[Research advances in the non-invasive diagnosis of metabolic dysfunction-associated fatty liver disease based on magnetic resonance technology].

Metabolic dysfunction-associated fatty liver disease is becoming the most common cause of chronic liver disease worldwide, with a disease spectrum including simple steatosis, steatohepatitis, hepatic fibrosis/cirrhosis, and liver cancer. Most metabolic dysfunction-associated fatty liver diseases progress slowly, but steatohepatitis, especially in patients accompanied by significant liver fibrosis, has a significantly increased risk of adverse liver disease outcomes and all-cause death. Therefore, early-stage identification of medium-and high-risk groups carried out by stratified management has important clinical significance. Pathological diagnosis is the gold standard for diagnosing steatohepatitis and liver fibrosis. However, its invasiveness, sampling errors, and unsuitability for dynamic monitoring limit its clinical application. In recent years, a large number of non-invasive diagnostic methods based on somatology, serology, and imaging have shown great development prospects in order to meet the clinical needs of assessing disease severity and risk stratification. This article reviews and summarizes the application and progress of magnetic resonance imaging technology in the non-invasive diagnosis of metabolic dysfunction-associated fatty liver disease.

[Improvement situation on indexes of the zebrafish disease model of non-alcoholic fatty liver disease with FGF21 analogues].

Objective: To detect the therapeutic efficacy of FGF21 analogues on the zebrafish model of non-alcoholic fatty liver disease. Methods: A zebrafish model of non-alcoholic fatty liver disease was established by providing the normal diet fed to wild-type zebrafish three times daily. PF-05231023 was administered exogenously at a final concentration of 0.5 µmol/L. Body length, body weight, triglycerides, and other indexes were measured after 20 days. Pathological changes were evaluated in liver tissue sections by HE staining. Quantitative PCR was used to identify expressional changes in genes related to lipid metabolism, endoplasmic reticulum stress, and inflammation. Results: QPCR and immunofluorescence staining results showed that FGF21 was highly expressed in the zebrafish model group. The addition of the FGF21 analogue PF-05231023 significantly reduced the body length and body weight (P < 0.01), and the triglyceride content (P < 0.05) in the zebrafish model group. The liver HE staining results showed that PF-05231023 had alleviated the large and tiny bullae fat, lesions, and others in the zebrafish model group. The quantitative PCR results demonstrated that PF-05231023 reduced the expression of lipogenic factors (P < 0.01), inflammatory-related factors (P < 0.001), and genes related to endoplasmic reticulum stress (P < 0.05), but raised lipid-oxidation-related factors (P < 0.05) in the zebrafish model group. The addition of PF-05231023 reduced oleic acid-induced lipid and triglyceride levels in HepG2 cells. Conclusion: FGF21 analogue addition can improve indexes in the zebrafish disease model of non-alcoholic fatty liver disease.

[Metabolic study of iron deposition based on magnetic resonance in patients with nonalcoholic fatty liver disease].

Objective: To explore the relationship between liver iron deposition and steatosis in patients with non-alcoholic fatty liver disease (NAFLD) through MRI. Methods: 163 cases of liver biopsy underwent MRI examination. R2* was used to measure liver iron content. Dixon-based proton density fat fraction (PDFF) was used to measure liver fat content. One-way ANOVA, r-correlation, ROC curve, and others were used to assess the relationship between clinical case data, serological indices, and imaging results in accordance with the pathological results of the liver biopsy. Results: R2* gradually increased as the pathological steatosis grade rose. The R2* that corresponded to no steatosis (< 5%), mild steatosis (14.95%±8.55%), moderate steatosis (46.30%±9.32%), and severe steatosis (73.86%±6.35%) were 27.56±4.40, 31.06±5.95, 38.06±4.80, and 48.10±5.55 (P < 0.001), respectively. There was a positive correlation between R2* and liver steatosis content (r= 0.769, P < 0.05). The area under the ROC curve and cut-off value were 0.88 and 31.77, respectively, and there was no distinct relationship with liver inflammation or fibrosis. Conclusion: R2* can quantitatively and non-invasively evaluate liver iron deposition in patients with NAFLD. A distinct relationship exists between liver steatosis and iron deposition, and iron deposition tends to increase as the steatosis aggravates.

[Application analysis of noninvasive prenatal testing for fetal chromosome copy number variations in Chinese laboratories].

Objective: To investigate the general situation, detection range, testing reagents, and clinical performance of non-invasive prenatal testing (NIPT) for fetal chromosomal copy number variations (CNVs) in Chinese laboratories. Methods: The National Center for Clinical Laboratories of the National Health Commission designed a questionnaire for the detection of CNVs by NIPT, which included the investigation of whether the laboratory has carried out NIPT to detect CNVs and its testing scope, reagents/platforms, intended uses, screening populations and clinical performance. The questionnaires were distributed to 355 laboratories in 31 provinces, autonomous regions, and municipalities across the country on October, 2020. Further, the feedbacks were statistical analyzed. Results: Two hundred and twenty-eight laboratories had performed NIPT to detect CNVs, including 116 types of CNVs, and more than 95% of laboratories chose to detect the CNVs of 5p15 deletion, 22q11.2 deletion, 1p36 deletion, and 15q11.2 deletion. All testing reagents used were laboratory-developed tests and were based on massive parallel sequencing, the minimum amount of sequencing data was 3-15 M reads, the detection limit of fetal fraction was 3%-5%, and the minimum size of variants that can be detected was 1-5 Mb. The proportion of laboratories that apply CNVs testing for daily project, voluntary requirements of patients, and scientific research were 58.8% (134/228), 57.5% (131/228), and 20.6% (47/228), respectively. One hundred and thirty-four laboratories were fully or partially aware of the clinical performance of NIPT to detect microdeletion/microduplication syndromes, and the laboratories' declared sensitivity of NIPT for Cri du Chat syndrome, 22q11.2 deletion syndrome, 1p36 deletion syndrome, and Angelman syndrome were 50.0%-100%, 60.0%-100%, 50.0%-100%, and 33.3%-100%, and the positive predictive values were 9.0%-50.0%, 18.0%-100%, 20.0%-30.0%, and 20.0%. Conclusion: The detection of CNVs by NIPT in Chinese laboratories need to be standardized. Laboratories should detect CNVs with clear clinical significance in accordance with the guidelines, conduct performance validation of the reagents, then perform NIPT test and provide adequate interpretation after mastering the clinical performance sufficiently.

[Metabolic-associated fatty liver disease-related liver cirrhosis and cryptogenic liver cirrhosis].

Metabolic-associated fatty liver disease (MAFLD) is currently the most common chronic liver disease in the world, which may eventually progress to cirrhosis, hepatocellular carcinoma and liver failure. Fatty liver disease was once considered to be the most common cause of cryptogenic cirrhosis. Recently, a new definition of MAFLD suggests that MAFLD-related liver cirrhosis is no longer a kind of cryptogenic cirrhosis, and it belong to two different concepts and may have different liver and extrahepatic adverse outcomes. In this paper, the definition, epidemiology, diagnosis, treatment and other aspects of MALFD-related liver cirrhosis and cryptogenic liver cirrhosis are described in order to facilitate clinical practice, improve the efficiency of clinical research, and benefit clinicians and patients.

[Role of gut-liver-immune axis in the pathogenesis of nonalcoholic steatohepatitis].

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in the world. Liver cirrhosis, liver cancer and a variety of extrahepatic chronic diseases are important risk factors for NAFLD. Currently, there is still a lack of effective therapeutic drugs. Liver inflammation is a key driving factor for the progression of NAFLD, so regulating liver inflammation may provide a potential means to delay and reverse the progression of nonalcoholic steatohepatitis (NASH). Studies have found that the gut-liver-immune axis plays an important role in the progression of NASH. Gut microbiota can use its metabolites to induce glycolipid toxicity, oxidative stress and intestinal barrier damage, while bacterial components such as lipopolysaccharides, peptidoglycans, bacterial DNA and extracellular vesicles can translocate into the liver through the damaged intestinal barrier, causing excessive activation of immune cells, thus aggravating liver inflammation and promoting the progress of NASH. This paper focuses on the gut-liver-immune axis to analyze the gut microbiota mediated liver immunity and its mechanism in the occurrence and development of NASH, so as to lay a theoretical foundation for the research and development of new therapeutic strategies for NASH.

[Studying the correlation between ferritin and non-alcoholic fatty liver disease].

Objective: To analyze the correlation between serum ferritin and steatosis in non-alcoholic fatty liver disease. Methods: Data of 167 cases who underwent liver biopsy in the Affiliated Hospital of Hangzhou Normal University were collected. Hydrogen proton magnetic resonance spectroscopy were performed within one week. The pathological results of liver biopsy were used as the gold standard to analyze the case data, serological indicators, magnetic resonance spectroscopy-proton density fat fraction. Results: Pathological monitoring result showed that the serum ferritin in patients without steatosis, and with mild, moderate and severe steatosis were (206.20 ± 189.83), (286.65 ± 200.80), (326.55 ± 214.71), (391.50 ± 184.93) ng/ml, respectively, P < 0.005. Serum ferritin was correlated to body mass index, PDFF, alanine aminotransferase, gamma glutamyltransferase, low-density lipoprotein, high-density lipoprotein. The area under ​​the receiver operating characteristic curve with ferritin for the diagnosis of non-alcoholic fatty liver disease was 0.716, and the optimal diagnostic threshold was 214.56 ng/ml. The sensitivity and specificity were 80.1%, and 68.8%, respectively. There was no statistically significant difference between the intralobular inflammation, fibrosis, and ferritin. Prussian blue iron staining had no apparent deposition of iron particles. Conclusion: Ferritin has significant positive correlation with the results of pathological and magnetic resonance imaging for liver steatosis. Therefore, it can be used as a non-invasive diagnostic method for liver steatosis evaluation.

[Study of liver fat and iron deposition quantification based on magnetic resonance imaging in rats with nonalcoholic fatty liver disease].

Objective: To investigate the accuracy of magnetic resonance imaging (MRI) for quantitative determination of liver fat and iron content through a rat model of non-alcoholic fatty liver disease (NAFLD) induced by methionine-choline deficient (MCD) diet. Methods: Sixty SD rats were randomly divided into experimental (MCD-diet group, n = 30) and normal control group (normal diet, n = 30). Rats were subjected to special MRI examinations at the ends of 2, 4, and 8 weeks. Proton density fat fraction (PDFF) and R2* value were obtained, and then the rats were sacrificed. The liver tissues were stained with HE, Prussian blue, etc. Liver tissue non-heme iron (NHI) homogenate was determined by flame atomic absorption spectrometry. According to different data, one-way analysis of variance, t-test or χ (2) test was used for statistical analysis. Results: PDFF and R2 * values in the MCD diet group at 2, 4 and 8 weeks were 23.37% ± 9.20%, 28.07% ± 6.84%, 25.40% ± 7.04% (P < 0.01) and 90.58 ± 15.92, 104.12 ± 13.47, 106.35 ± 15.76 (P < 0.05), respectively, which were significantly higher than the normal control group PDFF (2.39% ± 0.50%, 2.45% ± 0.45%, 3.26% ± 0.80%) and R2* (48.93 ± 7.90, 54.71 ± 5.91, 64.25 ± 15.76). Additionally, with the disease progression, R2 * had gradually increased, which was consistent with the NHI trend in liver tissue homogenates of each group. Conclusion: MRI, as a non-invasive quantitative method, can accurately assess liver fat and iron content in fatty liver disease, and with the degree of severity of fat changes, iron deposits tend to increase.

[The effect of welding ultraviolet on the antioxidant function and DNA of workers].

Objective: To explore the skin damage, oxidative damage and DNA damage of welding workers caused by ultra violet. Methods: From 1 May to 30 June 2017, 125 welding workers were selected as the contact group, which was divided into 2, 4, 6, 8 and 10-year-working age groups with 25 workers in each group and 25 healthy volunteers as the control group. The workers in the contact group were investigated with questionnaires on the distribution and wearing of protective equipment; the subjects in the two groups were examined in dermatology, and the peripheral blood was collected to detect the total antioxidant capacity (T-AOC) and DNA damage of mononuclear cells, and the influence of ultraviolet on the above indexes was analyzed. Results: The distribution rates of welding mask, dust mask and goggles were 100.0% (125/125) , 96.0% (120/125) , 98.4% (123/125) , respectively, and the distribution rates of welding gloves were 64.8% (81/125) ; the wearing rates of welding mask and goggles were 100.0% (125/125) , 90.4% (113/125) , and the wearing rates of dust mask and welding gloves were 89.6% (112/125) and 64.0% (80/125) , respectively. The acute skin injuries such as hand and face peeling and skin pruritus were serious in the 2-year-working age group, and the chronic skin injuries such as insensitive and numbness and verrucous vegetations were the main manifestations in the 10-year-working age group. Compared with the control group, T-AOC in peripheral blood of workers exposed to 6, 8 and 10 years decreased significantly, and DNA content in comet tail of workers exposed to various working years increased significantly (P<0.05) , while DNA content in comet tail increased with working years (F=1501.130, P<0.05) . Conclusion: UV welding can cause skin damage, reduce the antioxidant capacity of the body, and increase DNA damage with the increase of working age.

[Clinical analysis of 8 cases with anti-GQ1b antibody syndrome].

Objective: To investigate the clinical characterization, treatment and prognosis of anti-GQ1b antibody syndrome. Methods: The clinical data of 8 patients with positive serum anti-GQ1b antibody from the Department of Neurology of Nanjing Brain Hospital between June 2016 and July 2018 were analyzed retrospectively. Their serums were tested by immunoblotting. Relevant literatures were reviewed to investigate possible pathogenesis. Results: Of the 8 cases, 4 cases were male, 4 cases were female; their age ranged from 16 to 76 (47±21) years old. Seven of them were with acute onset, the time course of the disease ranged from 2 to 15 (7±4) days. Six cases had a history of influenza prior to the onset of the presenting symptoms. In terms of the clinical manifestations of the eight patients, two were affected with Guillain-Barre syndrome (GBS), two with Cavernous sinus syndrome, one with Miller Fisher syndrome, one with both GBS and spinal cord demyelination, one with Bulbar paralysis, and one with chronic inflammatory demyelinating polyneuropathy (CIDP). The anti-GQ1b antibody IgG in serum was positive in 6 patients, two of whom were combined with positive IgG of anti-GD1b antibody in serum. The anti-GQ1b antibody IgM in serum was positive in 1 patient, and the anti-GQ1b antibody IgM and anti-GT1b antibody IgM in cerebrospinal fluid (CSF) were both positive in the other patient. In terms of the treatment, 3 patients (3/8) received vitamin B treatment only, 2 patients (2/8) received steroid plus vitamin B treatment, 2 patients (2/8) received intravenous immunoglobulin (IVIG) plus vitamin B treatment, and 1 patient (1/8) received steroid plus IVIG treatment. During the 8-33 months' follow-up after discharge, 6 patients were significantly improved in their symptoms, one with mild diplopia, one with limbs weakness, numbness and difficulty in walking. The symptoms of one patient (case 3) fluctuated twice and recovered again after treatment. Conclusions: The disease spectrum of anti-GQ1b antibodies syndrome is broad, and main symptom is ophtalmoplegia. Immunotherapy with IVIG and steroid would be beneficial to prognosis.

[Study on the correlation between small intestinal dendritic cells and non-alcoholic fatty liver disease in mice].

Objective: To preliminary, explore the effect of small intestinal epithelial dendritic cells on the occurrence and development of nonalcoholic fatty liver disease in mice. Methods: Thirty-two (half male and half female) 4-week-old C57BL/6 mice were randomly divided into two groups. The mice were fed with normal diet (SD group) and high-fat diet (HFD group). Eight mice (half male and half female) were randomly killed from each group over the 14 and 20-weeks feeding period to observe their body weight, liver and small-intestine wet weight. Alanine aminotransferase, aspartate aminotransferase, blood glucose, high-density lipoprotein, low-density lipoprotein, total cholesterol and triglyceride were determined by eyeball blood samples. Pathological diagnosis and alcoholic fatty liver disease activity score were collected. The number of mice small intestinal dendritic cells was observed under a microscope. Statistical analysis was performed to compare two groups of independent samples with homogeneity test of variance, t test, and covariance analysis. Results: The body weight, liver wet weight, serum alanine aminotransferase and aspartate aminotransferase of mice in HFD group were significantly higher than those of control group at 20 weeks (P < 0.05), and the serum high density lipoprotein of mice in HFD group was significantly higher than that of SD group at 14 weeks (P < 0.05). At 14th weeks, the liver tissue of mice in HFD group had early pathological manifestations of hepatitis (fatty degeneration, punctate necrosis and balloon-like degeneration). Of which 87.5% (7/8) of them were diagnosed as non-alcoholic steatohepatitis or non-alcoholic fatty liver disease, while only a few mice in SD group had early pathological manifestations of hepatitis. At 20th weeks, all mice in HFD group were diagnosed with non-alcoholic steatohepatitis or non-alcoholic fatty liver disease, while none of the mice in SD group was diagnosed with non-alcoholic fatty liver disease. At both time points, the percentage of small intestinal dendritic cells in HFD group was significantly higher than that in SD group (14 weeks: 4.181 ± 4.314 vs. 15.099 ± 10.349; 20 weeks: 9.615 ± 8.267 vs. 32.839 ± 24.475, both P < 0.05). Statistical analysis combined with the alcoholic fatty liver disease activity score showed that there was no linear correlation between the two groups (regression coefficient was 20.196%). Conclusion: The number and different staging of small intestinal dendritic cells in mice is associated with the occurrence and development of NAFLD.

[Treatment and prognosis of immunoglobulin G4-related hepatobiliary disease].

Currently, there is no randomized controlled clinical trial of immunoglobulin (Ig) G4-related diseases in the world. Therefore, the best-known evidence-based medical treatment plan for this disorder is unavailable. The goal of IgG4-related hepatobiliary diseases treatment is to alleviate symptoms, prevent disease-related complications and fibrosis progression. A definite diagnosis is warranted before treatment. Hormonal therapy has become the basis of induction of remission in IgG4-related hepatobiliary disease. An initial prednisone dose is 30 ~ 40mg/d or 0.6 mg.kg-1.d-1 for 2 to 4 weeks, thereafter, gradually the dose is reduced within 2-3 months. Maintenance therapy with low-dose glucocorticoids hormone (prednisone 2.5 to 5.0 mg/d) is recommended for 1 to 3 years to prevent disease recurrence. In addition, immunosuppressive agents are equally effective, and in most cases, hormone combined immunosuppressive therapy may respond. Rituximab, a monoclonal antibody is a promising drug for treatment of this kind of diseases.

[Establishment of zebrafish model for non-alcoholic steatohepatitis].

Objective: To establish overfed zebrafish model for non-alcoholic steatohepatitis. Methods: The wild-type zebrafish was fed 3 times a day with normal diet. Body length, weight, and triglyceride levels were measured after 20 days of feeding. The changes in expression of genes associated with cholesterol metabolism, lipid metabolism, endoplasmic reticulum stress, and inflammation were detected by quantitative PCR. Liver tissue sections were stained with H&E. Statistical analyses between groups were compared using t-test. Results: The body length (0.71±0.014) cm and body weight (44.83±1.833) mg of model group were higher than that of control group (0.50±0.009) cm and body weight (19.33±2.753) mg (total (body length) = 12.36, total (body weight) = 7.71, P < 0.01). Triglyceride content in the model group was (59.15 ± 0.5612) µmol / L, higher than the control group (16.71 ± 0.3562) µmol / L (t = 63.84, P < 0.001). Quantitative PCR results showed that the expression of genes related to cholesterol synthesis in the model group was higher than that in the control group (P < 0.01). The expression levels of lipid production and lipid oxidation related factors in the model group were higher than the control group. The difference between the two groups was statistically significant (P < 0.05). The expression of inflammation-related factors in the model group was higher than that in the control group (P < 0.001), and the expression of genes related to endoplasmic reticulum stress in the model group was higher than that to control group (P<0.001). Liver H&E staining showed that the model group had pathological changes such as large bulla and vesicles compared to the control group. Conclusion: A continuous 3 times 20 days of normal diet can simulate the disease characteristics of human non-alcoholic steatohepatitis in a zebrafish.

[Value of hydrogen proton magnetic resonance spectroscopy in the determination of liver triglyceride in patients with fatty liver disease and its influencing factors].

Objective: To investigate the value of 1H-magnetic resonance spectroscopy ((1)H-MRS) in determining the content of liver triglyceride in patients with fatty liver disease (FLD), as well as its influencing factors. Methods: A total of 124 patients with nonalcoholic fatty liver disease (NAFLD), chronic hepatitis B (CHB), or hepatitis B complicated by FLD who underwent liver biopsy in the Affiliated Hospital of Hangzhou Normal University were enrolled, and the clinical data, serological markers, FibroScan results, and (1)H-MRS results were collected. A correlation analysis was performed with the results of liver biopsy as the gold standard, and the influence of factors including hepatitic B virus (HBV) infection and obesity on accuracy was analyzed. A one-way analysis of variance was used for comparison of means between the three groups, and the LSD or SNK test (for homogeneity of variance) or the Tamhane's or Dunnett's test (heterogeneity of variance) was used for comparison between any two groups. The t-test was used for comparison of continuous data between groups, and the chi-square test was used for comparison of categorical data. The MRS-PDFF receiver operating characteristic (ROC) curve was plotted, the area under the ROC curve (AUC) was calculated, the optimal cut-off points for the diagnosis of NAFLD were estimated, and sensitivity and specificity were calculated. Results: The NAFLD group (42 patients) and the CHB + NAFLD group (40 patients) had a significantly higher proton density fat fraction (PDFF, the content of triglyceride in the liver) than the CHB group (42 patients) (16.84±9.76/9.39 ± 5.50 vs 3.45 ± 1.63, P < 0.001). The results were significantly correlated with the degree of steatosis confirmed by liver biopsy (P < 0.001), but it was not significantly correlated with inflammation or fibrosis grade. The correlation analysis showed that the MRS-PDFF value measured by 1H-MRS was significantly correlated with body mass index (BMI), blood lipids, alkaline phosphatase, and blood glucose, while it was not significantly correlated with age, sex, or the presence or absence of hepatitis B. The ROC curve analysis showed that the AUCs of PDFF measured by 1H-MRS were 0.93, 0.974, and 0.976, respectively, for the diagnosis of steatosis S1(≥5%), S2(≥34%), and S3(≥66%), and the corresponding optimal thresholds were 5.14%, 11.16%, and 16.7%, respectively. Conclusion: 1H-MRS has a high diagnostic value in quantitative evaluation of the degree of liver steatosis in patients with FLD and is not affected by the factors such as HBV infection, age, and sex, while it is correlated with BMI and lipid metabolism.

[Sivelestat alleviates nonalcoholic steatohepatitis in mice through inhibiting activation of Kupffer cells].

Objective: To investigate the role of neutrophil elastase inhibitor, sivelestat, in preventing and treating nonalcoholic steatohepatitis (NASH) and its underling mechanisms. Methods: A total of forty 4-week-old male C57BL/6J ApoE-/-mice were equally divided into the following four groups: standard chow (SC)+isotonic saline; SC+sivelestat; high-fat, high-cholesterol (HFHC) diet+isotonic saline; and HFHC+sivelestat. These mice were treated with above methods for 12 weeks. Blood and liver tissue samples were collected to measure biochemical parameters, hepatic steatosis and non-alcoholic fatty liver disease (NAFLD) activity score (inflammation) were evaluated by oil red O staining and HE staining, respectively. The mRNA and protein expression levels of hepatic inflammatory cytokines, CD68, and F4/80 were determined by quantitative RT-PCR and immunohistochemistry, respectively. Comparison of means between the four groups was made by one-way analysis of variance, and comparison between any two groups was made by the LSD or SNK method (for data with homogeneity of variance) or the Tamhane or Dunnett method (for data with heterogeneity of variance). Results: Mice fed with an HFHC diet for 12 weeks developed typical pathological features of NASH compared with those fed with SC. Compared with mice fed with HFHC diet without sivelestat, those treated with HFHC and sivelestat exhibited the following features: (1) significantly reduced fast blood glucose, blood cholesterol, and hepatic biochemical parameters, as well as increased insulin sensitivity; (2) significantly reduced NAFLD activity score (5.71±1.11 vs 3.16±1.16, P < 0.05); (3) reduced monocyte chemoattractant protein-1 and tumor necrosis factor -α; (4) significantly reduced mRNA levels of CD68 and F4/80; and (5) reduced expression of CD68 in the liver. Conclusion: Sivelestat alleviates the hepatic steatosis and inflammation of NASH in mice by inhibiting the activation of Kupffer cells.

[Establishment and evaluation of a mouse model of nonalcoholic steatohepatitis-related hepatocellular carcinoma].

OBJECTIVE: To establish an apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) double-knockout (ApoE(-/-)/LDLR(-/-)) mouse model of nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) induced by high-fat and high-cholesterol (HFHC) diet. METHODS: ApoE(-/-) knockout mice were crossed with LDLR(-/-) knockout mice to obtain ApoE(-/-)/LDLR(-/-) mice. The ApoE(-/-)/LDLR(-/-) mice mated with each other, and the offspring were injected with low-dose streptozotocin (STZ) at 2-3 days after birth. Some mice were fed with HFHC diet after weaning as the model group (n = 15), and some mice were fed with normal diet as the control group (n = 15). Mice were sacrificed at the end of weeks 10, 16, and 20 (5 mice at each time point). The body weight was measured. Liver tissue and blood were collected to measure biochemical parameters, evaluate the pathological changes in the liver tissue by HE staining, oil red O staining, and Masson staining, and detect the expression of glypican-3 (a marker of HCC) by immunohistochemical staining. RESULTS: The model group had significantly higher levels of fasting blood glucose and total cholesterol than the control group (P < 0.01). Serum levels of alanine aminotransferase, aspartate aminotransferase, and total triglyceride gradually increased with time in the model group; at week 20, there were significant differences in above three indices between the two groups (P < 0.05). HE staining showed that compared with the control group at the corresponding time point, the model group developed sequential histological changes: NASH at week 10, dysplastic nodules at week 16, and early HCC at week 20. Oil red O staining showed that in the model group, the degree of liver steatosis increased within 10 weeks and gradually decreased later. Masson staining demonstrated that the model group developed pathological changes: mild perisinusoidal fibrosis at week 16 and bridging fibrosis around tumors at week 20. HE staining, oil red O staining, and Masson staining showed that no histological or pathological changes were found in the control group. Glypican-3 was detected in the nodules at week 16 and in the cytoplasm of HCC cells at week 20 in the model group. CONCLUSION: The mouse model of NASH-related HCC can be developed by giving STZ injection to neonatal ApoE(-/-)/LDLR(-/-) mice and feeding them with HFHC diet after weaning for 20 weeks. Early HCC may develop directly from NASH.

[Value of a two-step approach with cytokeratin-18 and controlled attenuation parameter in noninvasive differential diagnosis of nonalcoholic steatohepatitis].

OBJECTIVE: To investigate the value of a two-step approach with cytokeratin-18 (CK-18) and controlled attenuation parameter (CAP) in the noninvasive diagnosis of nonalcoholic steatohepatitis (NASH). METHODS: A total of 65 patients with biopsy-proven nonalcoholic fatty liver disease were enrolled, including 30 patients with NASH. The M30 and M65 enzyme-linked immunosorbent assay kits were used to measure serum CK-18, and FibroScan was used to measure CAP. The receiver operating characteristic (ROC) curve was plotted, and the area under the ROC curve (AUROC) was used to determine the value of noninvasive diagnosis. The binary logistic regression model was used to calculate the predicted probability of combined diagnosis. The maximum Youden index, a sensitivity of >90%, and a specificity of > 90% were used to determine the optimal cut-off value, the low value, and the high value, respectively. RESULTS: The results of the multivariate analysis showed that M65 (OR = 1.004, 95% CI 1.002-1.007, P = 0.003) and CAP (OR = 1.017, 95% CI 1.001-1.033, P = 0.036) were independent predictors of NASH. The AUROC of M65+CAP was 0.851 (95% CI 0.761-0.942), higher than 0.808 (95% CI 0.702-0.913) of M65 and 0.677 (95% CI 0.545-0.808) of CAP alone. A two-step approach with high (820.8 U/L) and low (527.7 U/L) values for M65 and the optimal cut-off value (293.5 dB/m) for CAP was used for the differential diagnosis of NASH, with a positive predictive value of 85.7%, a negative predictive value of 100%, and a coincidence rate of 92.0%. CONCLUSION: A two-step approach with M65 and CAP can improve the value of noninvasive diagnosis of NASH, and a high negative predictive value can avoid unnecessary liver biopsy.

Investigation of single nucleotide polymorphisms in phosphodiesterase 4D gene in Mongol and Han patients with ischemic stroke in Inner Mongolia.

We investigated single nucleotide polymorphisms (SNP) at 87 sites of the phosphodiesterase 4D (PDE4D) gene in Mongol and Han patients with ischemic stroke in Inner Mongolia. SNPs in 226 patients with ischemic stroke (case group, 110 Mongol patients, 116 Han patients) and 220 patients without neurological disease (control group, 102 Mongol patients, 118 Han patients) were detected by polymerase chain reaction-restriction fragment length polymorphism and gene sequencing. The genotype and allele frequencies of all groups were compared. There were no statistically significant differences in genotypes in the PDE4D gene at 87 sites between the case and control groups (P > 0.05). The C allele frequency in the case group was significantly higher than that in the control group (P < 0.05). The CC genotype and C allele frequencies in the Mongol case subgroup were higher than those in the Mongol control subgroup (P < 0.05). The CC genotype and C allele frequencies in the Han case subgroup were higher than those in the Han control subgroup (P < 0.05). In the case group, there were no significant differences at 87 sites for genotypes and allele frequencies between the Mongol and Han subgroups. In the control group, there were no significant differences at 87 site genotypes and allele frequencies between the Mongol and Han subgroups. The increase in the C allele frequency at 87 SNP sites in PDE4D may increase ischemic stroke risk. We found no differences in the risk between Mongol and Han populations in Inner Mongolia.

Meta-analysis of differentially expressed genes in autism based on gene expression data.

The purpose of this study was to identify differentially expressed (DE) genes and biological processes associated with changes in gene expression in autism. We performed a meta-analysis using new publicly available Gene Expression Omnibus (GEO) datasets of autism. We performed Gene Ontology (GO) enrichment analyses and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Ten GEO datasets, including 364 cases and 248 controls, were available for the meta-analysis. We identified 3105 genes that were consistently DE in autism (1425 upregulated and 1680 downregulated genes). We also found that 7 genes were associated with phospholipase A2 (PLA2), including LYPLA2P1, PLA2G4D, PNPLA2, LYPLA2, PLA2G6, PLA2G7, and PLA2G5. We found GO terms for molecular functions significantly enriched in structural constituent of ribosome (GO: 0003735, P = 1.87-E06) and transcription regulator activity (GO: 0030528, P = 8.86E-04), while for biological processes, the enriched GO terms were involved in translational elongation (GO: 0006414, P = 1.74E-12) and the response to cytokine stimuli (GO: 0034097, P = 2.76E-05). The most significant pathway in our KEGG analysis was the ribosome pathway (P = 7.90E-12). Our meta-analysis identified genes that were consistently DE and biological pathways associated with gene expression changes in autism.

Overlapping nucleotide determinants for specific aminoacylation of RNA microhelices.

A seven-base pair microhelix that recapitulates a glycine transfer RNA (tRNA) acceptor helix can be specifically aminoacylated with glycine. A single base pair and the single-stranded discriminator base near the attachment site are essential for aminoacylation. These nucleotide sequence elements, and those in microhelices that can be charged with histidine and alanine, occur in the same positions and therefore overlap. Studies on a systematic set of sequence variants showed that no microhelix could be charged with more than one amino acid. Also, none of the three cognate aminoacyl-tRNA synthetases (aaRSs) gave a detectable amount of aminoacylation of the CCA trinucleotide that is common to the 3' ends of all tRNAs, showing that the specific acceptor stem nucleotide bases confer aminoacylation. An analysis of the relative contributions of these microhelices to overall tRNA recognition indicated that their interaction with aaRSs constitutes a substantial part of the recognition of the whole tRNAs.