miR-155 promotes Th17 differentiation by targeting FOXP3 to aggravate inflammation in MRSA pneumonia.

BACKGROUND: Previous researches have clarified that miR-155 is increased in methicillin-resistant Staphylococcus aureus (MRSA) pneumonia, and modulates Th9 differentiation. Like Th9 cells, Th17 cells were also a subset of CD4+ T cells and involved in MRSA pneumonia progression. This work aimed to investigate the role and mechanism of miR-155 in Th17 differentiation. METHODS: Bronchoalveolar lavage fluid (BALF) was collected from children with MRSA pneumonia and bronchial foreign bodies. MRSA-infected murine model was established followed by collecting BALF and lung tissues. qRT-PCR, ELISA and flow cytometry were performed to examine the mRNA expression and concentration of IL-17 and the number of Th17 cells in above samples. HE and ELISA were used to evaluate inflammatory responses in lung. Furthermore, CD4+ T cells were isolated from BALF of children for in vitro experiments. After treatments with miR-155 mimic/inhibitor, the roles of miR-155 in Th17/IL-17 regulation were determined. The downstream of miR-155 was explored by qRT-PCR, western blotting, dual luciferase reporter analysis and RIP assay. RESULTS: The levels of IL-17 and the proportion of Th17 cells were increased in children with MRSA pneumonia. A similar pattern was observed in MRSA-infected mice. On the contrary, IL-17 neutralization abolished the activation of Th17/IL-17 induced by MRSA infection. Furthermore, IL-17 blockade diminished the inflammation caused by MRSA. In vitro experiments demonstrated miR-155 positively regulated IL-17 expression and Th17 differentiation. Mechanistically, FOXP3 was a direct target of miR-155. miR-155 inhibited FOXP3 level via binding between FOXP3 and Argonaute 2 (AGO2), the key component of RNA-induced silencing complex (RISC). FOXP3 overexpression reversed elevated IL-17 levels and Th17 differentiation induced by miR-155. CONCLUSIONS: miR-155 facilitates Th17 differentiation by reducing FOXP3 through interaction of AGO2 and FOXP3 to promote the pathogenesis of MRSA pneumonia. IL-17 blockade weakens the inflammation due to MRSA, which provides a nonantibiotic treatment strategy for MRSA pneumonia.

IL-9 promotes methicillin-resistant Staphylococcus aureus pneumonia by regulating the polarization and phagocytosis of macrophages.

In this study, we examined the effect of Il9 deletion on macrophages in methicillin-resistant Staphylococcus aureus (MRSA) infection. MRSA-infected mice were employed for the in vivo experiments, and RAW264.7 cells were stimulated with MRSA for the in vitro experiments. Macrophage polarization was determined by flow cytometry and quantitative real-time PCR; macrophage phagocytosis was assessed by flow cytometry and laser scanning confocal microscopy; cell apoptosis was assessed by flow cytometry and western blotting. Il9 deletion markedly elevated macrophage phagocytosis and M2 macrophages in MRSA infection, which was accompanied by elevated expression of Il10 and Arg1 and reduced expression of Inos, tumor necrosis factor-α (Tnfα), and Il6. Il9 deletion also inhibited macrophage apoptosis in MRSA infection, which was manifested by elevated B-cell lymphoma 2 (BCL-2) protein level and reduced protein levels of cleaved cysteine protease 3 (CASPASE-3) and BCL2-Associated X (BAX). Both the in vivo and in vitro experiments further showed the activation of phosphoinositide 3-kinase (PI3K)/AKT (also known as protein kinase B, PKB) signaling pathway in MRSA infection and that the regulation of Il9 expression may be dependent on Toll-like receptor (TLR) 2/PI3K pathway. The above results showed that Il9 deletion exhibited a protective role against MRSA infection by promoting M2 polarization and phagocytosis of macrophages and the regulation of Il9 partly owing to the activation of TLR2/PI3K pathway, proposing a novel therapeutic strategy for MRSA-infected pneumonia.

Ad5-nCoV booster and Omicron variant breakthrough infection following two doses of inactivated vaccine elicit comparable antibody levels against Omicron variants.

Little information is available for antibody levels against SARS-CoV-2 variants of concern induced by Omicron breakthrough infection and a third booster with an inactivated vaccine (InV) or Ad5-nCoV in people with completion of two InV doses. Plasma was collected from InV pre-vaccinated Omicron-infected patients (OIPs), unvaccinated OIPs between 0 and 22 days, and healthy donors (HDs) 14 days or 6 months after the second doses of an InV and 14 days after a homogenous booster or heterologous booster of Ad5-nCoV. Anti-Wuhan-, Anti-Delta-, and Anti-Omicron-receptor binding domain (RBD)-IgG titers were detected using enzyme-linked immunosorbent assay. InV pre-vaccinated OIPs had higher anti-Wuhan-, anti-Delta-, and anti-Omicron-RBD-IgG titers compared to unvaccinated OIPs. Anti-Wuhan-RBD-IgG titers sharply increased in InV pre-vaccinated OIPs 0-5 days postinfection (DPI), while the geometric mean titers (GMTs) of anti-Delta- and anti-Omicron-RBD-IgG were 3.3-fold and 12.0-fold lower. Then, the GMT of anti-Delta- and anti-Omicron-RBD-IgG increased to 35 112 and 28 186 during 11-22 DPI, about 2.6-fold and 3.2-fold lower, respectively, than the anti-Wuhan-RBD-IgG titer. The anti-Wuhan-, anti-Delta-, and anti-Omicron-RBD-IgG titers declined over time in HDs after two doses of an InV, with 25.2-fold, 5.6-fold, and 4.5-fold declination, respectively, at 6 months relative to the titers at 14 days after the second vaccination. Anti-Wuhan-, anti-Delta-, and anti-Omicron-RBD-IgG titers elicited by a heterologous Ad5-nCoV booster were significantly higher than those elicited by an InV booster, comparable to those in InV pre-vaccinated OIPs. InV and Ad5-nCoV boosters could improve humoral immunity against Omicron variants. Of these, the Ad5-nCoV booster is a better alternative.

Clinical characteristics and host immunity responses of SARS-CoV-2 Omicron variant BA.2 with deletion of ORF7a, ORF7b and ORF8.

BACKGROUND: The pathogenicity and virulence of the Omicron strain have weakened significantly pathogenesis of Omicron variants. Accumulating data indicated accessory proteins play crucial roles in host immune evasion and virus pathogenesis of SARS-CoV-2. Therefore, the impact of simultaneous deletion of accessory protein ORF7a, ORF7b and ORF8 on the clinical characteristics and specific immunity in Omicron breakthrough infected patients (BIPs) need to be verified. METHODS: Herein, plasma cytokines were identified using a commercial Multi-cytokine detection kit. Enzyme-linked immunosorbent assay and pseudovirus neutralization assays were utilized to determine the titers of SARS-CoV-2 specific binding antibodies and neutralizing antibodies, respectively. In addition, an enzyme-linked immunospot assay was used to quantify SARS-CoV-2 specific T cells and memory B cells. RESULTS: A local COVID-19 outbreak was caused by the Omicron BA.2 variant, which featured a deletion of 871 base pairs (∆871 BA.2), resulting in the removal of ORF7a, ORF7b, and ORF8. We found that hospitalized patients with ∆871 BA.2 had significantly shorter hospital stays than those with wild-type (WT) BA.2. Plasma cytokine levels in both ∆871 BA.2 and WT BA.2 patients were within the normal range of reference, and there was no notable difference in the titers of SARS-CoV-2 ancestor or Omicron-specific binding IgG antibodies, neutralizing antibody titers, effector T cells, and memory B cells frequencies between ∆871 BA.2 and WT BA.2 infected adult patients. However, antibody titers in ∆871 BA.2 infected adolescents were higher than in adults. CONCLUSIONS: The simultaneous deletion of ORF7a, ORF7b, and ORF8 facilitates the rapid clearance of the BA.2 variant, without impacting cytokine levels or affecting SARS-CoV-2 specific humoral and cellular immunity in Omicron-infected individuals.

PPP2R5D promotes hepatitis C virus infection by binding to viral NS5B and enhancing viral RNA replication.

BACKGROUND: Hepatitis C virus (HCV) infection increased the risk of hepatocellular carcinoma. Identification of host factors required for HCV infection will help to unveil the HCV pathogenesis. Adaptive mutations that enable the replication of HCV infectious clones could provide hints that the mutation-carrying viral protein may specifically interact with some cellular factors essential for the HCV life cycle. Previously, we identified D559G mutation in HCV NS5B (RNA dependent RNA polymerase) important for replication of different genotype clones. Here, we searched for the factors that potentially interacted with NS5B and investigated its roles in HCV infection. METHODS: Wild-type-NS5B and D559G-NS5B of HCV genotype 2a clone, J6cc, were ectopically expressed in hepatoma Huh7.5 cells, and NS5B-binding proteins were pulled down and identified by mass spectrometry. The necessity and mode of action of the selected cellular protein for HCV infection were explored by experiments including gene knockout or knockdown, complementation, co-immunoprecipitation (Co-IP), colocalization, virus infection and replication, and enzymatic activity, etc. RESULTS: Mass spectrometry identified a number of cellular proteins, of which protein phosphatase 2 regulatory subunit B'delta (PPP2R5D, the PP2A regulatory B subunit) was one of D559G-NS5B-pulled down proteins and selected for further investigation. Co-IP confirmed that PPP2R5D specifically interacted with HCV NS5B but not HCV Core and NS3 proteins, and D559G slightly enhanced the interaction. NS5B also colocalized with PPP2R5D in the endoplasmic reticulum. Knockdown and knockout of PPP2R5D decreased and abrogated HCV infection in Huh7.5 cells, respectively, while transient and stable expression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that in wild-type Huh7.5 cells. Replicon assay revealed that PPP2R5D promoted HCV replication, but the phosphatase activity and catalytic subunit of PP2A were not affected by NS5B. CONCLUSIONS: PPP2R5D interactes with HCV NS5B and is required for HCV infection in cultured hepatoma cells through facilitating HCV replication.

Development of full-length cell-culture infectious clone and subgenomic replicon for a genotype 3a isolate of hepatitis C virus.

Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5'UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3'UTR by constructing an intragenotypic recombinant using 5'UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3'UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5'UTR-NS5A and NS5B-3'UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 104.2 f.f.u. ml-1. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.

Nutritional Signaling Regulates Vitellogenin Synthesis and Egg Development through Juvenile Hormone in Nilaparvata lugens (Stål).

Insect female reproduction which comprises the synthesis of vitellogenein (Vg) in the fat body and its incorporation into developing oocytes, needs a large amount of energy and food resources. Our previous studies found that juvenile hormone (JH) regulates vitellogenesis in the brown planthopper, Nilaparvata lugens. Here, we report on the role of JH in nutrient-regulated Vg synthesis and egg development. We first cloned the genes coding for juvenile hormone acid methyltransferase (JHAMT) which is involved in JH biosynthesis and methoprene-tolerant (Met) for JH action. Amino acids (AAs) induced the expression of jmtN, while showing no effects on the expression of met using an artificial diet culture system. Reduction in JH biosynthesis or its action by RNA interference (RNAi)-mediated silencing of jmtN or met led to a severe inhibition of AAs-induced Vg synthesis and oocyte maturation, together with lower fecundity. Furthermore, exogenous application of JH III partially restored Vg expression levels in jmtN RNAi females. However, JH III application did not rescue Vg synthesis in these met RNAi insects. Our results show that AAs induce Vg synthesis in the fat body and egg development in concert with JH biosynthesis in Nilaparvata lugens (Stål), rather than through JH action.

The frequency of Th17 and Th22 cells in patients with colorectal cancer at pre-operation and post-operation.

T helper 17 (Th17) and Th22 cells regulate the development of tumors. However, their roles in the development of colorectal cancer (CRC) are still unclear. A total of 49 patients with CRC and 18 healthy controls (HC) were evaluated for the percentages of circulating Th17 and Th22 cells by flow cytometry. The concentrations of serum interleukin-17A (IL-17A), IL-22 and carcinoembryonic antigen (CEA) were examined. The levels of IL-17A and IL-22 in tumors were determined by real-time PCR. We found that the percentages of Th17 and Th22 cells in the CRC patients were significantly lower than that in the HC and were associated negatively with the pathological stages of CRC. The levels of IL-17A and IL-22 mRNA transcripts were lower in the tumor tissues, particularly in the advanced CRC. After the tumor resection, the percentages of circulating Th17 and Th22 cells increased. These data suggest that decreased Th17 and Th22 responses may be associated with the development of CRC.

Effective reduction on flame soot via plasma coupled with carbon dioxide.

This study explored the impact of non-thermal plasma and CO2 on the flame soot characteristics within the diffusion flames. We analyzed on flame structures that were diluted with either CO2 or N2, temperature distributions, and soot characteristics, both in the presence and absence of plasma. Due to the higher specific heat capacity of CO2 compared to N2, the optical observations consistently showed lower temperatures in flames diluted with CO2 as compared to those diluted with N2. The inclusion of plasma and carbon dioxide resulted in the lowest soot concentration, indicating that plasma coupled with CO2 has a synergistic inhibitory effect on soot emissions. The findings revealed that when CO2 was used to dilute the flames and the oxygen concentration was low, the soot nanostructure appeared amorphous. Raman results showed that the level of graphitization observed in soot particles from CO2 dilution flames was lower than that from N2 dilution flames. In the presence of plasma and CO2, the soot obtained exhibited the shortest fringe length and the highest fringe tortuosity. Significant correlations were observed between the nanostructure of soot and its reactivity. The combined application of plasma and CO2 proved to be effective in reducing the soot carbonization degree.

SCAC: A Semi-Supervised Learning Approach for Cervical Abnormal Cell Detection.

Cervical abnormal cell detection plays a crucial role in the early screening of cervical cancer. In recent years, some deep learning-based methods have been proposed. However, these methods rely heavily on large amounts of annotated images, which are time-consuming and labor-intensive to acquire, thus limiting the detection performance. In this paper, we present a novel Semi-supervised Cervical Abnormal Cell detector (SCAC), which effectively utilizes the abundant unlabeled data. We utilize Transformer as the backbone of SCAC to capture long-range dependencies to mimic the diagnostic process of pathologists. In addition, in SCAC, we design a Unified Strong and Weak Augment strategy (USWA) that unifies two data augmentation pipelines, implementing consistent regularization in semi-supervised learning and enhancing the diversity of the training data. We also develop a Global Attention Feature Pyramid Network (GAFPN), which utilizes the attention mechanism to better extract multi-scale features from cervical cytology images. Notably, we have created an unlabeled cervical cytology image dataset, which can be leveraged by semi-supervised learning to enhance detection accuracy. To the best of our knowledge, this is the first publicly available large unlabeled cervical cytology image dataset. By combining this dataset with two publicly available annotated datasets, we demonstrate that SCAC outperforms other existing methods, achieving state-of-the-art performance. Additionally, comprehensive ablation studies are conducted to validate the effectiveness of USWA and GAFPN. These promising results highlight the capability of SCAC to achieve high diagnostic accuracy and extensive clinical applications.

Hepatitis C virus hypervariable region 1 antibodies interrupt E2-SR-B1 interaction to suppress viral infection.

Whether hypervariable region 1 (HVR1)-targeting antibodies elicited during natural hepatitis C virus (HCV) infection contribute to virus clearance and what is the mechanism underlying remain unclear. Here, we demonstrated that treatment of HCV-infected hepatoma Huh7.5 cells with the IgGs purified from 2 of 28 (7.1%) chronic hepatitis C (CHC) patients efficiently controlled the infection, for which genotype 1b HVR1 (1bHVR1)-binding antibody was critical. Moreover, we found that 1bHVR1 peptide was superior to 2aHVR1 in rabbit immunization to elicit antibodies neutralizing genotypes 1a, 2a, 3a, and 4a. The neutralization effect of 1bHVR1 IgG could be augmented by HH-1, an antibody constructed from CHC memory B cells but without binding to HVR1 peptide. Mechanistic studies showed that 1bHVR1 antisera and IgGs disrupted the interaction of E2-SR-B1 receptor. This study highlights the neutralizing activity of HVR1 antibody elicited by CHC patients and generated by HVR1-immunization against the established infections of multiple HCV genotypes.

Omicron variants breakthrough infection elicited higher specific memory immunity than third dose booster in healthy vaccinees.

Homologous booster, heterologous booster, and Omicron variants breakthrough infection (OBI) could improve the humoral immunity against Omicron variants. Questions concerning about memory B cells (MBCs) and T cells immunity against Omicron variants, features of long-term immunity, after booster and OBI, needs to be explored. Here, comparative analysis demonstrate antibody and T cell immunity against ancestral strain, Delta and Omicron variants in Omicron breakthrough infected patients (OBIPs) are comparable to that in Ad5-nCoV boosted healthy volunteers (HVs), higher than that in inactivated vaccine (InV) boosted HVs. However, memory B cells (MBCs) immunity against Omicron variants was highest in OBIPs, followed by Ad5-nCoV boosted and InV boosted HVs. OBIPs and Ad5-nCoV boosted HVs have higher classical MBCs and activated MBCs, and lower naïve MBCs and atypical MBCs relative to both vaccine boosted HVs. Collectively, these data indicate Omicron breakthrough infection elicit higher MBCs and T cells against SARS-CoV-2 especially Omicron variants relative to homologous InV booster and heterologous Ad5-nCoV booster.

Diagnostic accuracy of red blood cell distribution width for neonatal sepsis.

INTRODUCTION: Red blood cell distribution width (RDW) is a biomarker for the diagnosis and prognosis of many diseases. However, the relevance between RDW and neonatal sepsis (NS) have not reached a consensus yet; the perform of RDW in the diagnosis of neonatal sepsis is still not clear. The aim of this meta-analysis was to estimate the significance of RDW in neonatal sepsis and the perform of RDW in diagnosis of neonatal sepsis. EVIDENCE ACQUISITION: We used Pubmed, Embase, Web of science, CNKI and Google academic database to find all articles that met the inclusion criteria until July 1, 2020. EVIDENCE SYNTHESIS: Fifteen eligible studies involving 1362 newborns were included in the meta-analysis after two independent investigators read the title, abstract and full text in detail. The pooled result of this meta-analysis showed that RDW was significantly higher in the NS group than in the control group (WMD=3.224; 95%CI: 2.359-4.090, P<0.001). In addition, the overall pooled sensitivity, specificity, PLR, NLR and DOR were 0.88 (95%CI:0.66-0.96), 0.90 (95%CI:0.65-0.98), 9.2 (95%CI:2.1-40.3), 0.14(95%CI:0.04-0.43) and 66.9 (95%CI:8.73-513.26), respectively. The area under the SROC curve (AUC) was 0.95 (95%CI:0.93-0.96). CONCLUSIONS: The meta-analysis demonstrated that newborns with sepsis had an elevated RDW level than healthy controls. RDW levels have significant correlated with neonatal sepsis; and RDW can be used as a cheap and satisfactory diagnostic biomarker for neonatal sepsis with a relatively high performance.

Immunity against Delta and Omicron variants elicited by homologous inactivated vaccine booster in kidney transplant recipients.

Background: A third mRNA vaccine booster is recommended to improve immunity against SARS-CoV-2 in kidney transplant recipients (KTRs). However, the immunity against SARS-CoV-2 Ancestral strain and Delta and Omicron variants elicited by the third dose of inactivated booster vaccine in KTRs remains unknown. Methods: The blood parameters related to blood cells count, hepatic function, kidney function, heart injury and immunity were explored clinically from laboratory examinations. SARS-CoV-2 specific antibody IgG titer was detected using an enzyme-linked immunosorbent assay. Cellular immunity was analyzed using interferon-γ enzyme-linked immunospot assay. Results: The results showed that there were no severe adverse effects and apparent changes of clinical laboratory biomarkers in KTRs and healthy volunteers (HVs) after homologous inactivated vaccine booster. A third dose of inactivated vaccine booster significantly increased anti-Ancestral-spike-trimer-IgG and anti-Ancestral-receptor binding domain (RBD)-IgG titers in KTRs and HVs compared with the second vaccination. However, the anti-Delta-RBD-IgG and anti-Omicron-RBD-IgG titers were significantly lower than anti-Ancestral-RBD-IgG titer in KTRs and HVs after the third dose. Notably, only 25.6% (10/39) and 10.3% (4/39) of KTRs had seropositivity for anti-Delta-RBD-IgG and anti-Omicron-RBD-IgG after booster, which were significantly lower than HVs (anti-Delta-RBD-IgG: 100%, anti-Omicron-RBD-IgG: 77.8%). Ancestral strain nucleocapsid protein and spike specific T cell frequency after booster was not significantly increased in KTRs compared with the second dose, significantly lower than that in HVs. Moreover, 33.3% (12/36), 14.3% (3/21) and 14.3% (3/21) of KTRs were positive for the Ancestral strain and Delta and Omicron spike-specific T cells, which were significantly lower than HVs (Ancestral: 80.8%, Delta: 53.8%, and Omicron: 57.7%). Conclusions: A third dose of inactivated booster vaccine may significantly increase humoral immunity against the Ancestral strain in KTRs, while humoral and cellular immunity against the Delta and Omicron variants were still poor in KTRs.

[Pathological changes of rectal cancer after irinotecan, 5-fluorouracil or combined short-term radiotherapy].

OBJECTIVE: To observe and evaluate the pathologic changes and curative effects of irinotecan (CPT-11), 5-fluorouracil (5-FU) and combined short-term radiotherapy before low-set rectal cancer operation so as to provide a theoretic basis for formulating a new effective adjuvant therapeutic regimen. METHODS: A total of 41 patients of low rectal cancer were treated with CPT-11, 5-FU therapy or CPT-11 plus 5-FU combined short-term radiotherapy from April 2002 to April 2009. They were divided into 2 groups according to different treatment schemes, including irinotecan group (n = 18) and irinotecan combined short-term radiotherapy group (n = 23). The pathologic changes before and after treatment were observed and the differences of two treatment approaches compared. RESULTS: Tumor cells had different degrees of degeneration and necrosis under microscope in two groups. Compared with computed tomographic findings before therapy, tumor sizes of two groups were reduced by an average of 33.1% (13.5 mm vs 20.2 mm) and 34.4% (12.8 mm vs 19.5 mm) respectively. Two groups were graded according to the RCRG (rectal cancer regression grade) score: RCRG1: 7 cases vs 18 cases, RCRG2: 4 cases vs 3 cases and RCRG3: 7 cases vs 2 cases. According to the pathologic evaluation standard, 3-degree necrosis, cell interstitial fibrosis and intimal thickening in vessels were observed in two groups: 7 cases vs 17 cases, 6 cases vs 17 cases and 3 cases vs 14 cases respectively (all P < 0.05). Five patients achieved complete pathological remission in the irinotecan combined short-term radiotherapy group. CONCLUSION: Based on the pathological changes and mitigation results after treatment, CPT-11 and 5-FU may be used as neoadjuvant drugs for rectal cancer. If the above two drugs can be used in combination with short-term radiation, the curative effect will be better.

Keyhole Limpet Hemocyanin-Conjugated Peptides from Hepatitis C Virus Glycoproteins Elicit Neutralizing Antibodies in BALB/c Mice.

Currently, no vaccine to prevent hepatitis C virus (HCV) infection is available. A major challenge in developing an HCV vaccine is the high diversity of HCV sequences. The purpose of immunization with viral glycoproteins is to induce a potent and long-lasting cellular and humoral immune response. However, this strategy only achieves limited protection, and antigen selection plays a crucial role in vaccine design. In this study, we investigated the humoral immune responses induced by intraperitoneal injection of keyhole limpet hemocyanin conjugated with 4 highly conserved peptides, including amino acids [aa]317-325 from E1 and aa418-429, aa502-518, and aa685-693 from E2, or 3 peptides from hypervariable region 1 (HVR1) of E2, including the N terminus of HVR1 (N-HVR1, aa384-396), C terminus of HVR1 (C-HVR1, aa397-410), and HVR1 in BALB/c mice. The neutralizing activity against HCV genotypes 1-6 was assessed using the cell culture HCV (HCVcc) system. The results showed that the 4 conserved peptides efficiently induced antibodies with potent neutralizing activity against 3 or 4 genotypes. Antibodies induced by aa685-693 conferred potent protection (>50%) against genotypes 2, 4, and 5. Peptide N-HVR1 elicited antibodies with the most potent neutralization activities against 3 HCV genotypes: TNcc(1a), S52(3a), and ED43(4a). These findings suggested that peptides within HCV glycoproteins could serve as potent immunogens for vaccine design and development.

The impact of cytochrome 450 and Paraoxonase polymorphisms on clopidogrel resistance and major adverse cardiac events in coronary heart disease patients after percutaneous coronary intervention.

BACKGROUND: Clopidogrel is an inactive prodrug, it catalyzed into its active form by Cytochrome 450 and Paraoxonase-1(PON-1). polymorphisms of genes encoding these enzymes will affect the efficacy of Clopidogrel. The main objective of our study was to investigate the association of CYP2C19*2, CYP2C19*3 and PON-1Q192R polymorphisms with Clopidogrel resistance and major adverse cardiac events in Jin Hua district in the middle of Zhe Jiang Province in China. METHODS: One hundred sixty coronary heart disease patients with percutaneous coronary intervention, who were followed-up for 1 year, were enrolled in our study. These patients were co-administered aspirin 100 mg/d and clopidogrel 75 mg/d following a loading dose of 300 mg. The ADP-induced platelet aggregation rate was measured by Platelet aggregator. Genotypes of CYP2C19*2, CYP2C19*3, PON-1Q192R were determined using Sanger sequencing in all patients. Various clinical data were collected. RESULTS: The frequencies of CYP2C19*2, CYP2C19*3 and PON-1Q192R homozygous mutant genotypes were significantly lower in non-responders than those in responders. After for all variables, CYP2C19*2, CYP2C19*3 and PON-1Q192R independently increased the risk of clopidogrel resistance with adjusted ORs 46.65(95% CI,1.77-25.04; p = 0.005); 22.74(95% CI, 3.11-166.27; p = 0.002); 5.69 (95% CI,1.06-30.47; p = 0.042). Over a follow-up of 12 months, the incidence of major adverse cardiac events (MACE) in CYP2C19*1/*2, *1/*3, *2/*2, *2/*3 was significantly higher than no mutant genotype (18/40vs.2/63,3/9vs.2/63, 11/6vs.2/63, 7/1vs2/63, respectively). There was no significant correlation between PON-1Q192R mutant allele and MACE. CONCLUSION: Our study was first time to report on CYP2C19 and PON-1 polymorphisms in Jin Hua population in the middle of Zhe Jiang province in China. The carriage of CYP2C19*2 or *3 mutant allele significantly reduced the platelet response to clopidogrel and increase the MACE. The carriage of PON-1 mutant allele also significantly reduced the platelet response to clopidogrel, but would not increase the major adverse cardiac events after 1 year follow-up. TRIAL REGISTRATION: ChiCTR, ChiCTR1800018316. Registered 11 September 2018 - prospective registered, http://www.chictr.org.cn/edit.aspx?pid=30927&htm=4.

miR-15a-3p Suppresses Prostate Cancer Cell Proliferation and Invasion by Targeting SLC39A7 Via Downregulating Wnt/ß-Catenin Signaling Pathway.

Background: MiR-15a-3p has been reported as a tumor suppressor in several kinds of cancer, including cervical cancer and gastric cancer. However, the precise molecular mechanisms underlying its role in prostate cancer (PCa) remain largely unknown. Methods: The expression of miR-15a-3p was determined in PCa tissues and cell lines using quantitative real time PCR. The biological function of miR-15a-3p in PCa cells was investigated using a MTT assay, Edu staining and transwell assay. Moreover, luciferase reporter assay, quantitative real time PCR and western blotting were used to identify and verify the direct downstream target of miR-15a-3p. Results: We found that the expression of miR-15a-3p was down-regulated in both PCa tissues and cell lines. The in vitro results showed that miR-15a-3p overexpression suppressed cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) via down-regulating Wnt/ß-catenin signaling in PCa cells. Moreover, SLC39A7 was a direct downstream target of miR-15a-3p. Furthermore, SLC39A7 overexpression attenuated the effects of miR-15a-3p on cell proliferation, invasion, Wnt/ß-catenin pathway and EMT molecules. Conclusions: In summary, our study indicated that miR-15a-3p inhibited the proliferation, invasion, and EMT process of PCa cells via targeting SLC39A7 and suppressing Wnt/ß-catenin signaling pathway, which may represent a new therapeutic objective for PCa treatment.

Serum­derived hepatitis C virus can infect human glioblastoma cell line SF268 and activate the PI3K­Akt pathway.

Extra­hepatic manifestations are frequently observed in hepatitis C virus (HCV)­infected patients; however the underlying mechanisms remain largely unknown. In the present study, the human glioblastoma SF268 cell line (the precise origin of the cell type is not clear) was infected with HCV using HCV­positive serum, and viral replication was assessed by immunofluorescence, reverse transcription­polymerase chain reaction (PCR), quantitative PCR and western blotting following infection. HCV core protein and HCV RNA were detected in HCV­positive serum­infected SF268 cells at day 4 post­infection, while no infection was observed in cells exposed to HCV­negative serum. The mean HCV RNA levels at day 4 post­infection were up to 5.00 IU/ml log10; however, HCV RNA and immunostaining for core protein were negative when cultured to day 6 or longer. The data suggest that human glioblastoma SF268 cells were transiently infected with HCV. AKT serine/threonine kinase phosphorylation was also detected in HCV­infected SF268 cells at day 4 post­infection. To the best of our knowledge, this is the first demonstration that a human glioblastoma cell line can be infected with serum­derived HCV. The results provide evidence that HCV infection can occur in cells of the central nervous system. Neurological disorder­associated phosphoinositide 3­kinase­AKT signaling pathway was activated in parallel with HCV infection, suggesting that SF268 may serve as an in vitro model for investigating HCV­nervous system cell interactions.

Identification of nucleotides in the 5'UTR and amino acids substitutions that are essential for the infectivity of 5'UTR-NS5A recombinant of hepatitis C virus genotype 1b (strain Con1).

Genotype 1b strain Con1 represents an important reference in the study of hepatitis C virus (HCV). Here, we aimed to develop an advanced infectious Con1 recombinant. We found that previously identified mutations A1226G/F1464L/A1672S/Q1773H permitted culture adaption of Con1 Core-NS5A (C-5A) recombinant containing 5'UTR and NS5B-3'UTR from JFH1 (genotype 2a), thus acquired additional mutations L725H/F886L/D2415G. C-5A containing all seven mutations (C-5A_7m) replicated efficiently in Huh7.5 and Huh7.5.1 cells and had an increased infectivity in SEC14L2-expressing Huh7.5.1 cells. Incorporation of Con1 NS5B was deleterious to C-5A_7m, however Con1 5'UTR was permissive but attenuated the virus. Nucleotides G1, A4, and G35 primarily accounted for the viral attenuation without affecting RNA translation. C-5A_7m was inhibited dose-dependently by simeprevir and daclatasvir, and substitutions at A4, A29, A34, and G35 conferred resistance to miR-122 antagonism. The novel Con1 5'UTR-NS5A recombinant, adaptive mutations, and critical nucleotides described here will facilitate future studies of HCV culture systems and virus-host interaction.