prevention may have major consequences if such prescribing practices become widespread for human health.

Expression of CD22 in Triple-Negative Breast Cancer: A Novel Prognostic Biomarker and Potential Target for CAR Therapy.

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer cases. Due to the lack of expression of well-known molecular targets [estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)], there is a need for more alternative treatment approaches in TNBC. Chimeric antigen receptor (CAR)-T cell-based immunotherapy treatment is one of the latest treatment technologies with outstanding therapeutic advances in the past decade, especially in the treatment of hematologic malignancies, but the therapeutic effects of CAR-T cells against solid tumors have not yet shown significant clinical benefits. Identification of highly specific CAR-T targets in solid tumors is also crucial for its successful treatment. CD22 is reported to be a multifunctional receptor that is mainly expressed on the surface of mature B-cells (lymphocytes) and is also highly expressed in most B-cell malignancies. This study aimed to investigate the expression of CD22 in TNBC. Bioinformatic analysis was performed to evaluate the expression of CD22 in breast carcinoma and normal tissues. RNA-seq data of normal and breast carcinoma patients were downloaded from The Cancer Genome Atlas (TCGA), and differential gene expression was performed using R language. Additionally, online bioinformatics web tools (GEPIA and TNM plot) were used to evaluate the expression of CD22 in breast carcinoma and normal tissues. Western blot (WB) analysis and immunofluorescence (IF) were performed to characterize the expression of CD22 in TNBC cell lines. Immunohistochemical (IHC) staining was performed on tumor specimens from 97 TNBC patients for CD22 expression. Moreover, statistical analysis was performed to analyze the association of clinical pathological parameters with CD22 expression. Correlation analysis between overall survival data of TNBC patients and CD22 expression was also performed. Differential gene expression analysis of TCGA data revealed that CD22 is among the upregulated differentially expressed genes (DEGs) with high expression in breast cancer, as compared to normal breast tissues. WB and IF analysis revealed high expression of CD22 in TNBC cell lines. IHC results also showed that approximately 62.89% (61/97) of TNBC specimens were stained positive for CD22. Cell membrane expression of CD22 was evident in 23.71% (23/97) of TNBC specimens, and 39.18% (38/97) of TNBC specimens showed cytoplasmic/membrane expression, while 37.11% (36/97) specimens were negative for CD22. Furthermore, significant associations were found between the size of tumors in TNBC patients and CD22 expression, which unveils its potential as a prognostic biomarker. No significant correlation was found between the overall survival of TNBC patients and CD22 expression. In conclusion, we demonstrated for the first time that CD22 is highly expressed in TNBC. Based on our findings, we anticipated that CD22 could be used as a prognostic biomarker in TNBC, and it might be a potential CAR-T target in TNBC for whom few therapeutic options exist. However, more large-scale studies and clinical trials will ensure its potential usefulness as a CAR-T target in TNBC.

Photodynamic therapy treats acne by altering the composition of the skin microbiota.

BACKGROUND: Acne is the eighth-most prevalent inflammatory skin disease with no optimal treatment. Photodynamic therapy (PDT) is an effective treatment for severe acne. AIMS: The effect of PDT on the composition and diversity of skin microflora in severe acne patients was studied. MATERIALS AND METHODS: A total of 18 patients with severe acne and 8 healthy individuals were selected for this study. Patients were treated with 5-aminolevulinic acid-mediated PDT once a week three times in total; the skin microbiome was measured by 16S ribosomal RNA gene sequencing before and after treatment (1 week after each PDT). RESULTS: The microflora composition was different between healthy controls and patients, and between patients before and after treatment. Alpha diversity indices were lower in patients than those in control. There were 15 bacterial genera with high relative abundance that had noticeable changes during treatment. At the genus level,particularly Cutibacterium acnes (C. acnes formerly Propionibacterium acnes), there was no statistically significant difference among different group. The abundances of Staphylococcus epidermidis and Staphylococcus aureus were low. DISCUSSION: The microbial composition is different between severe acne patients acne patients and healthy individuals. The therapeutic efficacy of severe acne treated with PDT is associated with the composition and diversity of skin microbiota. CONCLUSION: The skin microbial composition changes after PDT treatment. PDT is an effective method for the treatment of severe acne.

Direct Differentiation of Human Embryonic Stem Cells to 3D Functional Hepatocyte-like Cells in Alginate Microencapsulation Sphere.

BACKGROUND: The lack of a stable source of hepatocytes is one of major limitations in hepatocyte transplantation and clinical applications of a bioartificial liver. Human embryonic stem cells (hESCs) with a high degree of self-renewal and totipotency are a potentially limitless source of a variety of cell lineages, including hepatocytes. Many techniques have been developed for effective differentiation of hESCs into functional hepatocyte-like cells. However, the application of hESC-derived hepatocyte-like cells (hESC-Heps) in the clinic has been constrained by the low yield of fully differentiated cells, small-scale culture, difficulties in harvesting, and immunologic graft rejection. To resolve these shortcomings, we developed a novel 3D differentiation system involving alginate-microencapsulated spheres to improve current hepatic differentiation, providing ready-to-use hESC-Heps. METHODS: In this study, we used alginate microencapsulation technology to differentiate human embryonic stem cells into hepatocyte-like cells (hESC-Heps). Hepatic markers of hESC-Heps were examined by qPCR and Western blotting, and hepatic functions of hESC-Heps were evaluated by indocyanine-green uptake and release, and ammonia removal. RESULTS: The maturity and hepatic functions of the hESC-Heps derived from this 3D system were better than those derived from 2D culture. Hepatocyte-enriched genes, such as HNF4α, AFP, and ALB, were expressed at higher levels in 3D hESC-Heps than in 2D hESC-Heps. 3D hESC-Heps could metabolize indocyanine green and had better capacity to scavenge ammonia. In addition, the 3D sodium alginate hydrogel microspheres could block viral entry into the microspheres, and thus protect hESC-Heps in 3D microspheres from viral infection. CONCLUSION: We developed a novel 3D differentiation system for differentiating hESCs into hepatocyte-like cells by using alginate microcapsules.

Tremella fuciformis Crude Polysaccharides Attenuates Steatosis and Suppresses Inflammation in Diet-Induced NAFLD Mice.

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disorder characterized by an enhanced accumulation of lipids, which affects around 40% of the world's population. The T. fuciformis fungus possesses immunomodulatory activity and other beneficial properties that may alleviate steatosis through a different mechanism. The present study was designed to evaluate the effect T. fuciformis crude polysaccharides (TFCP) on inflammatory and lipid metabolism gene expression, oxidative stress, and lipid profile. Mice were divided into groups receiving (a) a normal chow diet (NCD), (b) a methionine-choline-deficient (MCD) diet, and (c) a MCD diet with TFCP. Liver histopathology was performed, and the hepatic gene expression levels were estimated using qRT-PCR. The lipid profiles, ALT, AST, and efficient oxidative enzymes were analyzed using ELISA. The TFCP administration in the MCD-fed mice suppressed hepatic lipid accumulation, lipid metabolism-associated genes (HMGCR, FABP, SREBP, ACC, and FAS), and inflammation-associated genes (IL-1ß, TLR4, TNF-α, and IL-6) whilst enhancing the expression of HNF4α genes. TFCP mitigated against oxidative stress and normalized healthy lipid profiles. These results highlighted that TFCP prevents NAFLD through the inhibition of oxidative stress and inflammation, suggesting TFCP would potentially be an effective therapeutic agent against NAFLD progression.

Incidence and molecular characterization of ESBL-producing and colistin-resistant Escherichia coli isolates recovered from healthy food-producing animals in Pakistan.

OBJECTIVES: To investigate the occurrence and molecular features of ESBL-producing and colistin-resistant Escherichia coli isolates recovered from healthy food-producing animals in Pakistan. METHODS: A total of 153 E. coli isolates were recovered from 250 faecal samples collected from livestock and poultry. The antibiotic susceptibility, resistant determinants and mobile genetic elements were determined for all the isolates. The clonal relatedness was analysed by MLST. Plasmids harbouring, localization and transferability of mcr-1 gene were carried out by Southern hybridization, S1-PFGE and transconjugation. RESULTS: Out of 153 E. coli strains, 49.01% isolates were ESBLs producers, whereas 18.95% were resistant to colistin and 84.31% of the isolates. Multidrug resistance was found in 84% of the isolates. The ESBL-producing E. coli in buffaloes, cattle, sheep, goat and broilers faecal samples were 60%, 74%, 54%, 50% and 68%, respectively. Among the ESBLs genes, blaCTX-M was the most prevalent group detected in 98.66%, while only mcr-1 of the colistin-resistant genes could be PCR amplified in 29 isolates. The common MGEs found were ISECP1 (35.13%), ISCR1 (33.78%), ISApl1 (20.27%) and Inti1 (58.10%). The most predominant Inc. types found were IncFIB 46.66%, followed by IncFIA 30.66%, IncFIC 26.66%, IncFrepB 26.66%, IncHI2 26.66%, IncP 22.66% and IncX4 21.33%. The most frequent sequence type detected was ST58. Southern blot and S1-PFGE confirmed the plasmid harbouring of mcr-1 gene. CONCLUSION: The co-occurrence of mcr-1 and ESBLs-encoding genes, along with MGEs in E. coli from healthy food animals in Pakistan, is a major concern. SIGNIFICANCE AND IMPACT OF STUDY: Antimicrobial resistance can be transferred from animals to humans by direct contact or via the food chain and environment. The prevalence and co-occurrence of ESBL and colistin resistance genes from food-producing animals is rare in Pakistan. To our knowledge, this is the first report to find ESBLs and mcr-1-harbouring E. coli from the faecal samples of the healthy food-producing animals in Pakistan. The presence of ARGs in association with MGEs, co-harbouring the virulence factors, as determined in the current study, is a severe threat to livestock and the human community as it has horizontally and food web transferability.

The Role of Fecal Fusobacterium nucleatum and pks+ Escherichia coli as Early Diagnostic Markers of Colorectal Cancer.

BACKGROUND: Accurate analysis of intestinal microbiota will facilitate establishment of an evaluating system for assessing colorectal cancer (CRC) risk and prognosis. This study evaluates the potential role of Fusobacterium nucleatum (F. nucleatum) and Escherichia coli with a pks gene (pks+ E. coli) in early CRC diagnosis. METHODS: We recruited 139 patients, including CRC (n = 60), colorectal adenomatous polyposis (CAP) (n = 37), and healthy individuals (n = 42) based on their colonoscopy examinations. We collected stool and serum samples from the participants and measured the relative abundance of F. nucleatum and pks+ E. coli in fecal samples by quantitative PCR. Receiver operating characteristic curve (ROC) analyses were used to analyze the diagnostic value of single or combined biomarkers. RESULTS: Fecal F. nucleatum and pks+ E. coli levels were higher in the CRC group in either the CAP group or healthy controls (P = 0.02; 0.01). There was no statistical difference in the distribution of F. nucleatum and pks+ E. coli in patients with different tumor sites (P > 0.05). The combination of F. nucleatum+pks+ E. coli+CEA+CA19-9+FOBT was chosen as the optimal panel in differentiating both CRC and CAP from the controls. The combination of F. nucleatum, pks+ E. coli, and FOBT improved diagnostic efficiency. However, there was difficulty in differentiating CRC from CAP. CONCLUSION: Our results suggested that combining bacterial markers with conventional tumor markers improves the diagnostic efficiency for noninvasive diagnosis of CRC.

Microbiomes and Resistomes in Biopsy Tissue and Intestinal Lavage Fluid of Colorectal Cancer.

Aim: The gut microbiome plays a crucial role in colorectal cancer (CRC) tumorigenesis, but compositions of microorganisms have been inconsistent in previous studies due to the different types of specimens. We investigated the microbiomes and resistomes of CRC patients with colonic biopsy tissue and intestinal lavage fluid (IVF). Methods: Paired samples (biopsy tissue and IVF) were collected from 20 patients with CRC, and their gut microbiomes and resistomes were measured by shotgun metagenomics. Clinical and laboratory data were recorded. Bioinformatics (KneadData, Kraken2, and FMAP) and statistical analysis were done using the R (v4.0.2) software. Results: Bacterial diversity in IVF was higher than in tissue samples, and bacterial operational taxonomic units (OTUs) were 2,757 in IVF vs. 197 in tissue. ß-diversity showed distinct clusters in paired samples. The predominant bacteria in IVF were phylum Proteobacteria, while the predominant bacteria of tissue were phylum Actinobacteria. Twenty-seven representative bacteria were selected to form six bacterial clusters, which showed only Firmicutes Cluster 1, and the Bacteroidetes Cluster 1 were significantly more abundant in the IVF group than those in the tissue group (p < 0.05). The Firmicutes Cluster 2, Bacteroidetes Cluster 2, Pathogen Cluster, and Prevotella Cluster were not significantly different between IVF and tissue (p > 0.05). Correlation analysis revealed that some bacteria could have effects on metabolic and inflammatory parameters of CRC patients. A total of 1,295 antibiotic resistance genes (ARGs) were detected in the gut microbiomes, which conferred multidrug resistance, as well as resistance to tetracycline, aminoglycoside, and more. Co-occurrence patterns revealed by the network showed mainly ARG-carrying bacteria to be similar between IVF and tissue, but leading bacteria located in the hub differed between IVF and tissue. Conclusion: Heterogeneity of microbiota is particularly evident when studied with IVF and tissue samples, but bacterial clusters that have close relationships with CRC carcinogenesis are not significantly different, using IVF as an alternative to tissue for gut microbiome, and resistome assessment may be a feasible method.

Antimicrobial resistance bacteria and genes detected in hospital sewage provide valuable information in predicting clinical antimicrobial resistance.

Extensive use of antibiotics is significantly associated with development of antibiotic-resistant (AR) bacteria. However, their causal relationships have not been adequately investigated, especially in human population and hospitals. Our aims were to understand clinical AR through revealing co-occurrence patterns between antibiotic-resistant bacteria and genes (ARB and ARGs), and their association with antibiotic use, and to consider impact of ARB and ARGs on environmental and human health. Antibiotic usage was calculated based on the actual consumption in our target hospital. ARB was identified by culture. In isolates collected from hospital sewage, bacterial-specific DNA sequences and ARGs were determined using metagenomics. Our data revealed that the use of culture-based single-indicator-strain approaches only captured ARB in 16.17% of the infectious samples. On the other hand, 1573 bacterial species and 885 types of ARGs were detected in the sewage. Furthermore, hospital use of antibiotics influenced the resistance profiles, but the strength varied among bacteria. From our metagenomics analyses, ARGs for aminoglycosides were the most common, followed by sulfonamide, tetracycline, phenicol, macrolides, and quinolones, comprising 82.6% of all ARGs. Association analyses indicated that 519 pairs of ARGs were significantly correlated with ARB species (r > 0.8). The co-occurrence patterns of bacteria-ARGs mirrored the AR in the clinic. In conclusion, our systematic investigation further emphasized that antibiotic usage in hospital significantly influenced the abundance and types of ARB and ARGs in dose- and time-dependent manners which, in turn, mirrored clinical AR. In addition, our data provide novel information on development of certain ARB with multiple antibiotic resistance. These ARB and ARGs from sewage can also be disseminated into the environment and communities to create health problems. Therefore, it would be helpful to use such data to develop improved predictive risk model of AR, to enhance effective use of antibiotics, and to reduce environmental pollution.

Hepatitis B virus surface protein induces sperm dysfunction through the activation of a Bcl2/Bax signaling cascade triggering AIF/Endo G-mediated apoptosis.

BACKGROUND: Hepatitis B virus (HBV) was found to exist in semen and male germ cells of patients with chronic HBV infection. Our previous studies demonstrated that HBV surface protein (HBs) could induce sperm dysfunction by activating a calcium signaling cascade and triggering caspase-dependent apoptosis. However, the relationship between sperm dysfunction caused by HBs and caspase-independent apoptosis has not been investigated. OBJECTIVES: To evaluate the effects of HBs exposure on sperm dysfunction by activating caspase-independent apoptosis. MATERIALS AND METHODS: Spermatozoa were exposed to HBs at concentrations of 0, 25, 50, and 100 µg/mL for 3 h. Flow cytometry, qRT-PCR, immunofluorescence assay, ELISA, and zona-free hamster oocyte penetration assays were performed. RESULTS: With increasing concentrations of HBs, various parameters of the spermatozoa changed. The number of Bcl2-positive cells declined and that of both Bax-positive cells and Apaf-1-positive cells increased. The transcription level of Bcl2 increased and that of both Bax and Apaf-1 declined. The average levels of AIF and Endo G declined in mitochondria and increased in the cytoplasm and nucleus. The sperm DNA fragmentation index increased. The mean percentages of live spermatozoa declined and that of both injured and dead spermatozoa increased; and the sperm penetration rate declined. For the aforementioned parameters, the differences between the test and the control groups were statistically significant. CONCLUSION: HBs exposure can activate the Bax/Bcl2 signaling cascade that triggers AIF/Endo G-mediated apoptosis, resulting in sperm DNA fragmentation, sperm injury, and death, and a decrease in the sperm fertilizing capacity. This new knowledge will help to evaluate the negative impact of HBV on male fertility in HBV-infected patients.

Hepatitis B virus surface protein induces oxidative stress by increasing peroxides and inhibiting antioxidant defences in human spermatozoa.

Hepatitis B virus (HBV) infection may affect sperm motility in patients with HBV. HBV surface protein (HBs) decreases mitochondrial membrane potential, impairs motility and induces apoptotic-like changes in human spermatozoa. However, little is known about how human spermatozoa respond to reactive oxygen species (ROS; mainly peroxides) induced by HBs. In this study, HBs induced supraphysiological ROS levels in human spermatozoa and reduced the formation of 2-cell embryos (obtained from hamster oocytes and human spermatozoa). HBs induced a pre-apoptotic status in human spermatozoa, as well as antioxidant defences by increasing glutathione peroxidase 4 (GPX4) and peroxiredoxin 5 (PRDX5) levels. These results highlight the molecular mechanism responsible for the oxidative stress in human spermatozoa exposed to HBV and the antioxidant defence response involving GPX4 and PRDX5.

Defined host factors support HBV infection in non-hepatic 293T cells.

Hepatitis B virus (HBV) is a human hepatotropic virus. However, HBV infection also occurs at extrahepatic sites, but the relevant host factors required for HBV infection in non-hepatic cells are only partially understood. In this article, a non-hepatic cell culture model is constructed by exogenous expression of four host genes (NTCP, HNF4α, RXRα and PPARα) in human non-hepatic 293T cells. This cell culture model supports HBV entry, transcription and replication, as evidenced by the detection of HBV pgRNA, HBV cccDNA, HBsAg, HBeAg, HBcAg and HBVDNA. Our results suggest that the above cellular factors may play a key role in HBV infection of non-hepatic cells. This model will facilitate the identification of host genes that support extrahepatic HBV infection.

The abundance and mineral-weathering effectiveness of Bacillus strains in the altered rocks and the soil.

In this study, we collected different levels of altered rocks of a rocky mountain and the adjacent soil and characterized the abundance and weathering effectiveness of Bacillus strains. Based on qPCR and culture-dependent approaches, the gene copies or the numbers of Bacillus strains were significantly higher in the soil than in the altered rocks, while the ratio of the gene copies or the numbers of Bacillus strains to those of total bacteria was higher in the less altered rock, followed by the more altered rock and the soil. The relative abundance of the highly active Al-solubilizing Bacillus strains was higher in the more altered rock, followed by the less altered rock and the soil. Among the Al-solubilizing Bacillus species, 30-36% of them were different between the altered rocks and the soil, however, similar Al-solubilizing Bacillus species were found in the less altered rocks and the more altered ones. The results showed the alteration-related changes in the abundance and mineral weathering effectiveness of Bacillus strains and suggested the ecological adaptation of the mineral-weathering Bacillus populations and their role in mineral weathering in the rock and soil environments.

Transcription and regulation of hepatitis B virus genes in host sperm cells.

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.

The Clinical Significance of FilmArray Respiratory Panel in Diagnosing Community-Acquired Pneumonia.

AIM: FilmArray Respiratory Panel (FilmArray RP) test is an emerging diagnostic method in fast detecting multiple respiratory pathogens; the methodology and clinical significance of FilmArray RP in community-acquired pneumonia (CAP) diagnosis were evaluated in this study. METHODS: Specimens from 74 patients with CAP were analyzed and compared using FilmArray RP, traditional multiple PCR assay, bacterial (or fungal) culture, and serological detection. RESULTS: FilmArray RP and multiplex PCR showed 100% coincidence rate in detecting coronaviruses 229E, OC43, HKU1, and NL63, human metapneumovirus, influenza A and B, and parainfluenza viruses (PIV1, PIV2, and PIV4). There were 15 viral specimens tested as disagreement positive results. FilmArray RP had higher detection rate in detecting dual viral and Mycoplasma pneumoniae infection. The positive bacteria (or fungi) were found in 25 specimens. CONCLUSIONS: This study demonstrated the capability of FilmArray RP for simultaneous detection of broad-spectrum respiratory pathogens and potential use in facilitating better patient care.

Host genes regulate transcription of sperm-introduced hepatitis B virus genes in embryo.

Hepatitis B virus (HBV) can invade the male germline, and sperm-introduced HBV genes could be transcribed in embryo. This study was to explore whether viral gene transcription is regulated by host genes. Embryos were produced by in vitro fertilization of hamster oocytes with human sperm containing the HBV genome. Total RNA extracted from test and control embryos were subjected to SMART-PCR, SSH, microarray hybridization, sequencing and BLAST analysis. Twenty-nine sequences showing significant identity to five human gene families were identified, with CSH2, EIF4G2, PCBD2, PSG4 and TTN selected to represent target genes. Using qRT-PCR, when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNAi, transcriptional levels of HBV s and x genes decreased (or increased). This is the first report that host genes participate in regulation of sperm-introduced HBV gene transcription in embryo, which is critical to prevent negative impact of HBV infection on early embryonic development.

Circulating tight junction proteins mirror blood-brain barrier integrity in leukaemia central nervous system metastasis.

The aim of this study was to evaluate the clinical significance of circulating tight junction (TJ) proteins as biomarkers reflecting of leukaemia central nervous system (CNS) metastasis. TJs [claudin5 (CLDN5), occludin (OCLN) and ZO-1] concentrations were measured in serum and cerebrospinal fluid (CSF) samples obtained from 45 leukaemia patients. Serum ZO-1 was significantly higher (p < 0.05), but CSF ZO-1 levels were not significantly higher in the CNS leukaemia (CNSL) compared to the non-CNSL. The CNSL patients also had a lower CLDN5/ZO1 ratio in both serum and CSF than in non-CNSL patients (p < 0.05). The TJ index was negatively associated with WBCCSF , ALBCSF and BBB values in leukaemia patients. Among all of the parameters studied, CLDN5CSF had the highest specificity in discriminating between CNSL and non-CNSL patients. Therefore, analysing serum and CSF levels of CLDN5, OCLN and the CLDN5/ZO1 ratio is valuable in evaluating the potential of leukaemia CNS metastasis. Copyright © 2016 John Wiley & Sons, Ltd.

CpG methylation participates in regulation of hepatitis B virus gene expression in host sperm and sperm-derived embryos.

AIM: This study was undertaken to investigate relationship between hepatitis B virus (HBV) CpG methylation and HBV gene transcription in sperm and sperm-derived embryos. METHODS: HBV-infected patient sperm and HBV plasmid-transfected donor sperm were subjected to interspecific in vitro fertilization, methylation-specific PCR, bisulfite sequencing PCR, reverse transcription PCR and real-time quantitative PCR. RESULTS: Positive methylation bands for CpG islands II and III in the HBV genome were observed in patient sperm but not in controls, and methylation percentages of CpG sites varied among different patient sperm samples. After fertilization, CpG sites were highly demethylated in embryos. Transcriptional levels of HBV X and S genes increased with decrease in CpG site methylation percentages. CONCLUSION: HBV CpG sites can be methylated in patient sperm before maturation. Methylation of CpG islands II and III participates in transcriptional regulation of HBV X and S genes, respectively, in sperm and sperm-derived embryos.