Inhibiting apoptosis and GSDME-mediated pyroptosis attenuates hepatic injury in septic mice.

BACKGROUND: Sepsis is characterized by severe inflammation and organ dysfunction resulting from a dysregulated organismal response to infection. Although pyroptosis has been presumably shown to be a major cause of multiple organ failure and septic death, whether gasdermin E (GSDME)-mediated pyroptosis occurs in septic liver injury and whether inhibiting apoptosis and GSDME-mediated pyroptosis can attenuate septic liver injury remain unclear. This study investigated the role of apoptosis and GSDME-mediated pyroptosis in septic liver injury. METHODS: Adult male C57BL/6 mice were randomly divided into four groups: sham, cecal ligation puncture (CLP), CLP + Z-DEVD-FMK (a caspase-3 inhibitor, 5 mg/kg), and CLP + Ac-DMLD-CMK (a GSDME inhibitor, 5 mg/kg). Sepsis severity was assessed using the murine sepsis score (MSS). Hepatic tissue damage was observed by the hematoxylin-eosin staining method, the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the levels of malondialdehyde (MDA), the concentrations of interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) were measured according to the related kits, and the changes in the hepatic tissue reactive oxygen species (ROS) levels were detected by immunofluorescence (IF). The protein expression levels of cleaved caspase-3, GSDME-N, IL-1ß, B-cell lymphoma-2 (Bcl-2), cytochrome C (Cyt-c), and acetaldehyde dehydrogenase 2 (ALDH2) were detected using western blotting. GSDME expression was detected by immunohistochemistry. RESULTS: Compared with the Sham group, CLP mice showed high sepsis scores and obvious liver damage. However, in the CLP + Z-DEVD-FMK and CLP + Ac-DMLD-CMK groups, the sepsis scores were reduced and liver injury was alleviated. Compared with the Sham group, the serum ALT and AST activities, MDA and ROS levels, and IL-1ß and TNF-α concentrations were increased in the CLP group, as well as the protein expression of cleaved caspase-3, GSDME-N, IL-1ß, Cyt-c, and GSDME positive cells (P < 0.05). However, the expression levels of Bcl-2 and ALDH2 protein were decreased (P < 0.05). Compared with the CLP group, the CLP + Z-DEVD-FMK and CLP + Ac-DMLD-CMK groups showed low sepsis scores, ALT and AST activities, MDA and ROS levels, decreased IL-1ß and TNF-α concentrations, and decreased expression of cleaved caspase-3, GSDME-N, IL-1ß protein expression, and GSDME positive cells (P < 0.05). The expression levels of Bcl-2 and ALDH2 protein were increased (P < 0.05). CONCLUSION: Apoptosis and GSDME-mediated pyroptosis are involved in the development of sepsis-induced hepatic injury. Inhibition of apoptosis and GSDME-mediated pyroptosis attenuates injury. ALDH2 plays a protective role by inhibiting apoptosis and pyroptosis.

Characterization of antibody responses to SARS-CoV-2 in convalescent COVID-19 patients.

The coronavirus disease 2019 (COVID-19) is a pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, little is known about the durability of the antibody response during COVID-19 convalescent phase. We investigated the prevalence of anti-SARS-CoV-2 specific antibodies including immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies and the dynamic changes in antibody levels in convalescent COVID-19 patients. A total of 159 blood samples were collected from 52 recovered COVID-19 patients up to six months after symptom onset for longitudinal serological tests. The positive rate of IgG and IgM antibodies was 92.3% and 90.4% in the first month after symptom onset, and the seropositivity of IgG antibody remained high at all follow-up time points, whereas the seropositivity of IgM antibody decreased to 22.73% by the sixth months after symptom onset. The level of IgG antibody was stable, the level of IgM antibody decreased slightly in the early convalescent phase and was detected in only five patients in the sixth month after symptom onset. The level of IgG antibody was higher in the severe and critical group than in the moderate group. The anti-SARS-CoV-2 specific antibodies have a long-term persistence in convalescent COVID-19 patients, whether they have long-term protection need to be further investigated.

[Clinical observation of 6 severe COVID-19 patients treated with plasma exchange or tocilizumab].

OBJECTIVE: To observe the clinical effect of plasma exchange and tocilizumab in treatment of patients with severe coronavirus disease 2019 (COVID-19). METHODS: Six patients with severe COVID-19 admitted in First Affiliated Hospital of Bengbu Medical College from January 25 to February 25, 2020. Three patients were treated with plasma exchange and three patients were treated with tocilizumab. The effect on excessive inflammatory reaction of plasma exchange and tocilizumab was observed. RESULTS: The C-reactive protein (CRP) and IL-6 levels were significantly decreased and the lymphocyte and prothrombin time were improved in 3 patients after treatment with plasma exchange; while inflammation level was not significantly decreased, and lymphocyte and prothrombin time did not improve in 3 patients treated with tocilizumab. CONCLUSIONS: For severe COVID-19 patients with strong inflammatory reaction, plasma exchange may be preferred.

Meta-analysis reveals an association between signal transducer and activator of transcription-4 polymorphism and hepatocellular carcinoma risk.

AIM: Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related mortality worldwide. Signal transducer and activator of transcription (STAT) proteins play a multitude of important functions in liver pathophysiology. Recent studies have indicated associations of rs7574865 single nucleotide polymorphism (SNP) in the STAT4 gene with various autoimmune diseases. The association between STAT4 polymorphism and the risk of HCC has been analyzed in several studies, but results remain inconsistent. This study used a meta-analysis approach to comprehensively investigate the correlation between STAT4 polymorphism and HCC risk based on previously published reports. METHODS: Studies were searched from the databases of PubMed, EMBase, Web of Science, and the Chinese National Knowledge Infrastructure up to 31 December 2015. The meta-analysis was carried out based on the statement of Preferred Reporting Items for Systematic Reviews and Meta-Analyses. RESULTS: Eight published studies, consisting of 7503 HCC patients (cases) and 13 831 individuals without HCC (controls), were included in the present study. Meta-analysis of the included studies revealed that STAT4 rs7574865 polymorphism contributed to the risk of HCC under all four genetic models, consisting of the allelic model (G vs. T: odds ratio [OR], 1.25; 95% confidence interval [CI], 1.19-1.30), the dominant effect model (GG + GT vs. TT: OR, 1.52; 95% CI, 1.26-1.84), the recessive effect model (GG vs. GT + TT: OR, 1.35; 95% CI, 1.21-1.50), and the co-dominant effect model (GG vs.. TT: OR, 1.72; 95% CI, 1.42-2.10) comparisons. No publication bias was indicated from either visualization of the funnel plot or Egger's test. CONCLUSION: A significantly increased risk of HCC associated with the rs7574865 G was found. The rs7574865 polymorphism might be used as one risk factor for HCC.

Expression profile of microRNAs in patients with decompensated cirrhosis by small RNA deep sequencing.

INTRODUCTION AND OBJECTIVE: Decompensated cirrhosis (DCC) is a more advanced stage of liver cirrhosis (LC). It is important to identify biomarkers to predict DCC progression. The aim of this study was to analyze microRNA (miRNA) profiles of whole blood involved in the DCC process to gain a better understanding of the molecular mechanisms underlying its development. MATERIALS AND METHODS: RNA-Seq analysis of blood samples from a discovery set, including four DCC patients and four LC individuals, was performed to identify differentially expressed miRNAs. The selected differentially expressed miRNAs were validated by using an independent validation set. RESULTS: In this study, a total of 1,036 miRNAs were identified in whole blood samples. Forty differentially expressed miRNAs were identified, including 24 upregulated and 16 downregulated miRNAs. The expression levels of three upregulated miRNAs (hsa-miR-20b-5p, hsa-miR-421, and hsa-miR-1307-3p) and two downregulated miRNAs (hsa-miR-139-5p and hsa-miR-150-5p) were validated by quantitative reverse transcriptase polymerase chain reaction. The receiver operator characteristic curve for the logistic regression model based on hsa-miR-20b-5p, hsa-miR-421, and hsa-miR-150-5p could distinguish DCC patients with excellent diagnostic accuracy (area under the curve: 0.981, p < 0.01). CONCLUSION: The miRNA expression profiles in patients with DCC and LC controls suggested that miR-20b-5p, miR-421, and miR-150-5p could be potential biomarkers and therapeutic targets for this condition.

Role of Ferroptosis in the Progression of COVID-19 and the Development of Long COVID.

OBJECTIVES: To examine the role of ferroptosis on the pathogenesis and progression of COVID-19. MATERIALS AND METHODS: A total of 127 patients who were hospitalized for COVID-19 were categorized into two groups according to the intensity of oxygen therapy (high-flow or low-flow). Clinical characteristics, laboratory parameters, plasma markers, and peripheral blood mononuclear cell (PBMC) markers were measured at baseline and one or two weeks after treatment. Telephone follow-up was performed 3 months after discharge to assess long COVID. RESULTS: Patients receiving high-flow oxygen therapy had greater levels of neutrophils; D-dimer; C reactive protein; procalcitonin; plasma protein levels of tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), IL-17, and acyl-CoA synthetase long-chain family member 4 (ACSL4); and PBMC mRNA level of TNF-α; but had lower levels of lymphocytes and plasma glutathione peroxidase 4 (GPX4). There were negative correlations of plasma GPX4 and cystine/glutamate transporter-11 (SLC7A11) with TNF-α, IL-6, and IL-17, and positive correlations of ACSL4 with inflammatory markers in plasma and PBMCs. The plasma levels of TNF-α, IL-6, IL-17, and ACSL4 were significantly lower after treatment than at baseline, but there were higher post-treatment levels of lymphocytes, GPX4, and SLC7A11. Patients with long COVID had a lower baseline level of plasma SLC7A11. CONCLUSION: Ferroptosis is activated during the progression of COVID-19, and a low baseline level of a ferroptosis marker (SLC7A11) may indicate an increased risk for long COVID-19. Ferroptosis has potential as a clinical indicator of long COVID and as a therapeutic target.

Targeted capture enrichment and sequencing identifies HLA variants associated with the severity of COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is currently a global pandemic. The pathogenesis of severe COVID-19 has been widely investigated, but it is still unclear. Human leukocyte antigen (HLA) plays a central role in immune response, and its variants might be related to COVID-19 progression and severity. OBJECTIVE: To investigate the hypothesis that individual HLA variations could alter the course of COVID-19 and might be associated with the severity of COVID-19. METHODS: In this study, we conducted an HLA targeted capture enrichment and sequencing of severe COVID-19 patients matched to mild cases. A total of 16 COVID-19 patients, confirmed by SARS-CoV-2 viral RNA polymerase-chain-reaction (PCR) test and chest computed tomography (CT) scan, were enrolled in this study. The HLA targeted capture enrichment and sequencing were conducted. HLA typing was performed by comparing contigs with IPD-IMGT/HLA Database. RESULTS: In this study, 139 four-digit resolution HLA alleles were acquired. The results showed that HLA-DRB3*01:01 allele was significantly associated with the severity of COVID-19 (odds ratio [OR] = 27.64, 95% confidence interval [CI] = 1.35-560.50, P = 0.0064). And HLA-K*01:01 might be a potential risk factor for COVID-19 severity (OR = 0.11, 95% CI = 0.017-0.66, P = 0.019), but HLA-K*01:02 might be a protective factor (OR = 7.50, 95% CI = 1.48-37.92, P = 0.019). CONCLUSION: Three non-classical HLA alleles, including HLA-DRB3*01:01, HLA-K*01:01, HLA-K*01:02 were identified to be associated with the severity of COVID-19 by comparing mild and severe patients. The current findings would be helpful for exploring the influence of HLA gene polymorphisms on the development and severity of COVID-19.

[Activation of Yes-associated protein (YAP) improves mouse acute liver failure by alleviating ferroptosis].

Objective To investigate the effects of YAP on the occurrence and progression of acute liver failure by regulating the ferroptosis pathway and its underlying mechanism. Methods A total of 20 8-week-old C57BL/6 mice were randomly divided into four groups: a control group, an acute liver failure model group, a YAP agonist XMU-MP-1 treatment group and a YAP inhibitor verteporfin treatment group, five mice for each group. HE staining was used to observe the pathological changes of hepatic inflammation and necrosis. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected by liver biochemistry. Iron (Fe), malondialdehyde (MDA), glutathione (GSH) determination kits were used to measure their levels in liver tissues of each group. The changes of hepatocyte mitochondrial in each group were observed by electron microscopy. Real time PCR and Western blot analysis were used to detect the mRNA and protein expressions of YAP, glutathione peroxidase 4 (GPX4) and 5-lipoxygenase (5-LOX). Results Compared with the control group, mice in the acute liver failure model group and the YAP inhibitor verteporfin treatment group showed severe liver tissue congestion with inflammatory cell infiltration and structural damage to hepatic lobules. Liver injury was alleviated in the XMU-MP-1 treatment group. With the occurrence of liver failure, plasma ALT and AST levels significantly increased, and liver function was improved in XMU-MP-1 treatment group. Electron microscopy showed that mitochondria in hepatocytes of mice with liver failure became smaller and bilayer membrane density increased, while mitochondria changes in the XMU-MP-1 group were alleviated. In addition, the acute liver failure model group showed an increase in Fe and MDA contents, decreased protein expressions of GPX4, and enhanced expression of 5-LOX, suggesting that ferroptosis was involved in acute liver failure in C57BL/6 mice. Ferroptosis was inhibited by activation of YAP. Conclusion Activation of YAP may ameliorate liver injury by inhibiting ferroptosis.

Case report: Successful immunomodulators combined with electromagnetic field therapy in a patient with methazolamide-induced Steven Johnson syndrome/toxic epidermal necrolysis overlap.

Methazolamide is used to treat patients with glaucoma. However, as a sulfonamide derivative, methazolamide shares the same adverse reaction profile as other sulfa-based medications. Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare delayed-type hypersensitivity cutaneous reactions with high morbidity and mortality. Here, we report a severe SJS/TEN overlap syndrome in an 85-year-old Chinese male patient who received methazolamide 25 mg twice daily for his left eye glaucoma. The causal relationship between SJS/TEN and methazolamide was categorized as "highly likely" on the algorithm for assessing drug causality for epidermal necrolysis. In addition to the treatments with methylprednisolone and immunoglobulin, we used a special electromagnetic spectrum therapeutic apparatus to provide skin wound care. The patient had a thoroughly satisfying recovery. This is the first case report to use electromagnetic field therapy in a patient with SJS/TEN. We share our experience here and suggest that electromagnetic field therapy can provide advanced skin wound care and facilitate the recovery of SJS/TEN.

Yes-associated protein inhibition ameliorates liver fibrosis and acute and chronic liver failure by decreasing ferroptosis and necroptosis.

Background/aims: This study aims to determine which cell death modes contribute most in the progression of cirrhosis and acute-on-chronic liver failure (ACLF), and to investigate whether Yes associated protein (YAP) affects the disease process by regulating cell death. Materials and methods: 30C57BL/6 male mice were divided into five groups: control, carbon tetrachloride (CCl4)-induced liver fibrosis model, CCl4+verteporfin, CCl4+lipopolysaccharides (LPS) combined with the D-(+)-Galactosamine (LPS/D-GalN)-induced ACLF model, and ACLF + verteporfin. Patients with chronic hepatitis B (CHB), hepatitis B virus (HBV) related liver cirrhosis or ACLF were enrolled. Histology, immunohistochemistry, transmission electron microscopy, Western blot and ELISA were conducted to assess the roles of YAP and cell death in liver cirrhosis and ACLF, and to explore the effect of YAP inhibition on cell deaths. Results: YAP was markedly increased in mice with liver fibrosis and ACLF, along with ferroptosis and necroptosis. Furthermore, YAP inhibition significantly suppressed fibrosis in CCl4-mediated liver fibrosis and ACLF-associated liver injury. Notably, CCl4 induced up-regulation of ACSL4 and RIPK3 and down-regulation of SLC7A11, key factors in ferroptosis and necroptosis. This was significantly abrogated by verteporfin treatment. Similar changes in ferroptosis and necroptosis were found in ACLF and ACLF + verteporfin groups. Consistent with the above findings in mice, we found that plasma YAP levels were gradually increased with the development of HBV-related liver fibrosis and ACLF. Conclusion: Ferroptosis and necroptosis are involved in the development of liver cirrhosis and ACLF. Inhibition of YAP improved liver fibrosis and liver damage in ACLF through a reduction in ferroptosis and necroptosis. Our findings may help better understanding the role of YAP in liver fibrosis and ACLF.

Research progress on the relationship between TM6SF2 rs58542926 polymorphism and non-alcoholic fatty liver disease.

INTRODUCTION: nonalcoholic fatty liver disease is a common liver disease with a global average prevalence of about 25%. In addition to the incidence of NAFLD being related to obesity, diabetes, hyperlipidemia, etc., genetic factors also have an important impact on the incidence of NAFLD. AREAS COVERED: Current experimental results and clinical studies show that the transmembrane 6 superfamily member 2 (TM6SF2) gene plays an important role in the pathogenesis of NAFLD. The research on genetic polymorphism of TM6SF2 gene mainly focuses on rs58542926 locus (rs58542926 c.449 C > T, p. Glu167Lys, E167K). The Mutations of this site might increase the risk of NAFLD in carriers. EXPERT OPINION: The mutation of this site causes the disorder of triglyceride metabolism in the liver, which leads to the deposition of a large amount of lipids in the liver, and further induces the incidence of NAFLD. With the study of the mechanism of TM6SF2 gene polymorphism in the pathogenesis of NAFLD, it is helpful to understand the molecular mechanism of the pathogenesis of NAFLD, which has a great value for the treatment of NAFLD.

Integrated Analysis of mRNA and lncRNA Expression Profiles Reveals Regulatory Networks Associated with Decompensated Cirrhosis.

The stage of decompensation is termed end-stage liver cirrhosis. Patients with decompensated cirrhosis (DCC) often have a variety of comorbidities that contribute to exacerbation of the disease and its high mortality rate. By comparing differential gene expression, transcriptomic analysis is useful for exploring relevant functional changes during disease progression. The purpose of this study was to identify differentially expressed long noncoding RNAs (lncRNAs) and mRNAs in patients with decompensated cirrhosis and to further explore the functions as well as interactions between lncRNAs and mRNAs. Four patients with decompensated cirrhosis and four controls with liver cirrhosis were recruited in this study. RNA was isolated from peripheral blood mononuclear cells, and RNA-seq was used for transcriptome analysis. The functions of differentially expressed mRNAs were revealed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and a regulatory network was also constructed. A total of 1046 differentially expressed mRNAs and 1175 lncRNAs were identified between the decompensated cirrhosis patients and cirrhosis controls. Functional enrichment analyses indicated enrichment of genes involved in pathways related to inflammation and cellular metabolic activities. In addition, the findings suggested that the phagosome/endosome/autophagy-lysosome pathway might play an important role in cirrhotic decompensation. In summary, this study identified differentially expressed mRNAs (DE-mRNAs) and DE-lncRNAs and predicted the biological processes and signaling pathways involved in cirrhotic decompensation, which might provide new ideas for further revealing the molecular mechanism of DCC pathogenesis.

Sex differences in hepatocellular carcinoma indicated BEX4 as a potential target to improve efficacy of lenvatinib plus immune checkpoint inhibitors.

Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer, and significant sex disparities have been observed in HCC. We aim to explore the potential sex-biased mechanisms involved in hepatocarcinogenesis. Methods: Based on TCGA data, we compared clinical features, genetic alterations, and immune cell infiltrations between male and female HCC patients. In addition, we performed sex-based differential expression analysis and functional enrichment analysis. Finally, GSE64041 dataset and another HCC cohort were engaged to validate our findings. Results: Significant differences of genetic alterations and TME were observed between male and female HCC patients. Enhanced metabolism of lipids was associated with hepatocarcinogenesis in men, while ECM-organization-related pathways were correlated to HCC development in women. BEX4 was upregulated in female but downregulated in male HCC patients, and was positively correlated with immune checkpoint molecules and infiltrated immune cell. These findings were further validated in dataset GSE64041 and our HCC cohort. More importantly, a negative correlation was found between BEX4 expression and lenvatinib sensitivity. Conclusion: Distinct biological processes were involved in sex-biased tumorigenesis of HCC. BEX4 can be targeted to improve the efficacy of lenvatinib plus immune checkpoint inhibitors.

Neutralizing activity of BBIBP-CorV vaccine-elicited sera against Beta, Delta and other SARS-CoV-2 variants of concern.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the generation of variants that may diminish host immune responses to vaccine formulations. Here we show a registered observational clinical trial (NCT04795414), we assess the safety and immunogenicity of the inactivated SARS-CoV-2 vaccine BBIBP-CorV in a cohort of 1006 vaccine recipients. No serious adverse events are observed during the term of the study. Detectable virus-specific antibody is measured and determined to be neutralizing in 698/760 (91.84%) vaccine recipients on day 28 post second vaccine dose and in 220/581 (37.87%) vaccine recipients on day 180 post second vaccine dose, whereas vaccine-elicited sera show varying degrees of reduction in neutralization against a range of key SARS-CoV-2 variants, including variant Alpha, Beta, Gamma, Iota, and Delta. Our work show diminished neutralization potency against multiple variants in vaccine-elicited sera, which indicates the potential need for additional boost vaccinations.

Comprehensive analysis of the MIR4435-2HG/miR-1-3p/MMP9/miR-29-3p/DUXAP8 ceRNA network axis in hepatocellular carcinoma.

A growing number of studies have shown that competitive endogenous RNA (ceRNA) regulatory networks might play important roles during the process of hepatocellular carcinoma (HCC). This study assessed the role of the ceRNA network in immune cell infiltration in HCC. Immune-related gene sets were downloaded from Molecular Signatures Database, and differentially expressed genes were screened based on TCGA HCC transcriptome data. The corresponding miRNAs with low expression and good prognostic implications, and the corresponding lncRNAs with high expression and poor prognostic were identified to construct ceRNA networks. The networks were utilized for clinical correlation analysis and risk model construction, and the CIBERSORT algorithm was applied to assess immune cell infiltration. In this study, the mRNA-miRNA-lncRNA model was used to construct a ceRNA network in HCC using immune-related differentially expressed mRNAs. Assessment of the MIR4435-2HG/hsa-miR-1-3p/MMP9/hsa-miR-29-3p/DUXAP8 ceRNA network axis in HCC showed that a high risk/poor prognosis was significantly correlated with tumor stage and invasion depth. MMP9 was positively correlated with resting M0 macrophages and NK cells and negatively correlated with activated mast cells, resting mast cells, monocytes and activated NK cells. DUXAP8 was positively correlated with M2 macrophages and negatively correlated with MIR4435-2HG, which was positively correlated with M2 macrophages and negatively correlated with activated mast cells, CD8 T cells and follicular helper T cells. The correlation of the MIR4435-2HG/hsa-miR-1-3p/MMP9/hsa-miR-29-3p/DUXAP8 ceRNA network axis with immune cell infiltration provides further information on the mechanism of HCC development. The result might improve our understanding the interactions between immune related genes and non-coding RNAs in the occurrence and development of HCC, and the relevant RNAs might be used as diagnostic and prognostic biomarkers and molecular targets in HCC patients.

[Establishment of chimeric antigen receptor NK92MI cells recognizing HBsAg].

Objective To construct a lentiviral expression vector of chimeric antigen receptor (CAR) recognizing hepatitis B surface antigen (HBsAg) and make it expressed in NK92MI cells. Methods The CAR was designed and synthesized with the sequence of single chain variable fragment (scFv). Next it was cloned into a lentivirus expression vector, followed by packaging, concentration and titer determination. Then HEK293T cells were transduced with the concentrated lentivirus. The expression of CD3 zeta chain and the infection efficiency in the NK92MI cells were detected by Western blotting. Results The lentiviral expression vector of CAR for HBsAg recognition was successfully constructed, and the titer was ≥107 transducing units (TU)/mL. The expression of protein CD3zeta was verified in the NK92MI cells after infected with the lentiviral vector. Conclusion CAR-NK92MI cells recognizing HBsAg has been established successfully.

Identification of microRNA hsa-miR-30c-5p as an inhibitory factor in the progression of hepatocellular carcinoma and investigation of its regulatory network via comprehensive analysis.

Hepatocellular carcinoma (HCC) is a primary liver cancer with high morbidity and mortality. An increasing number of abnormal gene expressions were identified to be associated with the progression of HCC. Previous studies showed that the hsa-miR-30 c-5p (miR-30 c), one of the miR-30 family members, might play a role in suppressing tumor progression in a variety of tumors. The present study aims to examine miR-30 c effects in the development of HCC. The role of miR-30 c in HCC was comprehensively investigated by using bioinformatics and experiments in vitro. The multiple databases were combined to predict and screen the target genes and upstream lncRNAs of miR-30 c, and then constructed a competitive endogenous RNA (ceRNA) regulatory network with miR-30 c as the central miRNA. The miR-30 c-related ceRNA regulatory network was also initially validated in vitro. The results showed that miR-30 c over-expression could inhibit proliferation, migration, invasion, induce apoptosis, and increase G0/G1 phase ratio of HCC cells. Three miR-30 c upstream lncRNAs and 12 miR-30 c target genes were expressed in HCC cells with increased expression and poor prognosis, and a miR-30 C-related ceRNA regulatory network was constructed. This study verified miR-30 c as an inhibitory factor in the progression of HCC and performed analyses on the miR-30 c regulatory network, which might provide potential target information for HCC prognoses and therapies. However, further experiments in vivo and studies including clinical trials will be conducted to validate our results.

Analysis of Risk Factors for 24 Patients With COVID-19 Developing From Moderate to Severe Condition.

Objective: The present study aimed at investigating the clinical risk factors for COVID-19 patients developing from moderate condition to severe condition, and providing reference for early intervention and prognosis. Methods: We collected the clinical data of 24 patients with moderate-to-severe COVID-19 who were admitted to the isolation ward of the First Affiliated Hospital of Bengbu Medical College from January, 2020 to February 20, 2020, and evaluated the data of clinical characteristics, blood test results, inflammatory index, chest CT imaging characteristics, and antiviral treatment, comparing this with the clinical data of 41 patients with moderate condition in the same period. From this comparison we thus summarized the current knowledge of potential risk factors for COVID-19 patients developing from moderate to severe condition. Results: (1) Clinical characteristics: The moderate-to-severe group and the moderate group in terms of combined common underlying diseases and respiratory frequency showed significant difference statistically (t-value were 13.32, 6.17, respectively, P < 0.05), while no significant difference between the two groups in gender, age, or clinical symptoms was statistically observed(P > 0.05). (2) Analysis of blood test results: The lymphocyte count and plasma albumin of the moderate-to-severe group were significantly lower than those of the moderate group (t-values were 4.16, 4.11, respectively, P < 0.05), and the blood glucose and urea of the moderate-to-severe group were significantly higher than those of the moderate group (t-value were 3.27, 4.19, respectively, P < 0.05). However, there was no significant difference in terms of white blood cell count (WBC), platelet count (PLT), and glutamic-pyruvic transaminase (GPT) (P > 0.05). (3) Comparison of inflammatory indicators: The level of IL-6 and CRP of the moderate-to-severe group were significantly higher than those of the moderate group (t-values were 2.84, 4.88, respectively, P < 0.05). (4) Imaging comparison: As for patients with moderate COVID-19, the imaging manifestations were the concurrence of ground-glass opacity, patchy shadow, and consolidation shadow in both lungs, diffuse ground-glass opacity in both lungs accompanied by air bronchogram, and large area consolidation of both lungs with pulmonary interstitial changes. The possibility for these patients to develop into severe condition increased, and the differences were statistically significant (t = 10.92, P < 0.05). (5) Clinical antiviral treatment: There was no statistically significant difference in the combination of two or three antiviral drugs between the two groups (χ2 = 0.05, P > 0.05). Conclusion: Current evidence suggested that the combination of common underlying diseases, respiratory frequency, lymphocyte count, blood glucose, albumin, urea level, inflammatory factors (CRP, IL-6), and imaging manifestations collectively contributed to the potential risk factors for the development of COVID-19 from moderate condition to severe condition. Particular attention should be paid to early detection and intervention during clinical work, which will be of vital significance to the ascent of the recovery rate as well as the reduction of mortality.

[Establishment of STO Cell Lines Expressing Green Fluorescent Protein and Mouse Leukemia Inhibitory Factor].

OBJECTIVE: To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer. METHODS: The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed. RESULTS: The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer. CONCLUSION: The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.

[Preparation and cytotoxic effects of scFv targeting human c-Met protein on lung adenocarcinoma A549 cells].

Objective To prepare and purify human single-chain variable fragment (scFv) targeting c-Met protein and identify its targeting to c-Met protein and its effects on lung adenocarcinoma cells. Methods The scFv gene was inserted into the pFuse vector to construct eukaryotic expression vector that expressed the Met-scFv fused with mouse Fc fragment. Met-scFv was purified by AKTA Protein Purification System and subjected to functional detection. ELISA was used to detect Met-scFv affinity. Flow cytometry and immunofluorescence staining were used to determine the recognition and binding ability of Met-scFv to lung adenocarcinoma cells. The impact of Met-scFv on the proliferation of A549 cells was evaluated by CCK-8 assay. The cytotoxic effect of Met-scFv on A549 cells was examined by the LDH release kit through CDC and ADCC assays. Results ELISA showed that Met-scFv had a dose-effect relationship with c-Met protein. Flow cytometry and immunofluorescence staining revealed that Met-scFv group signal was positive compared with the control group, which suggested that Met-scFv could specifically bind A549 cells. Met-scFv reduced the proliferation of A549 cells and had cytotoxicity to A549 cells, and the toxicity was positively correlated with the dose of Met-scFv. Conclusion Met-scFv targeting c-Met protein has been prepared and it can recognize and mediate to kill A549 cells in vitro.