C-type Lectin Domain Family 4 Member G (CLEC4G) Is a Negative Marker for CD34 in the Evolution of Liver Pathogenesis.

OBJECTIVE: This study aimed to explore the expression of the C-type lectin domain family 4 member G (CLEC4G) gene in the liver sinusoidal endothelial cells (LSECs) during liver pathogenesis, and to evaluate its correlation with CD34 and clinical significance in hepatocellular carcinoma patients. METHODS: We conducted bioinformatics analysis of the differential expression of CLEC4G in various human organs, carcinomatous and adjacent tissues. Then, mRNA and protein expression levels of CD34 in hepatocellular carcinoma (HCC) samples were detected via real-time quantitative reverse transcription PCR (qRT-PCR) and immunohistochemical (IHC), respectively. ELISA was applied to detect serum levels of CLEC4G in healthy controls, liver fibrosis and HCC patients. RESULTS: The expressions of mRNA and protein levels of CLEC4G were higher in normal liver tissues, moderately expressed in cirrhotic and para-cancerous tissues (P<0.001), and lowest in HCC tissues (P<0.001). We also found high CD34 expression in tumors, which was negatively correlated with CLEC4G at both mRNA and protein levels. Compared to the healthy controls, the CLEC4G levels in liver fibrosis patients and HCC patients gradually became lower (P<0.001). CONCLUSIONS: The low expression of CLEC4G is potentially correlated with LSEC capillarization and the appearance of micro-vessels. Such a phenomenon may serve as a reliable diagnostic marker for hepatocellular carcinoma.

Trends in prevalence and all-cause mortality of metabolic dysfunction-associated fatty liver disease among adults in the past three decades: Results from the NHANES study.

BACKGROUND: Given the escalating epidemic of obesity and diabetes coupled with redefined diagnostic criteria, it is critical to identify the prevalence of metabolic dysfunction-associated fatty liver disease (MAFLD). We sought to determine the prevalence and mortality outcomes of MAFLD subtypes based on diagnostic criteria in the USA over the past three decades. METHODS: Eleven cycles of the National Health and Nutrition Examination Surveys (NHANES; 1988-1994 and 1999-2020) were used, and 72,224 participants were included. MAFLD was defined according to the 2020 International Expert Consensus. Based on diagnostic criteria and risk factors, MAFLD was categorized into seven subtypes: type 1 (obesity subtype), 2 (metabolic unhealthy subtype), 3 (diabetes subtype), 4 (metabolic unhealthy non-diabetes subtype), 5 (obesity and diabetes subtype), 6 (metabolic unhealthy non-obesity subtype), and 7 (mixed subtype). RESULTS: Over the study period, the estimated prevalence of MAFLD increased significantly from 22% in 1988-1994 to 36% in 2017-2020. The prevalence of Type 4 was the highest, followed by that of Type 7, whereas other types were low and almost unchanged over time. Individuals with MAFLD had 19% and 38% increased mortality risks from all causes and cardiovascular disease, respectively. Among them, the metabolically unhealthy participants with normal weight demonstrated a 116% higher risk for all-cause mortality [hazard ratio (HR): 2.16, 95% CI: 1.52-3.08] and a 222% higher risk for cardiovascular mortality (HR: 3.22, 95% CI: 1.72-6.04). Interestingly, stratification and interaction analyses demonstrated a significant impact of metabolic parameters on the relationship between MAFLD and all-cause mortality. CONCLUSIONS: In conclusion, our study identified an increase in MAFLD prevalence and a significant association between metabolic derangements in MAFLD and all-cause or cardiovascular mortality.

Capsaicin suppresses hepatocarcinogenesis by inhibiting the stemness of hepatic progenitor cells via SIRT1/SOX2 signaling pathway.

BACKGROUND & AIMS: Capsaicin, a functional component of chili pepper, possesses anti-inflammatory, analgesic, and anti-cancer properties. This study aimed to determine the property of capsaicin against hepatocarcinogenesis in vivo and investigate the role of the SIRT1/SOX2 pathway in the mode of action of capsaicin in hepatic progenitor cells (HPCs), which is related to hepatocarcinogenesis. MATERIALS & METHODS: We prepared a diethylnitrosamine-induced liver cancer model in rats to examine hepatocarcinogenesis, and delivered liposomal capsaicin through the subcutaneous transposition of the spleen to the liver. Liver sections from rats and hepatocarcinoma patients were stained for the markers of HPCs or SIRT1/SOX2 signaling. SIRT1/SOX2 signalling expression was measured using immunoprecipitation and western blot. RESULTS: We found that capsaicin significantly inhibited hepatocarcinogenesis. Notably, capsaicin inhibited HPCs activation in vivo but did not induce apoptosis in the normal hepatic progenitor cell line in rats in vitro. This suggests that capsaicin suppresses hepatocarcinogenesis by inhibiting the stemness of HPCs. Moreover, capsaicin can induce this inhibition by reducing the stability of SOX2. SIRT1 is overexpressed in liver cancer and acts as a tumor promoter via SOX2 deacetylation. Using immunoprecipitation, we identified direct binding between SIRT1 and SOX2. The capsaicin treatment resulted in SIRT1 downregulation which reduced deacetylation, and increased nuclear export as well as subsequent ubiquitous degradation of SOX2. CONCLUSIONS: Altogether, we report that capsaicin suppresses hepatocarcinogenesis by inhibiting the stemness of HPCs via SIRT1/SOX2 signaling. It may serve as a promising therapeutic candidate for liver cancer.

Association of Cholecystectomy With Liver Fibrosis and Cirrhosis Among Adults in the USA: A Population-Based Propensity Score-Matched Study.

Background and Aims: Cholecystectomy is the "gold standard" for treating diseases of the gallbladder. In addition, non-alcoholic fatty liver disease (NAFLD), liver fibrosis or cirrhosis, are major causes of morbidity and mortality across the world. However, the association between cholecystectomy and these diseases is still unclear. We assessed the association among US adults and examined the possible risk factors. Methods: This cross-sectional study used data from 2017 to 2018 National Health and Nutrition Examination Survey, a population-based nationally representative sample of US. Liver fibrosis and cirrhosis were defined by median stiffness, which was assessed by transient elastography. Furthermore, patients who had undergone cholecystectomy were identified based on the questionnaire. In addition, Propensity Score Matching (PSM, 1:1) was performed based on gender, age, body mass index (BMI) and diabetes. Results: Of the 4,497 included participants, cholecystectomy was associated with 60.0% higher risk of liver fibrosis (OR:1.600;95% CI:1.278-2.002), and 73.3% higher risk of liver cirrhosis (OR:1.733, 95% CI:1.076-2.792). After PSM based on age, gender, BMI group and history of diabetes, cholecystectomy was associated with 139.3% higher risk of liver fibrosis (OR: 2.393;95% CI: 1.738-3.297), and 228.7% higher risk of liver cirrhosis (OR: 3.287, 95% CI: 1.496-7.218). Conclusions: The present study showed that cholecystectomy is positively associated with liver fibrosis and cirrhosis in US adults. The discovery of these risk factors therefore provides new insights on the prevention of NAFLD, liver fibrosis, and cirrhosis.

Dynamic Changes in the Expression of Interferon-Stimulated Genes in Joints of SPF Chickens Infected With Avian Reovirus.

Avian reovirus (ARV) can induce many diseases as well as immunosuppression in chickens, severely endangering the poultry industry. Interferons (IFNs) play an antiviral role by inducing the expression of interferon-stimulated genes (ISGs). The effect of ARV infection on the expression of host ISGs is unclear. Specific-pathogen-free (SPF) chickens were infected with ARV strain S1133 in this study, and real time quantitative PCR was used to detect changes in the dynamic expression of IFNs and common ISGs in joints of SPF chickens. The results showed that the transcription levels of IFNA, IFNB, and several ISGs, including myxovirus resistance (MX), interferon-induced transmembrane protein 3 (IFITM3), protein kinase R (PKR), oligoadenylate synthase (OAS), interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), interferon-stimulated gene 12 (ISG12), virus inhibitory protein (VIPERIN), interferon-alpha-inducible protein 6 (IFI6), and integrin-associated protein (CD47), were upregulated in joints on days 1-7 of infection (the levels of increase of MX, IFIT5, OAS, VIPERIN, ISG12, and IFI6 were the most significant, at hundreds-fold). In addition, the expression levels of the ISGs encoding zinc finger protein 313 (ZFP313), and DNA damage-inducible transcript 4 (DDIT4) increased suddenly on the 1st or 2nd day, then decreased to control levels. The ARV viral load in chicken joints rapidly increased after 1 day of viral challenge, and the viral load remained high within 6 days of viral challenge. The ARV viral load sharply decreased starting on day 7. These results indicate that in SPF chicken joints, many ISGs have mRNA expression patterns that are basically consistent with the viral load in joints. IFNA, IFNB, and the ISGs MX, IFITM3, PKR, OAS, IFIT5, ISG12, VIPERIN, IFI6, and CD47 play important roles in defending against ARV invasion, inhibiting ARV replication and proliferation, and promoting virus clearance. These results enrich our understanding of the innate immune response mechanisms of hosts against ARV infection and provide a theoretical basis for prevention and control of ARV infection.

Co-Infection with Common Respiratory Pathogens and SARS-CoV-2 in Patients with COVID-19 Pneumonia and Laboratory Biochemistry Findings: A Retrospective Cross-Sectional Study of 78 Patients from a Single Center in China.

BACKGROUND This retrospective study aimed to investigate co-infections with common respiratory pathogens and SARS-CoV-2 and laboratory biochemistry findings in patients with COVID-19 in the Zhuzhou area of China, in order to provide a reference for the disease assessment and clinical treatment of COVID-19. MATERIAL AND METHODS The clinical data of COVID-19 patients admitted to the hospital of Zhuzhou City from January 28 to March 15, 2020, as well as laboratory test results for respiratory pathogens and biochemical indicators, were collected to conduct correlation analyses. All patients were diagnosed based on fluorescence-based PCR assay for SARS-CoV-2. RESULTS Eleven of the 78 patients (14.1%) were co-infected with other respiratory pathogens, among which Mycoplasma pneumoniae (n=5, 45.5%) and respiratory syncytial virus (n=4, 36.4%) were the most frequent. There were 8 patients co-infected with 1 other pathogen and 3 patients co-infected with 2 other pathogens. Compared with mono-infected COVID-19 patients, patients with co-infections had significantly higher levels of procalcitonin (P=0.002). CONCLUSIONS The findings showed that Mycoplasma pneumonia and respiratory syncytial virus were the most common co-infections in patients with COVID-19 pneumonia. Increased levels of PCT in patients with COVID-19 pneumonia were associated with co-infection.

Association of Serum Vitamin C With NAFLD and MAFLD Among Adults in the United States.

BACKGROUND AND AIMS: Despite the remarkable progress of metabolic dysfunction-associated fatty liver disease (MAFLD), formerly named non-alcoholic fatty liver disease (NAFLD), the disease remains poorly improved. Since increased oxidative stress and inflammation contribute to the initiation and progression of fatty liver disorders, vitamin C (VC), an antioxidant agent, might be a suitable treatment option for MAFLD. However, the lack of clinically confirmed benefits makes clinicians challenging to recommend antioxidant supplements for MAFLD individuals. METHODS: Herein, the nationally representative National Health and Nutrition Examination Survey 2017-2018 data were collected to evaluate the potential association between the serum VC levels with the risk of different categories of NALFD and the newly proposed MAFLD terminology. Hepatic steatosis was defined as controlled attenuated parameter scores ≥ 263 dB/m, whereas liver fibrosis (LF) status was defined as F0-F4, with the cutoff values of median liver stiffness being 6.3, 8.3, 10.5, and 12.5 (KPa), respectively. A cross-sectional analysis was performed to calculate the odds rate and determine the potential beneficial effects of VC. RESULTS: A total of 4,494 participants aged more than 18 years and conducted transient elastography examinations were included. Our findings demonstrated that participants with increased serum VC status were more likely to be female predominant, more educated, and moderate drinkers. Interestingly, female participants tended to have a lower prevalence of NAFLD, MAFLD, LF, and liver cirrhosis (LC) after stratification by gender. Moreover, our results revealed that participants from the quartile three group (quartile 3: 50.5-67.0 µmol/L) experienced a slightly lower risk of MAFLD than the risk of NAFLD. Of note, the serum concentration of VC (quartile 2: 30.9-50.5 µmol/L) inversely associated with LF and LC was lower than the serum VC level (quartile 3) associated with NAFLD and MAFLD. Notably, individuals from the quartile 3 group experienced a statistically significant 32.5, 42.0, 45.7, and 71% decrease in risk of NAFLD, MAFLD, LF, and LC, respectively. CONCLUSION: In summary, our findings suggested an inverse association between serum VC levels and NAFLD, MAFLD, LF, or LC. Additionally, adjustment of VC supplementation according to age, gender, and ethnicity may be a promising candidate for these diseases.

GeXP analyzer-based multiplex reverse-transcription PCR assay for the simultaneous detection and differentiation of eleven duck viruses.

BACKGROUND: Duck viral pathogens primarily include the avian influenza virus (AIV) subtypes H5, H7, and H9; duck hepatitis virus (DHV); duck tembusu virus (DTMUV); egg drop syndrome virus (EDSV); duck enteritis virus (DEV); Newcastle disease virus (NDV); duck circovirus (DuCV); muscovy duck reovirus (MDRV); and muscovy duck parvovirus (MDPV). These pathogens cause great economic losses to China's duck breeding industry. RESULT: A rapid, specific, sensitive and high-throughput GeXP-based multiplex PCR assay consisting of chimeric primer-based PCR amplification with fluorescent labeling and capillary electrophoresis separation was developed and optimized to simultaneously detect these eleven viral pathogens. Single and mixed pathogen cDNA/DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. Corresponding specific DNA products were amplified from each pathogen. Other pathogens, including duck Escherichia coli, duck Salmonella, duck Staphylococcus aureus, Pasteurella multocida, infectious bronchitis virus, and Mycoplasma gallisepticum, did not result in amplification products. The detection limit of GeXP was 10(3)copies when all twelve pre-mixed plasmids containing the target genes of eleven types of duck viruses were present. To further evaluate the reliability of GeXP, 150 clinical field samples were evaluated. Comparison with the results of conventional PCR methods for the field samples, the GeXP-multiplex PCR method was more sensitive and accurate. CONCLUSION: This GeXP-based multiplex PCR method can be utilized for the rapid differential diagnosis of clinical samples as an effective tool to prevent and control duck viruses with similar clinical symptoms.

Epidemiological surveillance of low pathogenic avian influenza virus (LPAIV) from poultry in Guangxi Province, Southern China.

Low pathogenic avian influenza virus (LPAIV) usually causes mild disease or asymptomatic infection in poultry. However, some LPAIV strains can be transmitted to humans and cause severe infection. Genetic rearrangement and recombination of even low pathogenic influenza may generate a novel virus with increased virulence, posing a substantial risk to public health. Southern China is regarded as the world "influenza epicenter", due to a rash of outbreaks of influenza in recent years. In this study, we conducted an epidemiological survey of LPAIV at different live bird markets (LBMs) in Guangxi province, Southern China. From January 2009 to December 2011, we collected 3,121 cotton swab samples of larynx, trachea and cloaca from the poultry at LBMs in Guangxi. Virus isolation, hemagglutination inhibition (HI) assay, and RT-PCR were used to detect and subtype LPAIV in the collected samples. Of the 3,121 samples, 336 samples (10.8%) were LPAIV positive, including 54 (1.7%) in chicken and 282 (9.1%) in duck. The identified LPAIV were H3N1, H3N2, H6N1, H6N2, H6N5, H6N6, H6N8, and H9N2, which are combinations of seven HA subtypes (H1, H3, H4, H6, H9, H10 and H11) and five NA subtypes (N1, N2, N5, N6 and N8). The H3 and H9 subtypes are predominant in the identified LPAIVs. Among the 336 cases, 29 types of mixed infection of different HA subtypes were identified in 87 of the cases (25.9%). The mixed infections may provide opportunities for genetic recombination. Our results suggest that the LPAIV epidemiology in poultry in the Guangxi province in southern China is complicated and highlights the need for further epidemiological and genetic studies of LPAIV in this area.

[Development of a GeXP assay for simultaneous differentiation of six chicken respiratory viruses].

A GeXP based multiplex PCR assay was developed to simultaneously detect six different chicken respiratory viruses including H5, H7, H9 subtypes of avian influenza virus(AIV), new castle disease virus (NDV), infectious bronchitis virus(IBV) and infectious laryngotracheitis virus(ILTV). According to the conserved sequences of genes of each pathogen, seven pairs of specific primers were designed, and the reaction conditions were optimized. The specificity and accuracy of GeXP were examined using samples of single and mixed infections of virus. The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of cloned plasmids. To further evaluate the reliability, thirty-four clinical samples were detected by GeXP. The corresponding specific fragments of genes were amplified. The detection limit of GeXP was 10(2) copies/microL when all of 7 pre-mixed plasmids containing target genes of six chicken respiratory viruses were present. In the detection of thirty-four clinical samples, the results of GeXP were accorded with the viral isolation completely. In conclusion, this GeXP assay is a rapid, specific, sensitive and high-throughput method for the detection of chicken respiratory virus infections. It can be applied in rapid differential diagnosis for clinical samples, and also provide an effective tool to prevent and control chicken respiratory diseases with similar clinical symptoms.

[Visual detection of H1 subtype and identification of N1, N2 subtype of avian influenza virus by reverse transcription loop-mediated isothermal amplification assay].

In order to visually detect H1, N1 and N2 subtype of avian influenza virus (AIV), three reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed. According to the sequences of AIV gene available in GenBank, three degenerate primer sets specific to HA gene of H1 subtype AIV, NA gene of N1 and N2 subtype AIV were designed, and the reaction conditions were optimized. The results showed that all the assays had no cross-reaction with other subtype AIV and other avian respiratory pathogens, and the detection limit was higher than that of conventional RT-PCR. These assays were performed in water bath within 50 minutes. Without opening tube, the amplification result could be directly determined by inspecting the color change of reaction system as long as these assays were fin-ished. Fourteen specimens of H1N1 subtype and eight specimens of H1N2 subtype of AIV were identified from the 120 clinical samples by RT-LAMP assays developed, which was consistent with that of virus isolation. These results suggested that the three newly developed RT-LAMEP assays were simple, specific and sensitive and had potential for visual detection of H1, N1 and N2 subtype of AIV in field.

A duplex quantitative real-time PCR assay for the detection of Haplosporidium and Perkinsus species in shellfish.

A duplex quantitative real-time polymerase chain reaction (dq-PCR) assay was optimized to simultaneously detect Haplosporidium spp. and Perkinsus spp. of shellfish in one reaction. Two sets of specific oligonucleotide primers for Haplosporidium spp. and Perkinsus spp., along with two hydrolysis probes specific for each parasite group, were used in the assay. The dq-PCR results were detected and analyzed using the Light Cycler 2.0 software system. The dq-PCR identified and differentiated the two protozoan parasite groups. The sensitivity of the dq-PCR assay was 200 template copies for both Haplosporidium spp. and Perkinsus spp. No DNA product was amplified when known DNA from Marteilia refringens, Toxoplasma gondii, Bonamia ostreae, Escherichia coli, Cymndinium spp., Mykrocytos mackini, Vibrio parahaemolyticus, and shellfish tissue were used as templates. A total of 840 oyster samples from commercial cultivated shellfish farms from two coastal areas in China were randomly collected and tested by dq-PCR. The detection rate of Haplosporidium spp. was 8.6% in the Qindao, Shandong coastal area, whereas Perkinsus spp. was 8.3% coastal oysters cultivated from shellfish farms of Beihai, Guangxi. The dqPCR results suggested that Haplosporidium spp. was prevalent in oysters from Qindao, Shandong, while Perkinsus spp. was prevalent in oysters from the coastal areas of Beihai, Guangxi. This dq-PCR could be used as a diagnostic tool to detect Haplosporidium spp. and Perkinsus spp. in cultivated shellfish.

Identification and characterization of a rat novel gene RSEP4 expressed specifically in central nervous system.

The low-abundantly expressed genes composed the majorities of the mRNAs expressed in the central nervous system (CNS), and were thought to be important for the normal brain functions. Through differential screening a low-abundance cDNA sublibrary with mRNA from neuropathic pain of chronic constriction injury (CCI) model, we have identified a novel rat gene, rat spinal-cord expression protein 4 gene (RSEP4). The total length of RSEP4 cDNA is 2006 bp, with a 501 nucleotide open reading frame (ORF) that encodes a 167 amino acid polypeptide. Northern blot revealed that RSEP4 was expressed specifically in the CNS. In situ hybridization showed that the mRNA of RSEP4 was strongly expressed in the CA1, CA2, CA3 and DG regions of hippocampus, the Purkinje cells of cerebellum, and the small sensory neurons of dorsal horn and large motor neurons of ventral horn of spinal cord. Over-expression of RSEP4-EGFP fusion protein in the human embryonic kidney 293T cells showed that RSEP4 protein was mainly localized in the cell cytoplasm. These results suggest that RSEP4 may play some roles in the CNS.

Localization of CCTbeta in rat brain and overexpression in insect cells.

AIM: To study the localization of CTP: phosphocholine cytidylyltransferase beta isoform (CCTbeta) in rat brain, its expression in insect cells and enzymatic properties. METHODS: Using digoxigenin-labeled CCTbeta probes, in situ hybridization was carried out in rat brain wax sections. CCTbeta was overexpressed in Trichoplusia Ni (Tn) cells using baculovirus expression system. CTP:phosphocholine cytidylyltransferase assay (CT assay) and [3H] metabolic labeling experiment were used to study its activity, properties, and the effect on phosphatidylcholine (PC) synthesis. RESULTS: (1) CCbeta was abundant in CA1, CA2, CA4, and dentate gyrus (DG) region of hippocampus. (2) The content of CCTbeta in transfected Tn cells was over 1 104 times of that in rat brain, and CCTbeta increased the PC synthesis of Tn cells. (3) Hexadecylphosphocholine as well as some ions like Zn2+ and PO3-4 could inhibit the activity of CCTbeta, dCTP was another adaptive substrate of CCTbeta besides CTP. CONCLUSION: CCTbeta showed a similar localization in rat brain with the memory enhancing peptide argipressin (4-8).