Implications of deduplication on the detection rates of multidrug-resistant organism (MDRO) in various specimens: insights from the hospital infection surveillance program.

BACKGROUND: Currently, different guidelines recommend using different methods to determine whether deduplication is necessary when determining the detection rates of multidrug-resistant organisms (MDROs). However, few studies have investigated the effect of deduplication on MDRO monitoring data. In this study, we aimed to investigate the influence of deduplication on the detection rates of MDROs in different specimens to assess its impact on infection surveillance outcomes. METHODS: Samples were collected from hospitalized patients admitted between January 2022 and December 2022; four types of specimens were collected from key monitored MDROs, including sputum samples, urine samples, blood samples, and bronchoalveolar lavage fluid (BALF) samples. In this study, we compared and analysed the detection rates of carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Escherichia coli (CRECO), carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and methicillin-resistant Staphylococcus aureus (MRSA) under two conditions: with and without deduplication. RESULTS: When all specimens were included, the detection rates of CRKP, CRAB, CRPA, and MRSA without deduplication (33.52%, 77.24%, 44.56%, and 56.58%, respectively) were significantly greater than those with deduplication (24.78%, 66.25%, 36.24%, and 50.83%, respectively) (all P < 0.05). The detection rates in sputum samples were significantly different between samples without duplication (28.39%, 76.19%, 46.95%, and 70.43%) and those with deduplication (19.99%, 63.00%, 38.05%, and 64.50%) (all P < 0.05). When deduplication was not performed, the rate of detection of CRKP in urine samples reached 30.05%, surpassing the rate observed with deduplication (21.56%) (P < 0.05). In BALF specimens, the detection rates of CRKP and CRPA without deduplication (39.78% and 53.23%, respectively) were greater than those with deduplication (31.62% and 42.20%, respectively) (P < 0.05). In blood samples, deduplication did not have a significant impact on the detection rates of MDROs. CONCLUSION: Deduplication had a significant effect on the detection rates of MDROs in sputum, urine, and BALF samples. Based on these data, we call for the Infection Prevention and Control Organization to align its analysis rules with those of the Bacterial Resistance Surveillance Organization when monitoring MDRO detection rates.

Hypoxia preconditioning of human amniotic mesenchymal stem cells enhances proliferation and migration and promotes their homing via the HGF/C-MET signaling axis to augment the repair of acute liver failure.

BACKGROUND: Transplantation of mesenchymal stem cells (MSCs) is a newly developed strategy for treating acute liver failure (ALF). Nonetheless, the low survival rate of MSCs after transplantation and their poor homing to damaged tissues limit the clinical application of MSCs. The research assessed whether hypoxic preconditioning (HPC) can improve the biological activity of human amniotic mesenchymal stem cells (hA-MSCs), promote their homing ability to the liver of mice with ALF, and influence liver tissue repair. METHODS: Flow cytometry, CCK8, Transwell, and Western blotting assays were conducted to assess the effects of hypoxic preconditioning on the phenotype, proliferation, and migration of hA-MSCs and the changes in the c-Met and CXCR4 gene expression levels were studied. To evaluate the effects of the transplantation of hypoxic preconditioning of hA-MSCs on the homing and repair of D-galactosamine (D-GalN)/LPS-induced ALF, the mechanism was elucidated by adding c-Met, CXCR4-specific blockers (SU11274 and AMD3100). RESULTS: After hypoxia pretreatment (1% oxygen volume fraction), hA-MSCs maintained the morphological characteristics of adherence and vortex colony growth and showed high CD44, CD90, and CD105 and low CD31, CD34, and CD45 expression levels. Hypoxic preconditioning of hA-MSCs significantly increased their proliferation and migration and highly expressed the c-Met and CXCR4 genes. In vivo and in vitro, this migration-promoting effect was suppressed by the c-Met specific blocker SU11274. In the acute liver failure mouse model, the HGF expression level was considerably elevated in the liver than that in the serum, lungs and kidneys. The transplantation of hypoxic preconditioned hA-MSCs introduced a remarkable improvement in the liver function and survival rate of mice with ALF and enhanced the anti-apoptosis ability of liver cells. The anti-apoptotic enhancing effect of hypoxic preconditioning was suppressed by the c-Met specific blocker SU11274. Hypoxic hA-MSCs administration was observed to have considerably increased the fluorescent cells in the liver than that recorded after administering normal oxygen-hA-MSCs. The number of hepatic fluorescent cells decreased remarkably after adding the c-Met inhibitor SU11274, compared to that recorded after hypoxic pretreatment, whereas the effect of c-Met inhibitor SU11274 on normal oxygen-hA-MSCs was not significant. CONCLUSIONS: Hypoxic preconditioning depicted no impact on the morphology and phenotype features of the human amniotic mesenchymal stem cells, but it can promote their proliferation, migration, anti-apoptotic effect, and homing rate and improve the repair of acute liver failure, which might be mediated by the HGF/c-Met signaling axis.

Nanopore sequencing of infectious fluid is a promising supplement for gold-standard culture in real-world clinical scenario.

Introduction: Infectious diseases are major causes of morbidity and mortality worldwide, necessitating the rapid identification and accurate diagnosis of pathogens. While unbiased metagenomic next-generation sequencing (mNGS) has been extensively utilized in clinical pathogen identification and scientific microbiome detection, there is limited research about the application of nanopore platform-based mNGS in the diagnostic performance of various infectious fluid samples. Methods: In this study, we collected 297 suspected infectious fluids from 10 clinical centers and detected them with conventional microbiology culture and nanopore platform-based mNGS. The objective was to assess detective and diagnostic performance of nanopore-sequencing technology (NST) in real-world scenarios. Results: Combined with gold-standard culture and clinical adjudication, nanopore sequencing demonstrated nearly 100% positive predictive agreements in microbial-colonized sites, such as the respiratory and urinary tracts. For samples collected from initially sterile body sites, the detected microorganisms were highly suspected pathogens, and the negative predictive agreements were relatively higher than those in the microbial-colonized sites, particularly with 100% in abscess and 95.7% in cerebrospinal fluid. Furthermore, consistent performance was also observed in the identification of antimicrobial resistance genes and drug susceptibility testing of pathogenic strains of Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii. Discussion: Rapid NST is a promising clinical tool to supplement gold-standard culture, and it has the potential improve patient prognosis and facilitate clinical treatment of infectious diseases.

Severe pneumonia with co-infection of H5N1 and SARS-CoV-2: a case report.

BACKGROUND: The H5N1 influenza virus is a cause of severe pneumonia. Co-infection of influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may lead to poor prognosis of patients during the COVID-19 epidemic. However, reports on patients co-infected with avian influenza virus and SARS-CoV-2 are scarce. CASE PRESENTATION: A 52-year-old woman presented with a fever, which has persisted for the past eight days, along with worsening shortness of breath and decreased blood pressure. Computed tomography (CT) revealed an air bronchogram, lung consolidation, and bilateral pleural effusion. The subsequent polymerase chain reaction (PCR) of the bronchoalveolar lavage fluid (BALF) revealed positivity for H5N1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). CONCLUSION: The H5N1 influenza virus is a cause of severe pneumonia. The clinical presentation of the patient had a predomination of H5N1 influenza rather than COVID-19. A PCR analysis for the identification of the virus is necessary to reveal the pathogen causing the severe pneumonia. The patient exhibited an excellent prognosis upon the use of the appropriate antiviral medicine.

Metformin exerts anti-liver fibrosis effect based on the regulation of gut microbiota homeostasis and multi-target synergy.

Liver fibrosis can progress to cirrhosis if left untreated. Therefore, identifying effective antifibrotic drugs is crucial. This study aimed to investigate the role and potential mechanism of metformin in treating hepatic fibrosis based on the synergistic effect of multiple targets and the "intestine-liver axis" theory. A CCl4-induced liver fibrosis mouse model was established. We measured liver function, liver fibrosis indicators, oxidative stress and inflammation indices. Hematoxylin and eosin and Masson's trichrome staining were used to detect collagen deposition. The expression of apoptotic proteins, TGF-ß/Smads and TIMP-1/MMPs was assessed. 16S rRNA and untargeted metabolomics (liquid chromatography-mass spectrometry) were used to assess mouse intestinal flora and metabolites, performing a comprehensive correlation analysis. Metformin improved the general status and liver function and decreased liver collagen deposition in CCl4-induced liver fibrotic mice. Compared with the control group, IL-6, TNF-α and COX-2 serum levels in the liver fibrosis group increased. Although not significantly different, the serum inflammatory marker levels in the metformin group were lower than those in the model group. Metformin decreased serum MDA and increased serum SOD activity, which increased and decreased, respectively, in the model group. Furthermore, metformin inhibited liver cell apoptosis, TGF-ß1 expression and TIMP-1, while promoting Smad7 expression, MMP-1 and MMP-2 in fibrotic mice. 16S rRNA analysis indicated that metformin significantly ameliorated the Bacteroides, Helicobacter, Parabacteroides and Parasutterella imbalance. We identified 385 differential metabolites between the metformin and model groups. Prevotella abundance significantly decreased in the metformin group and positively correlated with decreased taurocholic acid levels. Metformin potentially reverses liver fibrosis by inhibiting inflammation, mitigating oxidative stress damage and suppressing hepatocyte apoptosis via intestinal flora metabolite regulation. Metformin also regulates the TGF-ß/Smads and TIMP-1/MMPs signalling pathways. This study provides a theoretical basis for the clinical use of metformin in patients with liver fibrosis.

Antibiotic prophylaxis after 48 h postoperatively are not associated with decreased surgical site infections and other healthcare associated infections in pancreatic surgery patients: a retrospective cohort study.

BACKGROUND: It is controversial whether antibiotic should be used prophylactically 48 h after pancreatic surgery. Hence, the association of antibiotic prophylaxis (AP) after 48 h postoperatively with the incidence of surgical site infections (SSIs) and other healthcare-associated infections (HAIs) in patients receiving pancreatic surgery was evaluated. METHODS: A retrospective cohort analysis was performed on 1073 patients who underwent pancreatic surgery. These patients were categorized into the non-AP after 48 h postoperatively group (n = 963) and the AP after 48 h postoperatively group (n = 110) based on whether or not they obtained AP from 48 h to 30 days after surgery. Outcomes included SSIs and other HAIs. RESULTS: The incidence of SSIs in the non-AP after 48 h postoperatively group (98/963, 10.2%) was notably lower than that in the AP after 48 h postoperatively group (22/110, 20.0%) (P = 0.002). Other HAIs incidence was not significantly different between the non-AP after 48 h postoperatively group (77/963, 8.0%) and the AP after 48 h postoperatively group (11/110, 10.0%) (P = 0.468). Multiple regression analysis demonstrated that AP after 48 h postoperatively was a risk factor for SSIs (OR = 2.14, 95% CI 1.28-3.59) but not for other HAIs (OR = 1.24, 95% CI 0.63-2.42) after adjustment for age, gender, and diabetes. Subsequent to adjustment for all confounding factors, AP after 48 h postoperatively was not a influence factor for SSIs (OR = 2.13, 95% CI 0.76-5.99) and other HAIs (OR = 3.69, 95% CI 0.99-13.81). CONCLUSIONS: AP after 48 h postoperatively following pancreatic surgery was not associated with the lower morbidity rate of SSIs and other HAIs. Nonetheless, this study may facilitate further development of strategies towards standardization of the duration of AP management of pancreatic surgery.

The change of Siglec-9 expression in peripheral blood NK cells of SFTS patients can affect the function of NK cells.

OBJECTIVES: To investigate the changes and mechanism of Siglec-9 on NK cells in peripheral blood of patients with severe fever with thrombocytopenia syndrome (SFTS). METHODS: First, we used single-cell RNA sequencing to analyze the frequency of NK cells in Peripheral Blood Mononuclear Cells (PBMCs) of SFTS patients and healthy controls (HCs), as well as the differences in the genes on NK cells. Secondly, we analyzed the expression of Siglec-9 and other receptors on NK cells by flow cytometry. Thirdly, we analyzed the correlation between Siglec-9 on NK cells and DBV viral load in plasma. RESULTS: Compared with HCs, the frequency of NK cells in peripheral blood of SFTS patients was significantly decreased, and the activating receptors on NK cells were reduced. The expression of Siglec-9 on NK cells and the frequency of Siglec-9+NK cells decreased significantly in SFTS patients. The expression of Siglec-9 on CD16+CD56dim NK cells was negatively correlated with DBV viral load. In addition, Siglec-9+NK cells expressed higher levels of activating receptors and exhibited stronger effector functions than Siglec-9-NK cells. CONCLUSIONS: The decreased expression of Siglec-9 on NK cells predicts NK cell dysfunction in SFTS patients, suggesting that Siglec-9 may be a potential marker for functional NK cell subsets in SFTS patients.

Role of type VI secretion system protein TssJ-3 in virulence and intracellular survival of Burkholderia pseudomallei.

TssJ-3 is an outer-membrane lipoprotein and is one of the key components of the type VI secretion system in Burkholderia pseudomallei. TssJ translocates effector proteins to target cells to induce innate immune response in the host. However, the tssJ gene has not been identified in B. pseudomallei and its function in this bacterium has not yet been characterized. tssJ-3 knockout and tssJ-3-complemented B. pseudomallei strains were constructed to determine the effects of tssJ-3 on bacterial growth, biofilm formation, flagellum synthesis, motility, host cell infection, and gene expression in B. pseudomallei. We found that the ΔtssJ-3 mutant strain of B. pseudomallei showed significantly suppressed biofilm formation, flagellum synthesis, bacterial growth, motility, and bacterial invasion into host cells (A549 cells). Furthermore, the ΔtssJ-3 mutation downregulated multiple key genes, including biofilm and flagellum-related genes in B. pseudomallei and induced interleukin-8 gene expression in host cells. These results suggest that tssJ-3, an important gene controlling TssJ-3 protein expression, has regulatory effects on biofilm formation and flagellum synthesis in B. pseudomallei. In addition, B. pseudomallei-derived tssJ-3 contributes to cell infiltration and intracellular replication. This study provides a molecular basis of tssJ-3 for developing therapeutic strategies against B. pseudomallei infections.

Inhibition of PRMT1 alleviates sepsis-induced acute kidney injury in mice by blocking the TGF-ß1 and IL-6 trans-signaling pathways.

Sepsis-induced acute kidney injury (SI-AKI) causes renal dysfunction and has a high mortality rate. Protein arginine methyltransferase-1 (PRMT1) is a key regulator of renal insufficiency. In the present study, we explored the potential involvement of PRMT1 in SI-AKI. A murine model of SI-AKI was induced by cecal ligation and perforation. The expression and localization of PRMT1 and molecules involved in the transforming growth factor (TGF)-ß1/Smad3 and interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) signaling pathways were detected in mouse kidney tissues by western blot analysis, immunofluorescence, and immunohistochemistry. The association of PRMT1 with downstream molecules of the TGF-ß1/Smad3 and IL-6/STAT3 signaling pathways was further verified in vitro in mouse renal tubular epithelial cells. Cecal ligation and perforation caused epithelial-mesenchymal transition, apoptosis, and inflammation in renal tissues, and this was alleviated by inhibition of PRMT1. Inhibition of PRMT1 in SI-AKI mice decreased the expression of TGF-ß1 and phosphorylation of Smad3 in the renal cortex, and downregulated the expression of soluble IL-6R and phosphorylation of STAT3 in the medulla. Knockdown of PRMT1 in mouse renal tubular epithelial cells restricted the expression of Cox-2, E-cadherin, Pro-caspase3, and phosphorylated Smad3 (involved in the TGF-ß1-mediated signaling pathway), and also blocked IL-6/soluble IL-6R, inducing the expression of Cox-2 and phosphorylated-STAT3. In conclusion, our findings suggest that inhibition of PRMT1 mitigates SI-AKI by inactivating the TGF-ß1/Smad3 pathway in the cortex and the IL-6/STAT3 pathway in the medulla. Our findings may aid in the identification of potential therapeutic target molecules for SI-AKI.

METTL14 reverses liver fibrosis by inhibiting NOVA2 through an m6A-YTHDF2-dependent mechanism.

BACKGROUND: N6-methyladenosine (m6A), the most prevalent internal RNA modification in eukaryotic cells, is dynamically regulated in response to a wide range of physiological and pathological states. Nonetheless, the involvement of METTL14-induced m6A in liver fibrosis (LF) has yet to be established. METHODS: In vitro, HSC cell lines with knock-down and overexpression of METTL14 were constructed, and the effects of METTL14 gene on the phenotypic function of activated HSCs were observed. The proliferation rate was measured by CCK8 and EDU, the cell proliferation cycle was measured by flow detector, the migration rate was measured by Transwell, and the contractility of F-actin was observed after phalloidin staining. The downstream target gene NOVA2 of METTL14 was screened by combined sequencing of MeRIP-seq and RNA-seq, combined with signal analysis. Adeno-associated virus (AAV) was injected into the tail vein in vivo to knock down the expression of METTL14, so as to further observe the role of METTL14 in the progress of LF. RESULTS: our research showed that the methylase METTL14 content was decreased in hepatic tissue from patients with LF, leading to a lowered degree of m6A modification. Functionally, we discovered that knocking down m6A methyltransferase METTL14 led to increased HSC activation and a substantial worsening of LF. Mechanically, as shown in a multiomics study of HSCs, depleting METTL14 levels decreased m6A deposition onNOVA2 mRNA transcripts, which prompted the activation of YTHDF2 to detect and degrade the decrease of NOVA2 mRNA. CONCLUSIONS: METTL14 functioned as a profibrotic gene by suppressing NOVA2 activity in a mechanism dependent on m6A-YTHDF2. Moreover, knocking down METTL14 exacerbated LF, while NOVA2 prevented its development and partly reversed the damage.

The Spliceosome Factor EFTUD2 Promotes IFN Anti-HBV Effect through mRNA Splicing.

Methods: Using a CRISPR/Cas9 gene-editing system, EFTUD2 single allele knockout HepG2.2.15 cells were constructed. Subsequently, the HBV biomarkers in EFTUD2+/- HepG2.2.15 cells and wild-type (WT) cells with or without IFN-α treatment were detected. And the EFTUD2-regulated genes were then identified using mRNA sequence. Selected gene mRNA variants and their proteins were examined by qRT-PCR and Western blotting. To confirm the effects of EFTUD2 on HBV replication and IFN-stimulated gene (ISG) expression, a rescue experiment in EFTUD2+/- HepG2.2.15 cells was performed by EFTUD2 overexpression. Results: IFN-induced anti-HBV activity was found to be restricted in EFTUD2+/- HepG2.2.15 cells. The mRNA sequence showed that EFTUD2 could regulate classical IFN and virus response genes. Mechanistically, EFTUD2 single allele knockout decreased the expression of ISG-encoded proteins, comprising Mx1, OAS1, and PKR (EIF2AK2), through mediated gene splicing. However, EFTUD2 did not affect the expression of Jak-STAT pathway genes. Furthermore, EFTUD2 overexpression could restore the attenuation of IFN anti-HBV activity and the reduction of ISG resulting from EFTUD2 single allele knockout. Conclusion: EFTUD2, the spliceosome factor, is not IFN-inducible but is an IFN effector gene. EFTUD2 mediates IFN anti-HBV effect through regulation of gene splicing for certain ISGs, including Mx1, OAS1, and PKR. EFTUD2 does not affect IFN receptors or canonical signal transduction components. Therefore, it can be concluded that EFTUD2 regulates ISGs using a novel, nonclassical mechanism.

TRIM3 attenuates cytokine storm caused by Dabie bandavirus via promoting Toll-like receptor 3 degradation.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease that was caused by the Dabie bandavirus (DBV), and it has become a global public health threat. Cytokine storm is considered to be an important pathogenesis of critical SFTS. Tripartite motif-containing 3 (TRIM3), as a member of the TRIM protein family, may contribute to the regulation of the immune and inflammatory responses after viral infection. However, whether TRIM3 plays a major role in the pathogenesis of SFTS has not yet been investigated. Methods: TRIM3 mRNA levels were detected in PBMCs between 29 SFTS patients and 29 healthy controls by qRT-PCR. We established the pathogenic IFNAR-/- SFTS mouse model successfully by inoculating subcutaneously with DBV and testing the expression levels of TRIM3 mRNA and protein by qRT-PCR and immunofluorescence in the livers, spleens, lungs, and kidneys. TRIM3OE THP-1 cells and peritoneal macrophages extracted from TRIM3-/- mice were infected with DBV. The effect of TRIM3 on cytokines was detected by qRT-PCR and ELISA. Then we examined Toll-like receptor 3 (TLR3) and protein phosphorylation in the MAPK pathway after DBV infection using Western blot. Flow cytometry was used to verify TLR3 expression on peripheral blood monocytes in SFTS patients. We further explored the interaction between TRIM3 and TLR3 using CO-IP and Western blot. Results: Compared to healthy controls, TRIM3 mRNA expression in PBMCs is decreased in SFTS patients, especially in severe cases. TRIM3 mRNA and protein were synchronously reduced in the livers, spleens, lungs, and kidney tissues of the IFNAR-/- SFTS mice model. In the DBV-infected cell model, TRIM3 overexpression can inhibit the DBV-induced release of IL-1ß, IL-6, and TNF-α, the expression of TLR3, and protein phosphorylation in the MAPK pathway, which plays an anti-inflammatory role, while TRIM3 deficiency exacerbates the pro-inflammatory effects. We further found that TRIM3 can promote TLR3 degradation through K48-linked ubiquitination. Conclusion: TRIM3 can inhibit the production of cytokines by regulating the degradation of TLR3 through K48-linked ubiquitination, which can be a therapeutic target for improving the prognosis of SFTS.

Macrophage PTEN controls STING-induced inflammation and necroptosis through NICD/NRF2 signaling in APAP-induced liver injury.

BACKGROUND: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling has been known to play a critical role in maintaining cellular and tissue homeostasis, which also has an essential role in the inflammatory response. However, it remains unidentified whether and how the macrophage PTEN may govern the innate immune signaling stimulator of interferon genes (STING) mediated inflammation and hepatocyte necroptosis in APAP-induced liver injury (AILI). METHODS: Myeloid-specific PTEN knockout (PTENM-KO) and floxed PTEN (PTENFL/FL) mice were treated with APAP (400 mg/kg) or PBS. In a parallel in vitro study, bone marrow-derived macrophages (BMMs) were isolated from these conditional knockout mice and transfected with CRISPR/Cas9-mediated Notch1 knockout (KO) or CRISPR/Cas9-mediated STING activation vector followed by LPS (100 ng/ml) stimulation. RESULTS: Here, we report that myeloid-specific PTEN knockout (PTENM-KO) mice were resistant to oxidative stress-induced hepatocellular injury with reduced macrophage/neutrophil accumulation and proinflammatory mediators in AILI. PTENM-KO increased the interaction of nuclear Notch intracellular domain (NICD) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) in the macrophage nucleus, reducing reactive oxygen species (ROS) generation. Mechanistically, it is worth noting that macrophage NICD and NRF2 co-localize within the nucleus under inflammatory conditions. Additionally, Notch1 promotes the interaction of immunoglobulin kappa J region (RBPjκ) with NRF2. Disruption of the Notch1 signal in PTEN deletion macrophages, reduced RBPjκ and NRF2 binding, and activated STING signaling. Moreover, PTENM-KO macrophages with STING activated led to ROS generation and TNF-α release, resulting in hepatocyte necroptosis upon co-culture with primary hepatocytes. CONCLUSIONS: Our findings demonstrate that the macrophage PTEN-NICD/NRF2-STING axis is critical to regulating oxidative stress-induced liver inflammation and necroptosis in AILI and implies the therapeutic potential for managing sterile liver inflammation. Video Abstract.

Exosomes from hepatitis B virus-infected hepatocytes activate hepatic stellate cells and aggravate liver fibrosis through the miR-506-3p/Nur77 pathway.

Cumulative evidence indicates the important role of Nur77 in organ fibrogenesis. However, the role of Nur77 in hepatitis B virus (HBV)-related liver fibrosis (LF) remains unclear. Cells were transfected with the microRNA mimic miRNA-506-3p or inhibitor, and pcDNA3.1-Nur77 or Nur77 guide RNA. Exosomes were isolated from HBV-infected HepG2-sodium taurocholate cotransporting polypeptide cells. The levels of miR-506-3p, Nur77, and LF-related genes and proteins were detected by quantitative polymerase chain reaction (qPCR) and western blot analysis, respectively. The pathology of the liver from HBV-infected patients was examined using hematoxylin-eosin and Masson's staining. The expression of Nur77 in liver tissue was determined by immunohistochemistry, and the LF score was assessed using the METAVIR system. The relationship between miR-506-3p/Nur77 and LF score was analyzed by correlation analysis. HBV infection downregulated miR-506-3p expression and upregulated Nur77 levels in hepatocytes. Exosomes from HBV-infected hepatocytes also displayed decreased gene expression of miR-506-3p and increased expressions of Nur77- and LF-related genes in stellate cells compared with exosomes from hepatocytes with mock infection. These changes were reversed by Nur77 guide RNA. Nur77 expression in liver tissue was strongly correlated with LF, whereas serum miR-506-3p was strongly negatively correlated with LF. Exosomes from HBV-infected hepatocytes activate stellate cells and aggravate LF through the miR-506-3p/Nur77 pathway. These exosomes may be the basis of a promising therapeutic strategy.

Cryptotanshinone induces apoptosis of activated hepatic stellate cells via modulating endoplasmic reticulum stress.

BACKGROUND: Cryptotanshinone (CPT) has wide biological functions, including anti-oxidative, antifibrosis, and anti-inflammatory properties. However, the effect of CPT on hepatic fibrosis is unknown. AIM: To investigate the effects of CPT treatment on hepatic fibrosis and its underlying mechanism of action. METHODS: Hepatic stellate cells (HSCs) and normal hepatocytes were treated with different concentrations of CPT and salubrinal. The CCK-8 assay was used to determine cell viability. Flow cytometry was used to measure apoptosis and cell cycle arrest. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analyses were used to measure mRNA levels and protein expression of endoplasmic reticulum stress (ERS) signaling pathway related molecules, respectively. Carbon tetrachloride (CCL4) was used to induce in vivo hepatic fibrosis in mice. Mice were treated with CPT and salubrinal, and blood and liver samples were collected for histopathological examination. RESULTS: We found that CPT treatment significantly reduced fibrogenesis by modulating the synthesis and degradation of the extracellular matrix in vitro. CPT inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in cultured HSCs. Furthermore, we found that CPT promoted apoptosis of activated HSCs by upregulating expression of ERS markers (CHOP and GRP78) and activating ERS pathway molecules (PERK, IRE1α, and ATF4), which were inhibited by salubrinal. Inhibition of ERS by salubrinal partially eliminated the therapeutic effect of CPT in our CCL4-induced hepatic fibrosis mouse model. CONCLUSION: CPT can promote apoptosis of HSCs and alleviate hepatic fibrosis through modulating the ERS pathway, which represents a promising strategy for treating hepatic fibrosis.

Characterizing the Wolbachia infection in field-collected Culicidae mosquitoes from Hainan Province, China.

BACKGROUND: Mosquitoes are vectors of many pathogens, such as malaria, dengue virus, yellow fever virus, filaria and Japanese encephalitis virus. Wolbachia are capable of inducing a wide range of reproductive abnormalities in their hosts, such as cytoplasmic incompatibility. Wolbachia has been proposed as a tool to modify mosquitoes that are resistant to pathogen infection as an alternative vector control strategy. This study aimed to determine natural Wolbachia infections in different mosquito species across Hainan Province, China. METHODS: Adult mosquitoes were collected using light traps, human landing catches and aspirators in five areas in Hainan Province from May 2020 to November 2021. Species were identified based on morphological characteristics, species-specific PCR and DNA barcoding of cox1 assays. Molecular classification of species and phylogenetic analyses of Wolbachia infections were conducted based on the sequences from PCR products of cox1, wsp, 16S rRNA and FtsZ gene segments. RESULTS: A total of 413 female adult mosquitoes representing 15 species were identified molecularly and analyzed. Four mosquito species (Aedes albopictus, Culex quinquefasciatus, Armigeres subalbatus and Culex gelidus) were positive for Wolbachia infection. The overall Wolbachia infection rate for all mosquitoes tested in this study was 36.1% but varied among species. Wolbachia types A, B and mixed infections of A × B were detected in Ae. albopictus mosquitoes. A total of five wsp haplotypes, six FtsZ haplotypes and six 16S rRNA haplotypes were detected from Wolbachia infections. Phylogenetic tree analysis of wsp sequences classified them into three groups (type A, B and C) of Wolbachia strains compared to two groups each for FtsZ and 16S rRNA sequences. A novel type C Wolbachia strain was detected in Cx. gelidus by both single locus wsp gene and the combination of three genes. CONCLUSION: Our study revealed the prevalence and distribution of Wolbachia in mosquitoes from Hainan Province, China. Knowledge of the prevalence and diversity of Wolbachia strains in local mosquito populations will provide part of the baseline information required for current and future Wolbachia-based vector control approaches to be conducted in Hainan Province.

Molecular detection of Enterocytozoon bieneusi in farmed Asiatic brush-tailed porcupines (Atherurus macrourus) and bamboo rats (Rhizomys pruinosus) from Hainan Province, China: Common occurrence, wide genetic variation and high zoonotic potential.

We investigated the occurrence and genotypic diversity of E. bieneusi in farmed Asiatic brush-tailed porcupines and bamboo rats from Hainan Province, China. Four hundred and sixty-seven fresh feces were collected from 164 Asiatic brush-tailed porcupines and 303 bamboo rats. DNA extraction from the feces and genotyping of E. bieneusi were performed by the amplification of the internal transcribed spacer (ITS) region of rDNA of E. bieneusi using PCR. A neighbor-joining tree was constructed based on the sequences obtained here and other sequences of E. bieneusi genotypes stored in Genbank. The total rate of infection with E. bieneusi was 32.5% (152/467), with 14.6% (24/164) in Asiatic brush-tailed porcupines and 42.2% (128/303) in bamboo rats infected. Seventeen genotypes of E. bieneusi were identified including 12 known genotypes, i.e., D (n = 78), Henan-III (n = 21), SHW7 (n = 19), KIN-1 (n = 11), ETMK5 (n = 7), TypeIV (n = 4), EbpD (n = 2), EbpA (n = 1), EbpC (n = 1), S7 (n = 1), HNPL-III (n = 1), HNR-VII (n = 1), and five novel genotypes named as HNZS-I (n = 1) and HNHZ-I to HNHZ-IV (n = 1 per genotype). Phylogenetic analysis revealed that all the genotypes found here except genotype S7 fell into Group 1. The present study demonstrated a relatively high prevalence of E. bieneusi infection (32.5%) and a large genetic variation of E. bieneusi (seventeen genotypes) in farmed Asiatic brush-tailed porcupines and bamboo rats in Hainan, China. The high proportion (78.3%) of zoonotic genotypes identified in the animals investigated here suggests that there is the potential for zoonotic or cross-species transmission which may pose a serious public health threat in the area. Public education on the management of Asiatic brush-tailed porcupines and bamboo rats should be implemented in the investigated areas.

KIR2DL4/HLA-G polymorphisms were associated with HCV infection susceptibility among Chinese high-risk population.

Killer-cell immunoglobulin-like receptors 2DL4 (KIR2DL4) and the human leukocyte antigen class I-G (HLA-G) display vital parts in immune responses against hepatitis C virus (HCV) infection. We select four potentially functional single nucleotide polymorphisms (SNPs) of KIR/HLA to explore the associations between KIR2DL4/HLA-G genetic variants and HCV infection results. In the present case-control study, a total of 2225 HCV-infected high-risk subjects, including 1778 paid blood donors (PBD) and 447 drug users were consecutively recruited before treatment from 2011 to 2018. KIR2DL4-rs660773, KIR2DL4-rs660437, HLA-G-rs9380142, and HLA-G-rs1707 SNPs were sorted as genotypes in the subdivided groups, involving 1095 uninfected controls subjects, 432 spontaneous HCV clearance subjects and 698 HCV persistent infection subjects. After genotyping experiments using the TaqMan-MGB assay, modified logistic regression was used to calculate the correlation among the SNPs and HCV infection. The SNPs were functionally annotated using bioinformatics analysis. Following adjusting by age, sex, alanine aminotransferase, aspartate aminotransferase, IFNL3-rs12979860, IFNL3-rs8099917, and the infection route, the logistic regression analysis discovered that KIR2DL4-rs660773 and HLA-G-rs9380142 were correlated with vulnerability to HCV infection (all p < 0.05). In a locus-dosage way, compared with subjects carrying the rs9380142-AA or rs660773-AA genotypes, subjects with rs9380142-AG or rs660773-AG/GG (all p < 0.05) were more vulnerable to HCV infection; the overall impact of their risk genotypes (rs9380142-AGrs660773-AG/GG) was correlated with an elevated incidence of HCV infection (ptrend < 0.001). In the Haplotype analysis, patients with haplotype AG were more likely to contract HCV compared to those with the highest common AA haplotype (p = 0.002) were higher in susceptibility to infect HCV. The SNPinfo web server estimated that rs660773 is a transcription factor binding site, whereas rs9380142 is a potential microRNA-binding site. In two Chinese high-risk population (PBD and drug uesrs), KIR2DL4 rs660773-G and HLA-G rs9380142-G alleles polymorphisms are related to HCV susceptibility. KIR2DL4/HLA-G pathway genes might affect the innate immune responses by regulating KIR2DL4/HLA-G transcription and translation play a potential role in HCV infection.

Plerixafor and resatorvid inhibit hepatitis B virus in vitro by upregulating elongation factor Tu GTP-binding domain containing 2.

Background: An increase in the demand for a functional cure has accelerated research on new methods of therapy for chronic hepatitis B, which is mainly focused on restoring antiviral immunity for controlling viral infections. Previously, we had described elongation factor Tu GTP-binding domain containing 2 (EFTUD2) as an innate immune regulator and suggested that it might be an antiviral target. Methods: In this study, we generated the Epro-LUC-HepG2 cell model for screening compounds that target EFTUD2. Plerixafor and resatorvid were screened from 261 immunity and inflammation-related compounds due to their ability to highly upregulate EFTUD2. The effects of plerixafor and resatorvid on hepatitis B virus (HBV) were examined in HepAD38 cells and HBV-infected HepG2-NTCP cells. Results: The dual-luciferase reporter assays showed that the EFTUD2 promoter hEFTUD2pro-0.5 kb had the strongest activity. In Epro-LUC-HepG2 cells, plerixafor and resatorvid significantly upregulated the activity of the EFTUD2 promoter and the expression of the gene and protein. In HepAD38 cells and HBV-infected HepG2-NTCP cells, treatment with plerixafor and resatorvid strongly inhibited HBsAg, HBV DNA, HBV RNAs, and cccDNA in a dose-dependent manner. Furthermore, the anti-HBV effect was enhanced when entecavir was administered along with either of the previous two compounds, and the effect could be blocked by knocking down EFTUD2. Conclusion: We established a convenient model for screening compounds that target EFTUD2 and further identified plerixafor and resatorvid as novel HBV inhibitors in vitro. Our findings provided information on the development of a new class of anti-HBV agents that act on host factors rather than viral enzymes.