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.

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.

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.

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.

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.

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.

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.

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.

Research on the Vertical Transmission of Hepatitis C Gene from Father-to-child via Human Sperm.

BACKGROUND: The aim was to develop a better experimental model which could facilitate further studies assessing the vertical HCV gene transmission via human spermatozoa, and verify the possibility of father-to-child transmission of the HCV gene. METHODS: The recombinant plasmid pIRES2-EGFP-HCV C was constructed. Fluorescence in situ hybridization was performed to detect the integration of the HCV C gene in human sperm genome and in zygote's pronucleus. RESULTS: Successful construction of recombinant plasmid pIRES2-EGFP-HCV C was confirmed by restriction mapping, PCR, and sequencing. Positive HCV C DNA signals were observed in sperm heads, human sperm chromosomes and two-cell embryos in transfected samples. No positive signal was found in normal control and HCV infected groups. CONCLUSIONS: The recombinant plasmid pIRES2-EGFP-HCV C was successfully constructed. The HCV C gene was able to pass through the sperm membrane and integrate into the sperm genome. Human sperm carrying the HCV C gene was able to achieve normal fertilization. The replication of the sperm-mediated HCV C gene was synchronized with that of the host genome. Our results provide direct evidence for vertical transmission of the HCV C gene from father-to-child via human sperm.

Effects of Hepatitis C Virus Infection on Human Sperm Chromosomes.

BACKGROUND: To determine the degree of chromosomal aberrations in the sperm of men with hepatitis C. METHODS: 36 subjects (20 in the healthy control group and 16 in the HCV infection group [genotype 1b]) were recruited. The cause of viral transmission was unknown in all patients. Sperm samples from the subjects were used for interspecies in vitro fertilization of zona-free golden hamster ova. The frequencies of spermatozoan aberrations were compared between the healthy control group and the HCV infection group. RESULTS: A total of 280 sperm chromosome complements were studied, including 129 complements from the 16 donors in the HCV infection group and 151 from the healthy control group. Of the 129 analyzable sperm metaphase spreads in the HCV infection group, 14 (10.85%) complements contained chromosomal aberrations, which was significantly higher than the number (9/151, 5.96%) in the healthy control group (p < 0.01). Moreover, in the HCV infection group, chromosomes frequently showed anomalies such as stickiness, clumping, and failure to stain, which prevented their analysis. CONCLUSIONS: HCV infection has mutagenic effects on the chromosomes in sperm and may lead to extensive heredi-tary effects owing to genetic alterations and/or chromosomal aberrations. In addition, there is the possibility of vertical transmission of HCV via the germ line.

Mutations and CpG islands among hepatitis B virus genotypes in Europe.

BACKGROUND: Hepatitis B virus (HBV) genotypes have a distinct geographical distribution and influence disease progression and treatment outcomes. The purpose of this study was to investigate the distribution of HBV genotypes in Europe, the impact of mutation of different genotypes on HBV gene abnormalities, the features of CpG islands in each genotype and their potential role in epigenetic regulation. RESULTS: Of 383 HBV isolates from European patients, HBV genotypes A-G were identified, with the most frequent being genotype D (51.96%) in 12 countries, followed by A (39.16%) in 7 countries, and then E (3.66%), G (2.87%), B (1.57%), F (0.52%) and C (0.26%). A higher rate of mutant isolates were identified in those with genotype D (46.7%) followed by G (45.5%), and mutations were associated with structural and functional abnormalities of HBV genes. Conventional CpG island I was observed in genotypes A, B, C, D and E. Conventional islands II and III were detected in all A-G genotypes. A novel CpG island IV was found in genotypes A, D and E, and island V was only observed in genotype F. The A-G genotypes lacked the novel CpG island VI. "Split" CpG island I in genotypes D and E and "split" island II in genotypes A, D, E, F and G were observed. Two mutant isolates from genotype D and one from E were found to lack both CpG islands I and III. CONCLUSIONS: HBV genotypes A-G were identified in European patients. Structural and functional abnormalities of HBV genes were caused by mutations leading to the association of genotypes D and G with increased severity of liver disease. The distribution, length and genetic traits of CpG islands were different between genotypes and their biological and clinical significances warrant further study, which will help us better understand the potential role of CpG islands in epigenetic regulation of the HBV genome.

The prognostic significance of serum and cerebrospinal fluid MMP-9, CCL2 and sVCAM-1 in leukemia CNS metastasis.

Metastasis to the central nervous system (CNS) is the primary obstacle in leukemia treatment. Matrix metalloproteinase-9 (MMP-9), chemokine ligand-2 (CCL2) and soluble vascular adhesion molecule-1 (sVCAM-1) play crucial roles in tumor cell adhesion, motivation and survival, but their roles in leukemia CNS metastasis remain to be elucidated. We investigated the prognostic significance of serum and cerebrospinal fluid (CSF) MMP-9, CCL2 and sVCAM-1 in leukemia patients to explore their potential as predictive biomarkers of the development of CNS leukemia (CNSL). MMP-9, CCL2 and sVCAM-1 were measured in paired CSF and serum samples collecting from 33 leukemia patients with or without CNS metastasis. Other risk factors related to CNSL prognosis were also analyzed. sVCAM-1Serum and CCL2Serum/CSF were significantly higher in the CNSL group than in the non-CNSL group and the controls (p < 0.05). MMP-9Serum was insignificantly lower in the CNSL group than in the non-CNSL group and the controls (p > 0.05). No differences were found for the sVCAM-1Serum, CCL2Serum, and MMP-9Serum levels between non-CNSL patients and controls (p > 0.05). MMP-9CSF was significantly higher in the CNSL group than both the non-CNSL and the control groups (p < 0.05). The indexes of sVCAM-1, CCL2, and MMP-9 in the CNSL group were lower than in the controls (p < 0.05). Positive correlations were determined between the MMP-9CSF and the ALBCSF/BBB value/WBCCSF, between sVCAM-1Serum and the WBCCSF/BBB value. Negative correlations existed between MMP-9Serum and the ALBCSF/BBB value/WBCCSF, and between the CCL2 index and ALBCSF. sVCAM-1Serum was positively associated with event-free survival (EFS), and patients with higher levels of ALBCSF, MMP-9CSF/Serum, CCL2CSF/Serum, and sVCAM-1CSF/Serum had shorter EFS. MMP-9CSF, CCL2CSF and sVCAM-1CSF are the first three principal components analyzed by cluster and principal component analysis. Our data suggest that MMP-9, CCL2 and sVCAM-1 in the CSF may be more potent than serum in predicting the possibility of leukemia metastatic CNS and the outcome of CNSL patients.

Factors affecting sperm fertilizing capacity in men infected with HIV.

Studies on the sperm-fertilizing capacity of HIV-seropositive men show conflicting results for reasons that are not yet clear. The aim of this study was to investigate the effects and relationships of some factors such as patient age, CD4(+) cells count, fathering offspring, concomitant sexually transmitted diseases (STD), and receipt of highly active anti-retroviral therapy (HAART) on sperm fertilizing capacity. Semen samples were collected from 33 HIV-seropositive men. Data on the above factors were acquired from a self-designed questionnaire. Computer-assisted sperm analysis, a hypo-osmotic swelling, and zona-free hamster oocyte penetration tests were performed according to criteria of the World Health Organization. CD4(+) cells in peripheral blood were examined using a flow cytometric (FCM) analyzer. Sperm vitality, sperm motility (grades a + b), total sperm motility, and sperm penetration rates were significantly higher in patients whose CD4(+) counts were ≧350/µl than in those whose CD4(+) counts were <350/µl (P < 0.05), and the parameters mentioned above were also significantly correlated with CD4(+) cell number (all P < 0.05). Significant differences in total sperm count and sperm tail swelling rate between patients co-infected with STD and without STD were observed (P < 0.05). Sperm penetration rate in patients receiving HAART was significantly higher than in those not receiving HAART (P < 0.05). Blood CD4(+) cell counts are an important indicator for evaluating sperm fertilizing capacity of HIV-seropositive men. After receiving HAART, the sperm penetration rate of HIV-seropositive men can be improved.

Highly efficient removal of chromium(VI) from aqueous solution using polyaniline/sepiolite nanofibers.

Polyaniline/sepiolite (PANI/sepiolite) nanofibers were prepared by in situ chemical oxidation polymerization in the presence of sepiolite. The effect of aniline/sepiolite weight ratio on the nanostructure of PANI/sepiolite composites was investigated by field-emission scanning electron microscopy. The adsorption of Cr(VI) onto the PANI/sepiolite nanofibers was highly dependent on pH values. The pseudo-second-order and Langmuir isothermal models can well describe the adsorption kinetics and adsorption isotherm, respectively. The maximum adsorption capacity of the PANI/sepiolite nanofibers for Cr(VI) was up to 206.6 mg/g at 25 °C and increased with the increase in temperature. Desorption experiments indicated that PANI/sepiolite can be regenerated and reused for two consecutive cycles with no loss of its removal efficiency. PANI/sepiolite nanofibers can be used as a highly efficient and economically viable adsorbent for Cr(VI) removal due to their excellent adsorption characteristics.

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.

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.

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.

In vitro study on vertical transmission of the HIV-1 gag gene by human sperm.

HIV/AIDS is a major public health problem worldwide. To explore the feasibility of HIV vertical transmission by human sperm, plasmid construction and transfection, interspecific in vitro fertilization of zona-free hamster ova by human sperm, fluorescence in situ hybridization (FISH), RT-PCR, and immunofluorescence assay (IFA) were carried out. The FISH signals for HIV-1 gag DNA were observed in the nuclei and chromosomes of transfected human sperm, male pronuclei of zygotes, and nuclei of blastomeres of two-cell embryos, indicating that the HIV-1 gag gene could be transmitted via the sperm membrane and integrated into the sperm genome. In contrast, human sperm carrying the target gene achieved normal fertilization, and replication of the sperm-mediated target gene was synchronized with the host genome. Using RT-PCR, the positive bands for the target gene were observed in the transfected human sperm and two-cell embryos. These results further confirm that the target gene can be transcribed into mRNA in human sperm and embryonic cells. Positive signals for the HIV-1 p24 gag protein were shown by IFA in two-cell embryos containing the sperm-mediated target gene and not in the transfected human sperm, which indicated that the sperm-mediated target gene could be translated to make HIV-1 p24 gag protein in embryonic cells, but not in sperm cells. The results provide evidence for possible vertical transmission of the HIV-1 gag gene to the embryo by fertilizing sperm in vitro.

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.