Proteomic and metabolomic analysis of GH deficiency-induced NAFLD in hypopituitarism: insights into oxidative stress.

Objective: Individuals with hypopituitarism (HPs) have an increased risk of developing non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) due to growth hormone deficiency (GHD). We aimed to investigate the possible mechanisms underlying the relationship between GHD and NAFLD using proteomic and metabolomic insights. Methods: Serum metabolic alternations were assessed in male HPs using untargeted metabolomics. A rat model of HP was established through hypophysectomy, followed by recombinant human growth hormone (rhGH) intervention. The mechanisms underlying GHD-mediated NAFLD were elucidated through the application of label-free proteomics and phosphorylation proteomics. Results: Metabolomic analysis revealed that biomarkers of mitochondrial dysfunction and oxidative stress, such as alanine, lactate, and creatine, were significantly elevated in HPs compared to age-matched controls. In rats, hypophysectomy led to marked hepatic steatosis, lipid peroxidation, and reduced glutathione (GSH), which were subsequently modulated by rhGH replacement. Proteomic analysis identified cytochrome P450s, mitochondrial translation elongation, and PPARA activating genes as the major distinguishing pathways in hypophysectomized rats. The processes of fatty acid transport, synthesis, oxidation, and NADP metabolism were tightly described. An enhanced regulation of peroxisome ß-oxidation and ω-oxidation, together with a decreased NADPH regeneration, may exacerbate oxidative stress. Phosphoproteome data showed downregulation of JAK2-STAT5B and upregulation of mTOR signaling pathway. Conclusions: This study identified proteo-metabolomic signatures associated with the development of NAFLD in pituitary GHD. Evidence was found of oxidative stress imbalance resulting from abnormal fatty acid oxidation and NADPH regeneration, highlighting the role of GH deficiency in the development of NAFLD.

Elucidating the changes in the heterogeneity and function of radiation-induced cardiac macrophages using single-cell RNA sequencing.

Purpose: A mouse model of irradiation (IR)-induced heart injury was established to investigate the early changes in cardiac function after radiation and the role of cardiac macrophages in this process. Methods: Cardiac function was evaluated by heart-to-tibia ratio, lung-to-heart ratio and echocardiography. Immunofluorescence staining and flow cytometry analysis were used to evaluate the changes of macrophages in the heart. Immune cells from heart tissues were sorted by magnetic beads for single-cell RNA sequencing, and the subsets of macrophages were identified and analyzed. Trajectory analysis was used to explore the differentiation relationship of each macrophage subset. The differentially expressed genes (DEGs) were compared, and the related enriched pathways were identified. Single-cell regulatory network inference and clustering (SCENIC) analysis was performed to identify the potential transcription factors (TFs) which participated in this process. Results: Cardiac function temporarily decreased on Day 7 and returned to normal level on Day 35, accompanied by macrophages decreased and increased respectively. Then, we identified 7 clusters of macrophages by single-cell RNA sequencing and found two kinds of stage specific macrophages: senescence-associated macrophage (Cdkn1ahighC5ar1high) on Day 7 and interferon-associated macrophage (Ccr2highIsg15high) on Day 35. Moreover, we observed cardiac macrophages polarized over these two-time points based on M1/M2 and CCR2/major histocompatibility complex II (MHCII) expression. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses suggested that macrophages on Day 7 were characterized by an inflammatory senescent phenotype with enhanced chemotaxis and inflammatory factors, while macrophages on Day 35 showed enhanced phagocytosis with reduced inflammation, which was associated with interferon-related pathways. SCENIC analysis showed AP-1 family members were associated with IR-induced macrophages changes. Conclusion: We are the first study to characterize the diversity, features, and evolution of macrophages during the early stages in an IR-induced cardiac injury animal model.

A high-dose of ursodeoxycholic acid treatment alleviates liver inflammation by remodeling gut microbiota and bile acid profile in a mouse model of non-alcoholic steatohepatitis.

Ursodeoxycholic acid (UDCA) is a hydrophilic bile acid commonly used for treating cholestatic liver disease. However, its efficacy on non-alcoholic steatohepatitis (NASH) was controversial. This study aimed to investigate the impact of a high dosage of UDCA on a mouse model of NASH. Forty 6-week-old mice were fed a high-fat high-cholesterol (HFHC) diet for 12 weeks to establish a mouse model of NASH, and then divided into four groups: two groups transitioned to a normal diet, and the other two groups maintained the HFHC diet. Each group was administered a daily dosage of 300 mg/kg of UDCA or saline for a period of 8 weeks. The 16 s ribosomal RNA genes extracted from mice fecal pellets were sequenced using next-generation sequencing techniques. Serum bile acid profiles were quantified using liquid chromatography electrospray ionization tandem mass spectrometry method. The results showed that UDCA treatment ameliorated liver inflammation, without affecting liver fibrosis. UDCA treatment reduced the relative abundance of the genera Bacteroides, Parabacteroides, and Intestinimonas, whereas increased the relative abundance of the genera norank_f_Muribaculaceae and Parasutterella in the HFHC-maintaining groups. The serum levels of total bile acids and total primary bile acids increased, whereas those of endogenous primary bile acids decreased after UDCA treatment. Correlation analysis showed that primary bile acids were negatively correlated with the genera norank_f_Christensenellaceae and unclassified_f_Ruminococcaceae. In conclusion, a high dosage of UDCA can alleviate liver inflammation, probably by modifying the composition of gut microbiota and serum bile acid profiles in NASH mice.

Poor vitamin D status was associated with increased appendicular fat deposition in US Adults: Data from 2011-2018 National Health and Nutrition Examination Survey.

The aim of the study was to explore the relationship between serum 25-hydroxyvitamin D [25(OH)D] concentrations and regional body fat deposition in 2011-2018 National Health and Nutrition Examination Survey participants aged 18 to 59 years. We hypothesized that serum 25(OH)D concentrations were negatively associated with total, appendicular, and truncal fat deposition. Serum 25(OH)D concentration was categorized into sufficient (≥75.0 nM), insufficient (50.0-74.9 nM), and deficient (<50.0 nM) groups. Fat mass (FM) was measured by dual-energy X-ray absorptiometry, and FM index (FMI) was calculated by dividing FM (kg) with height2 (m2). Multivariant linear regression and Granger causal analysis were performed to assess the causal relationship between vitamin D status and regional FMIs. Overall serum 25(OH)D concentrations were negatively associated with total (ß = -0.029, standard error [SE] = 0.002), trunk (ß = -0.015, SE = 0.001), arms (ß = -0.004, SE = 3.09 × 10-4), and legs (ß = -0.010, SE = 0.001) FMIs in all participants (P < .001, respectively); however, after stratified by vitamin D status and BMI, the negative associations were only observed in individuals with vitamin D deficiency and obesity. The causal analysis indicated that serum 25(OH)D concentrations may causally reduce the arms (F = 4.917, probability [P] = 0.007), legs (F = 5.783, P = 0.003), and total (F = 3.202, P = 0.041) FMIs except for trunk FMI but not vice versa. In conclusion, poor vitamin D status was associated with increased total and appendicular body fat deposition in US adults, particularly in participants with obesity.

Effects of obesity with reduced 25(OH)D levels on bone health in elderly Chinese people: a nationwide cross-sectional study.

Background: Obesity is often accompanied by lower 25(OH)D levels, whereas these two parameters exhibit opposite effects on bone health. It is uncertain what are the effects of lower 25(OH)D levels in obesity on bone health in elderly Chinese people. Methods: A nationally representative cross-sectional analysis of China Community-based Cohort of Osteoporosis (CCCO) was performed from 2016 to 2021, which consisted of 22,081 participants. Demographic data, disease history, Body mass index (BMI), bone mineral density (BMD), the levels of the biomarkers of vitamin D status and those of bone metabolism markers were measured for all participants (N = 22,081). The genes (rs12785878, rs10741657, rs4588, rs7041, rs2282679 and rs6013897) related to 25(OH)D transportation and metabolism were performed in a selected subgroup (N = 6008). Results: Obese subjects exhibited lower 25(OH)D levels (p < 0.05) and higher BMD (p < 0.001) compared with those of normal subjects following adjustment. The genotypes and allele frequency of rs12785878, rs10741657, rs6013897, rs2282679, rs4588 and rs7041 indicated no significant differences among three BMI groups following correction by the Bonferroni's method (p > 0.05). The levels of total 25(OH)D (ToVD) were significantly different among the GC1F, GC1S and GC2 haplotype groups (p < 0.05). Correlation analysis indicated that ToVD levels were significantly correlated with parathyroid hormone levels, BMD, risk of osteoporosis (OP) and the concentration levels of other bone metabolism markers (p < 0.05). Generalized varying coefficient models demonstrated that the increasing BMI, ToVD levels and their interactions were positively associated with BMD outcomes (p < 0.001), whereas the reduced levels of ToVD and BMI increased the risk of OP, which was noted notably for the subjects with reduced ToVD levels (less than 20.69 ng/ml) combined with decreased BMI (less than 24.05 kg/m2). Conclusion: There was a non-linear interaction of BMI and 25(OH)D. And higher BMI accompanied by decreased 25(OH)D levels is associated with increased BMD and decreased incidence of OP, optimal ranges exist for BMI and 25(OH)D levels. The cutoff value of BMI at approximately 24.05 kg/m2 combined with an approximate value of 25(OH)D at 20.69 ng/ml are beneficial for Chinese elderly subjects.

Associations between polyunsaturated fatty acid concentrations and Parkinson's disease: A two-sample Mendelian randomization study.

Introduction: Observational studies demonstrated controversial effect of polyunsaturated fatty acids (PUFAs) on Parkinson's disease (PD) with limited causality evidence. Randomized control trials showed possible improvement in PD symptoms with PUFA supplement but had small study population and limited intervention time. Methods: A two-sample Mendelian randomization was designed to evaluate the causal relevance between PUFAs and PD, using genetic variants of PUFAs as instrumental variables and PD data from the largest genome-wide association study as outcome. Inverse variance weighted (IVW) method was applied to obtain the primary outcome. Mendelian randomization Egger regression, weighted median and weighted mode methods were exploited to assist result analyses. Strict Mendelian randomization and multivariable Mendelian randomization (MVMR) were used to estimate direct effects of PUFAs on PD, eliminating pleiotropic effect. Debiased inverse variance weighted estimator was implemented when weak instrument bias was introduced into the analysis. A variety of sensitivity analyses were utilized to assess validity of the results. Results: Our study included 33,674 PD cases and 449,056 controls. Higher plasma level of arachidonic acid (AA) was associated with a 3% increase of PD risk per 1-standard deviation (SD) increase of AA (IVW; Odds ratio (OR)=1.03 [95% confidence interval (CI) 1.01-1.04], P = 2.24E-04). After MVMR (IVW; OR=1.03 [95% CI 1.02-1.04], P =6.15E-08) and deletion of pleiotropic single-nucleotide polymorphisms overlapping with other lipids (IVW; OR=1.03 [95% CI 1.01-1.05], P =5.88E-04), result was still significant. Increased level of eicosapentaenoic acid (EPA) showed possible relevance with increased PD risk after adjustment of pleiotropy (MVMR; OR=1.05 [95% CI 1.01-1.08], P =5.40E-03). Linoleic acid (LA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA) and alpha-linolenic acid (ALA) were found not causally relevant to PD risk. Various sensitivity analyses verified the validity of our results. In conclusion, our findings from Mendelian randomization suggested that elevated levels of AA and possibly EPA might be linked to a higher risk of PD. No association between PD risk and LA, DHA, DPA, or ALA was found. Discussion: The odds ratio for plasma AA and PD risk was weak. It is important to approach our results with caution in clinical practice and to conduct additional studies on the relationship between PUFAs and PD risk.

Single-cell transcriptomic analysis of tumor heterogeneity and intercellular networks in human urothelial carcinoma.

BACKGROUND: Heterogeneity of tumor cells and the tumor microenvironment (TME) is significantly associated with clinical outcomes and treatment responses in patients with urothelial carcinoma (UC). Comprehensive profiling of the cellular diversity and interactions between malignant cells and TME may clarify the mechanisms underlying UC progression and guide the development of novel therapies. This study aimed to extend our understanding of intra-tumoral heterogeneity and the immunosuppressive TME in UC and provide basic support for the development of novel UC therapies. METHODS: Seven patients with UC were included who underwent curative surgery at our hospital between July 2020 and October 2020. We performed single-cell RNA sequencing (scRNA-seq) analysis in seven tumors with six matched adjacent normal tissues and integrated the results with two public scRNA-seq datasets. The functional properties and intercellular interactions between single cells were characterized, and the results were validated using multiplex immunofluorescence staining, flow cytometry, and bulk transcriptomic datasets. All statistical analyses were performed using the R package with two-sided tests. Wilcoxon-rank test, log-rank test, one-way analysis of variance test, and Pearson correlation analysis were used properly. RESULTS: Unsupervised t-distributed stochastic neighbor embedding clustering analysis identified ten main cellular subclusters in urothelial tissues. Of them, seven urothelial subtypes were noted, and malignant urothelial cells were characterized with enhanced cellular proliferation and reduced immunogenicity. CD8 + T cell subclusters exhibited enhanced cellular cytotoxicity activities along with increased exhaustion signature in UC tissues, and the recruitment of CD4 + T regulatory cells was also increased in tumor tissues. Regarding myeloid cells, coordinated reprogramming of infiltrated neutrophils, M2-type polarized macrophages, and LAMP3 + dendritic cells contribute to immunosuppressive TME in UC tissues. Tumor tissues demonstrated enhanced angiogenesis mediated by KDR + endothelial cells and RGS5 + /ACTA2 + pericytes. Through deconvolution analysis, we identified multiple cellular subtypes may influence the programmed death-ligand 1 (PD-L1) immunotherapy response in patients with UC. CONCLUSION: Our scRNA-seq analysis clarified intra-tumoral heterogeneity and delineated the pro-tumoral and immunosuppressive microenvironment in UC tissues, which may provide novel therapeutic targets.

Prognostic Significance of Iron Metabolism Related Genes in Human Lung Adenocarcinoma.

Background: Iron metabolism related genes participate in cell proliferation, cell growth, and redox cycling in multiple cancers. Limited studies have revealed the roles and clinical significance of iron metabolism in the pathogenesis and prognosis of lung cancer. Methods: A total of 119 iron metabolism related genes were extracted from MSigDB database and their prognostic values were determined in The Cancer Genome Atlas lung adenocarcinoma (TCGA-LUAD) dataset and the Gene Expression Profiling Interactive Analysis 2 (GEPIA 2) database. Immunohistochemistry technique and correlations with immune cell infiltration, gene mutation and drug resistance were used to identify the potential and underlying mechanisms of STEAP1 and STEAP2 as prognostic biomarkers of LUAD. Results: The expression of STEAP1 and STEAP2 are negatively associated with the prognosis of LUAD patients both at the mRNA and protein level. The expression of STEAP1 and STEAP2 was not only negatively correlated with the trafficking degree of CD4+ T immune cells and positively related to most immune cells' trafficking degree, but also significantly associated with gene mutation status, particularly with mutations on TP53 and STK11. Four types of drug resistance showed significant correlation with the expression level of STEAP1 while 13 types of drug resistance were associated with the expression level of STEAP2. Conclusion: Multiple iron metabolism related genes including STEAP1 and STEAP2 are significantly associated with the prognosis of LUAD patients. STEAP1 and STEAP2 might affect the prognosis of LUAD patients partially through immune cell infiltration, gene mutation and drug resistance, which indicated they were independent prognostic factors for LUAD patients.

Plasma 25(OH)D Level is Associated with the Nucleic Acid Negative Conversion Time of COVID-19 Patients: An Exploratory Study.

Purpose: Vitamin D, an essential nutrient and a pleiotropic steroid hormone, has been reported to be associated with the risk and severity in patients infected with Coronavirus Disease-2019 (COVID-19). The role of vitamin D in predicting clinical outcome for COVID-19 patients is unknown. Here, we aimed to determine the prognostic value of plasma 25(OH)D level in COVID-19 patients. Patients and Methods: A total of 158 patients infected with novel COVID-19 Omicron variants in Shanghai were recruited in this study and were categorized into three groups by the tertile levels of plasma 25(OH)D. Plasma 25(OH)D level was determined along with routine blood tests related to liver and renal functions in newly diagnosed COVID-19 patients at admission. The nucleic acid negative conversion time of throat swab samples was evaluated as the primary clinical outcome. The prognostic value of clinical characteristics and plasma 25(OH)D level was assessed using the Kaplan-Meier plot and Cox proportional hazards regression tests. Results: Higher level of plasma 25(OH)D level in COVID-19 patients was independently associated with shorter nucleic acid negative conversion time from COVID-19 infection (multivariate adjusted HR: 0.54, 95%CI: 0.35-0.82, P=0.004, tertile 2 vs 1; multivariate adjusted HR: 0.60, 95%CI: 0.39-0.90, P=0.014, tertile 3 vs 1). Conclusion: Plasma 25(OH)D level may serve as an independent prognostic factor in COVID-19 patient. Our findings indicate the protective roles of vitamin D supplementation in the regiment of patients with COVID-19.

Major depression disorder may causally associate with the increased breast cancer risk: Evidence from two-sample mendelian randomization analyses.

INTRODUCTION: Major depression disorder (MDD) has been associated with increased breast cancer risk in epidemiological studies; however, it is still unknown whether this association is causal or not. The aim of this study is to determine the causal relationship between MDD and breast cancer risk. METHODS: Two-sample Mendelian randomization (MR) analyses with 92 single-nucleotide polymorphisms (SNPs) significantly associated with MDD as instrumental variables (IVs) were performed. Effects of these SNPs on breast cancer in women were estimated in the Breast Cancer Association Consortium (122,977 cases and 105,974 controls) using inverse variance weighted (IVW), weighted median and multivariable MR models. Heterogeneity and pleiotropy effects were assessed based on IVW and MR-Egger regression model, respectively. RESULTS: An 8.7% increased risk of overall breast cancer [odds ratio (OR) = 1.087; 95% confidence interval (CI) 1.011-1.170; P = 0.025] per log-odds ratio increment of MDD risk based on the IVW model was noticed. Similar results were obtained with the multivariable MR model (OR = 1.118, 95% CI = 1.010-1.237; P = 0.031). An increment but not statistically significant causality association was noticed between MDD and risk of ER+ (OR = 1.098, 95% CI = 0.984-1.227; P = 0.093) or ER- (OR = 1.129, 95% CI = 0.982-1.297; P = 0.089) breast cancer under multivariable MR model. No significant pleiotropy effects were observed for the IVs in the two-sample MR studies. CONCLUSIONS: The results suggested that a genetic predisposition of MDD is causally associated with overall breast cancer risk; however, the underlying biological mechanisms are worthy of further study.

VV116 versus Nirmatrelvir-Ritonavir for Oral Treatment of Covid-19.

BACKGROUND: Nirmatrelvir-ritonavir has been authorized for emergency use by many countries for the treatment of coronavirus disease 2019 (Covid-19). However, the supply falls short of the global demand, which creates a need for more options. VV116 is an oral antiviral agent with potent activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We conducted a phase 3, noninferiority, observer-blinded, randomized trial during the outbreak caused by the B.1.1.529 (omicron) variant of SARS-CoV-2. Symptomatic adults with mild-to-moderate Covid-19 with a high risk of progression were assigned to receive a 5-day course of either VV116 or nirmatrelvir-ritonavir. The primary end point was the time to sustained clinical recovery through day 28. Sustained clinical recovery was defined as the alleviation of all Covid-19-related target symptoms to a total score of 0 or 1 for the sum of each symptom (on a scale from 0 to 3, with higher scores indicating greater severity; total scores on the 11-item scale range from 0 to 33) for 2 consecutive days. A lower boundary of the two-sided 95% confidence interval for the hazard ratio of more than 0.8 was considered to indicate noninferiority (with a hazard ratio of >1 indicating a shorter time to sustained clinical recovery with VV116 than with nirmatrelvir-ritonavir). RESULTS: A total of 822 participants underwent randomization, and 771 received VV116 (384 participants) or nirmatrelvir-ritonavir (387 participants). The noninferiority of VV116 to nirmatrelvir-ritonavir with respect to the time to sustained clinical recovery was established in the primary analysis (hazard ratio, 1.17; 95% confidence interval [CI], 1.01 to 1.35) and was maintained in the final analysis (median, 4 days with VV116 and 5 days with nirmatrelvir-ritonavir; hazard ratio, 1.17; 95% CI, 1.02 to 1.36). In the final analysis, the time to sustained symptom resolution (score of 0 for each of the 11 Covid-19-related target symptoms for 2 consecutive days) and to a first negative SARS-CoV-2 test did not differ substantially between the two groups. No participants in either group had died or had had progression to severe Covid-19 by day 28. The incidence of adverse events was lower in the VV116 group than in the nirmatrelvir-ritonavir group (67.4% vs. 77.3%). CONCLUSIONS: Among adults with mild-to-moderate Covid-19 who were at risk for progression, VV116 was noninferior to nirmatrelvir-ritonavir with respect to the time to sustained clinical recovery, with fewer safety concerns. (Funded by Vigonvita Life Sciences and others; ClinicalTrials.gov number, NCT05341609; Chinese Clinical Trial Registry number, ChiCTR2200057856.).

Characterization of the tumour microenvironment phenotypes in malignant tissues and pleural effusion from advanced osteoblastic osteosarcoma patients.

PURPOSE: Malignant pleural effusion (MPE) is an adverse prognostic factor in patients with osteoblastic osteosarcoma; however, the cellular contexts of MPE are largely unknown. EXPERIMENTAL DESIGN: We performed single-cell RNA-sequencing (scRNA-seq) on 27 260 cells from seven MPE samples and 91 186 cells from eight osteosarcoma tissues, including one recurrent, one lung metastasis and six primary tumour (PT) samples, to characterize their tumour microenvironment. RESULTS: Thirteen main cell groups were identified in osteosarcoma tumour and MPE samples. Immune cells dominate the cellular contexts in MPE with more T/NK cells and less osteoclasts compared to PT samples. Of T/NK cells, CD8+ GNLY+ , CD8+ KLRC2+ T cells and FCGR3A+ NK cells were enriched in MPE but CD4+ FOXP3+ Tregs were enriched in PT samples. Naïve IGHD+ B and immune regulatory IGHA1+ B cells were largely identified in MPE, whereas bone metabolism-related CLEC11A+ B cells were significantly enriched in osteosarcoma PT. M2-type TAMs, including CLEC11A_TAM, C1QC_TAM and Prolif_TAMs, among myeloid cells were enriched in PT, which may suppress cytotoxicity activities of T cells through multiple ligand-receptor interactions. Mature LAMP3+ DCs were transformed from CD1C+ DC and CLEC9A+ DC sub-clusters when exposure to tumour alloantigens, which may improve T cell cytotoxicity activities on tumour cells under anti-PD-L1 treatments. In further, immune cells from MPE usually present up-regulated glycolysis and down-regulated oxidative phosphorylation and riboflavin metabolism activities compared to those in PT samples. CONCLUSIONS: Our study provided a novel cellular atlas of MPE and PT in patients with advanced osteosarcoma, which may provide potential therapeutic targets in the future.

Patient-derived rectal cancer organoids-applications in basic and translational cancer research.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and among the leading causes of death in both men and women. Rectal cancer (RC) is particularly challenging compared with colon cancer as the treatment after diagnosis of RC is more complex on account of its narrow anatomical location in the pelvis adjacent to the urogenital organs. More and more existing studies have begun to refine the research on RC and colon cancer separately. Early diagnosis and multiple treatment strategies optimize outcomes for individual patients. However, the need for more accurate and precise models to facilitate RC research is underscored due to the heterogeneity of clinical response and morbidity interrelated with radical surgery. Organoids generated from biopsies of patients have developed as powerful models to recapitulate many aspects of their primary tissue, consisting of 3-D self-organizing structures, which shed great light on the applications in both biomedical and clinical research. As the preclinical research models for RC are usually confused with colon cancer, research on patient-derived RC organoid models enable personalized analysis of cancer pathobiology, organizational function, and tumor initiation and progression. In this review, we discuss the various applications of patient-derived RC organoids over the past two years in basic cancer biology and clinical translation, including sequencing analysis, drug screening, precision therapy practice, tumor microenvironment studies, and genetic engineering opportunities.

A Novel Non-Invasive Approach Based on Serum Ceruloplasmin for Identifying Non-Alcoholic Steatohepatitis Patients in the Non-Diabetic Population.

Background and Aim: Few non-invasive models were established to identify patients with non-alcoholic steatohepatitis (NASH). Liver biopsy remains the gold standard in the clinic. Decreased serum ceruloplasmin (CP) is reported in patients with non-alcoholic fatty liver disease (NAFLD). We aimed to develop a non-invasive model incorporating CP for identifying NASH from NAFLD without type 2 diabetes mellitus (T2DM). Methods: A total of 138 biopsy-proven patients with NAFLD without T2DM were enrolled. The CP ratio was calculated for standardization as the CP value divided by the lower limit of normal. The clinical, anthropometric, biochemical, and histological parameters were compared between the low and high CP ratio groups divided by the median value. Multivariate logistic regression analysis was performed to develop a model for identifying NASH in patients with NAFLD. Results: The medians of the high (n = 69) and low (n = 69) CP ratio groups were 1.43 (1.28-1.61) and 1.03 (0.94-1.12), respectively. A comparison of the two groups showed that the severity of steatosis, hepatocellular ballooning, inflammation activity, fibrosis, and liver iron deposition decreased along with the CP ratio (p < 0.05). The median CP ratio of patients with NASH was significantly lower than those with NAFL [1.15 (1.01-1.41) vs. 1.33 (1.24-1.54), p = 0.001]. A novel model which consists of the CP ratio, BMI, and aspartate aminotransferase (AST) was developed. The AUCs of the model in discriminating NASH from NAFLD was 0.796 (0.694-0.899) and 0.849 (0.713-0.984) in the training and validation groups, and 0.836 (0.659-1.000), 0.833 (0.705-0.962), and 0.821 (0.612-1.000) in patients with normal serum alanine aminotransferase, AST, and both levels, respectively. Conclusions: Decreased CP ratio is associated with more severe histological activity, a diagnosis of NASH, and hepatic iron deposition among patients with NAFLD without T2DM. The CP ratio model could be served as a non-invasive approach to identifying patients with NASH, which might reduce the need for liver biopsy.

Hyperferritinemia Correlates to Metabolic Dysregulation and Steatosis in Chinese Biopsy-Proven Nonalcoholic Fatty Liver Disease Patients.

Purpose: Elevated serum ferritin (SF), also defined as hyperferritinemia, is commonly seen in patients with nonalcoholic fatty liver disease (NAFLD). However, the clinical significance of SF in NAFLD remains controversial. The aim of this study was to characterize the NAFLD patients with elevated SF and to explore the association of hyperferritinemia with the severity of NAFLD proved by liver biopsy in the Chinese population. Patients and Methods: A total of 136 NAFLD patients proved by liver biopsy were enrolled. The demographic, anthropometric, clinical historic, laboratory, and histological characteristics were compared between elevated and normal SF groups. The independent factors for elevated SF were determined using multivariate logistic regression analysis. Results: The median age and body mass index were 41.00 (33.00-57.75) years and 28.28 (26.28-31.34) kg/m2, respectively. Hyperferritinemia was detected in 57 (41.9%) patients. Patients in the elevated SF group presented with more severe lipo- and glucometabolic disorder, and higher aminotransferases compared to those in the normal SF group (p < 0.05). In terms of histopathology, elevated SF was associated with worse steatosis and a higher proportion of positive iron staining (p < 0.05). Multivariate logistic regression analysis identified homeostasis model assessment of insulin resistance (OR: 1.170, 95% CI: 1.036-1.322, p = 0.012), alanine aminotransferase (OR: 1.012, 95% CI: 1.005-1.019, p < 0.001), and positive Perl's staining (OR: 4.880, 95% CI: 2.072-11.494, p < 0.001) as independent risk factors of hyperferritinemia. Conclusion: NAFLD patients with hyperferritinemia were characterized as more severe metabolic dysfunction and liver injury. More attention should be paid to the metabolism state of NAFLD patients with elevated SF. Hyperferritinemia was correlated to hepatic steatosis in Chinese NAFLD patients.

Identification and Characterization of Extrachromosomal Circular DNA in Plasma of Lung Adenocarcinoma Patients.

Background: Chromosome is the basic framework for eukaryotic cells to store genetic information, but certain genes exist in circulation, such as extrachromosomal circular DNA (eccDNA). The unique genetic characteristics and structure of eccDNA provide a new vision on the early diagnosis of cancer; however, whether eccDNA contributes to the early diagnosis and progression of lung cancer remains unclear. Methods: We performed next-generation sequencing (NGS) analysis of eccDNA from the plasma of 6 lung adenocarcinoma (LUAD) patients. The data of plasma eccDNA of healthy people were obtained from public available database. We compared size distribution, chromosome origin, formation and expression patterns of eccDNA between LUAD patients and those of 6 healthy people and 4 healthy gravidas. Results: A total number of 716,059 eccDNA ranging from 22 bp to 3,297,519 bp were detected with an average size less than 800bp and distinctive bimodality in size around 191 bp and 320 bp. After comparison of eccDNA abundance in each sequencing sample, nine eccDNA were ranked on top with higher frequency in lung adenocarcinoma patients than healthy people. Among them, four eccDNA (DOCK1, PPIC, TBC1D16, and RP11-370A5.1) were uniquely expressed in lung adenocarcinoma patients, which may serve as potential biomarkers for early diagnosis LUAD. Conclusion: Cancer-specific eccDNA was presented in LUAD compared to normal people, which might serve as a promising biomarker in LUAD.

Ursodeoxycholic Acid Treatment Restores Gut Microbiota and Alleviates Liver Inflammation in Non-Alcoholic Steatohepatitic Mouse Model.

Gut microbiota dysbiosis plays an important role in the progression of non-alcoholic fatty liver disease (NAFLD), and no approved drugs are available for NAFLD treatment. In this study, we aimed to explore the dynamic changes of gut microbiota at the different stages of NAFLD and determine whether ursodeoxycholic acid (UDCA) could improve liver histopathological features of non-alcoholic steatohepatitis (NASH) mice induced by a high-fat high-cholesterol (HFHC) diet and its impact on gut microbiota. 6-week-old male C57BL/6 mice were fed with a HFHC or normal diet for 12, 18, and 24 weeks, respectively, to simulate the different stages of NAFLD. 16s ribosomal RNA genes from mice fecal samples at the different time points were sequenced to evaluate the dynamic changes of the gut microbiota. Then, C57BL/6 mice were fed with a HFHC diet for 24 weeks to establish the NASH model. Different doses of UDCA were administered intragastrically for additional 4 weeks. Normal diet-fed mice were taken as control. Serum samples, liver, and intestine tissues were harvested for biochemical tests and histopathological examinations. 16s ribosomal RNA genes from mice fecal samples were sequenced to assess the structural changes of gut microbiota. HFHC diet-fed mice developed simple steatosis, steatohepatitis, and fibrosis at 12, 18, and 24 weeks, respectively. The profile of gut microbiota dynamically changed with the different stages of NAFLD. NASH mice had significantly higher abundance of Fecalibaculum, Coriobacteriaceae_UCG-002, and Enterorhabdus, and lower abundance of norank_f_Muribaculaceae, Bacteroides, and Alistipes, which were partially restored by UDCA treatment. UDCA treatment significantly attenuated hepatic inflammation of NASH mice as indicated by the sum of ballooning and lobular inflammation of the NALFD activity score (3.2 ± 0.8 vs 1.8 ± 0.8, p = 0.029), and partially restored gut microbiota dysbiosis, and increased the expression of Claudin-1 and ZO-1 in the intestine, but did not activate the suppressed Farnesoid X receptor signal pathway. Conclusions: The gut microbiota dynamically changes with the different stages of NAFLD. UDCA treatment (120 mg/kg) could partially restore gut microbiota, repair gut barrier integrity, and attenuate hepatic inflammation in the NASH mouse model.

Untargeted LC/MS-Based Metabolic Phenotyping of Hypopituitarism in Young Males.

Objective: Hypopituitarism (Hypo-Pit) is partial or complete insufficiency of anterior pituitary hormones. Besides hormone metabolism, the global metabolomics in Hypo-Pit are largely unknown. We aimed to explore potential biomarkers to aid in diagnosis and personalized treatment. Methods: Using both univariate and multivariate statistical methods, we identified 72 differentially abundant features through liquid chromatography coupled to high-resolution mass spectrometry, obtained in 134 males with Hypo-Pit and 90 age matched healthy controls. Results: Hypopituitarism exhibits an increased abundance of metabolites involved in amino acid degradation and glycerophospholipid synthesis, but decreased content of metabolites in steroid hormone synthesis and fatty acid beta-oxidation. Significantly changed metabolites included creatine, creatinine, L-alanine, phosphocholines, androstenedione, hydroprenenolone, and acylcarnitines. In Hypo-Pit patients, the increased ratio of creatine/creatinine suggested reduced creatine uptake and impaired creatine utilization, whereas the decreased level of beta-hydroxybutyrate, acetylcarnitine (C2) and a significantly decreased ratio of decanoylcarnitine (C10) to free carnitine suggested an impaired beta-oxidation. Furthermore, the creatine/creatinine and decanoylcarnitine/carnitine ratio were identified as diagnostic biomarkers for Hypo-Pit with AUCs of 0.976 and 0.988, respectively. Finally, we found that the creatinine and decanoylcarnitine/carnitine ratio could distinguish cases that were sensitive vs. resistant to human chorionic gonadotropin therapy. Conclusion: We provided a global picture of altered metabolic pathways in Hypo-Pit, and the identified biomarkers in creatine metabolism and beta-oxidation might be useful for the preliminary screening and diagnosis of Hypo-Pit.

Diversity and intratumoral heterogeneity in human gallbladder cancer progression revealed by single-cell RNA sequencing.

BACKGROUND: Gallbladder cancer (GC) is a malignant disease characterized with highly cellular heterogeneity and poor prognosis. Determining the intratumoral heterogeneity and microenvironment (TME) can provide novel therapeutic strategies for GC. METHODS: We performed the single-cell RNA sequencing on the primary and lymph node metastatic gallbladder tumors and the adjacent normal tissues of five patients. The transcriptomic atlas and ligand-receptor-based intercellular communication networks of the single cells were characterized. RESULTS: The transcriptomic landscape of 24,887 single cells was obtained and characterized as 10 cellular clusters, including epithelial, neuroendocrine tumor cells, T&NK cells, B cells, RGS5+ fibroblasts, POSTN+ fibroblasts, PDGFRA+ fibroblasts, endothelial, myeloid cells, and mast cells. Different types of GC harbored distinct epithelial tumor subpopulations, and squamous cell carcinoma could be differentiated from adenocarcinoma cells. Abundant immune cells infiltrated into adenocarcinoma and squamous cell carcinoma, rather than neuroendocrine neoplasms, which showed significant enrichment of stromal cells. CD4+/FOXP3+ T-reg and CD4+/CXCL13+ T helper cells with higher exhausting biomarkers, as well as a dynamic lineage transition of tumor-associated macrophages from CCL20hi /CD163lo , CCL20lo /CD163hi to APOE+, were identified in GC tissues, suggesting the immunosuppressive and tumor-promoting status of immune cells in TME. Two distinct endothelial cells (KDR+ and ACKR1+), which were involved in angiogenesis and lymphangiogenesis, showed remarkable ligand-receptor interactions with primary GC cells and macrophages in gallbladder tumors. CONCLUSIONS: This study reveals a widespread reprogramming across multiple cell populations in GC progression, dissects the cellular heterogeneity and interactions in gallbladder TME, and provides potential therapeutic targets for GC.

Serum Levels of ITGBL1 as an Early Diagnostic Biomarker for Hepatocellular Carcinoma with Hepatitis B Virus Infection.

PURPOSE: Early diagnostic biomarkers of hepatocellular carcinoma (HCC) are needed to distinguish hepatitis B virus (HBV) associated HCC (HBV-HCC) patients from at-risk patients. We assessed the diagnostic values of serum Integrin beta-like 1 (ITGBL1) for early-stage HBV-HCC. PATIENTS AND METHODS: We recruited 716 participators including 299 in the training and 417 in the validation stage, (HBV-HCC, chronic hepatitis B (CHB), HBV-related liver cirrhosis (HBV-LC), and healthy controls) between 2017 and 2020 from three centers. Serum ITGBL1 was measured by ELISA. Receiver operating characteristic (ROC) was used to calculate diagnostic accuracy. RESULTS: The serum levels of ITGBL1 in HBV-HCC patients were significantly lower than those in CHB and HBV-LC patients. This result was confirmed in the follow-up patients who progressed from HBV-LC to HCC. The optimum diagnostic cutoff value of serum ITGBL1 was 47.93ng/mL for detection of early-stage HBV-HCC. The serum ITGBL1 has higher diagnostic accuracy than AFP20 in differentiating the early-stage HBV-HCC from the at-risk patients (area under curve [AUC] 0.787 vs 0.638, p<0.05). For AFP-negative (<20ng/mL) HBV-HCC patients, serum ITGBL1 maintained diagnostic accuracy (training cohort: AUC 0.756, 95% confidence interval [CI] 0.683-0.819, sensitivity 68.18%, and specificity 68.85%; validation cohort: 0.744, 0.686-0.796, 81.13%, and 55.88%). Combination ITGBL1 with AFP20 significantly increased diagnostic accuracy in differentiating the HBV-HCC from at-risk patients (AUC 0.840; 0.868) than ITGBL1 (AUC 0.773, p<0.05; 0.732, p<0.0001) or AFP20 (AUC 0.705, p<0.0001; 0.773, p<0.0001) alone. CONCLUSION: The serum level of ITGBL1 improved identification of AFP-negative HBV-HCC patients, and increased diagnostic accuracy with AFP20 together in the early detection of HBV-HCC.