Genetic drivers of heterogeneity in type 2 diabetes pathophysiology.

Type 2 diabetes (T2D) is a heterogeneous disease that develops through diverse pathophysiological processes1,2 and molecular mechanisms that are often specific to cell type3,4. Here, to characterize the genetic contribution to these processes across ancestry groups, we aggregate genome-wide association study data from 2,535,601 individuals (39.7% not of European ancestry), including 428,452 cases of T2D. We identify 1,289 independent association signals at genome-wide significance (P < 5 × 10-8) that map to 611 loci, of which 145 loci are, to our knowledge, previously unreported. We define eight non-overlapping clusters of T2D signals that are characterized by distinct profiles of cardiometabolic trait associations. These clusters are differentially enriched for cell-type-specific regions of open chromatin, including pancreatic islets, adipocytes, endothelial cells and enteroendocrine cells. We build cluster-specific partitioned polygenic scores5 in a further 279,552 individuals of diverse ancestry, including 30,288 cases of T2D, and test their association with T2D-related vascular outcomes. Cluster-specific partitioned polygenic scores are associated with coronary artery disease, peripheral artery disease and end-stage diabetic nephropathy across ancestry groups, highlighting the importance of obesity-related processes in the development of vascular outcomes. Our findings show the value of integrating multi-ancestry genome-wide association study data with single-cell epigenomics to disentangle the aetiological heterogeneity that drives the development and progression of T2D. This might offer a route to optimize global access to genetically informed diabetes care.

Identification of type 2 diabetes loci in 433,540 East Asian individuals.

Meta-analyses of genome-wide association studies (GWAS) have identified more than 240 loci that are associated with type 2 diabetes (T2D)1,2; however, most of these loci have been identified in analyses of individuals with European ancestry. Here, to examine T2D risk in East Asian individuals, we carried out a meta-analysis of GWAS data from 77,418 individuals with T2D and 356,122 healthy control individuals. In the main analysis, we identified 301 distinct association signals at 183 loci, and across T2D association models with and without consideration of body mass index and sex, we identified 61 loci that are newly implicated in predisposition to T2D. Common variants associated with T2D in both East Asian and European populations exhibited strongly correlated effect sizes. Previously undescribed associations include signals in or near GDAP1, PTF1A, SIX3, ALDH2, a microRNA cluster, and genes that affect the differentiation of muscle and adipose cells3. At another locus, expression quantitative trait loci at two overlapping T2D signals affect two genes-NKX6-3 and ANK1-in different tissues4-6. Association studies in diverse populations identify additional loci and elucidate disease-associated genes, biology, and pathways.

Hepatic GDP-fucose transporter SLC35C1 attenuates cholestatic liver injury and inflammation by inducing CEACAM1 N153 fucosylation.

BACKGROUND AND AIMS: Inflammatory response is crucial for bile acid (BA)-induced cholestatic liver injury, but molecular mechanisms remain to be elucidated. Solute Carrier Family 35 Member C1 (SLC35C1) can transport Guanosine diphosphate-fucose into the Golgi to facilitate protein glycosylation. Its mutation leads to the deficiency of leukocyte adhesion and enhances inflammation in humans. However, little is known about its role in liver diseases. APPROACH AND RESULTS: Hepatic SLC35C1 mRNA transcripts and protein expression were significantly increased in patients with obstructive cholestasis and mouse models of cholestasis. Immunofluorescence revealed that the upregulated SLC35C1 expression mainly occurred in hepatocytes. Liver-specific ablation of Slc35c1 ( Slc35c1 cKO ) significantly aggravated liver injury in mouse models of cholestasis induced by bile duct ligation and 1% cholic acid-feeding, evidenced by increased liver necrosis, inflammation, fibrosis, and bile ductular proliferation. The Slc35c1 cKO increased hepatic chemokine Ccl2 and Cxcl2 expression and T cell, neutrophil, and F4/80 macrophage infiltration but did not affect the levels of serum and liver BA in mouse models of cholestasis. Liquid chromatography with tandem mass spectrometry analysis revealed that hepatic Slc35c1 deficiency substantially reduced the fucosylation of cell-cell adhesion protein CEACAM1 at N153. Mechanistically, cholestatic levels of conjugated BAs stimulated SLC35C1 expression by activating the STAT3 signaling to facilitate CEACAM1 fucosylation at N153, and deficiency in the fucosylation of CEACAM1 at N135 enhanced the BA-stimulated CCL2 and CXCL2 mRNA expression in primary mouse hepatocytes and Primary Liver Carcinoma/Poliomyelitis Research Foundation/5- ASBT cells. CONCLUSIONS: Elevated hepatic SLC35C1 expression attenuates cholestatic liver injury by enhancing CEACAM1 fucosylation to suppress CCL2 and CXCL2 expression and liver inflammation.

Divergent evolution of extreme production of variant plant monounsaturated fatty acids.

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Creating yellow seed Camelina sativa with enhanced oil accumulation by CRISPR-mediated disruption of Transparent Testa 8.

Camelina (Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well-organized seed coat mucilage layer. Transcriptomic analysis of CsTT8-edited seeds revealed significantly increased expression of the lipid-related transcription factors LEC1, LEC2, FUS3, and WRI1 and their downstream fatty acid synthesis-related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced-oil germplasm in camelina. The resulting lines may directly contribute to future net-zero carbon energy production or be combined with other traits to produce desired lipid-derived bioproducts at high yields.

Runt-related transcription factor-1 ameliorates bile acid-induced hepatic inflammation in cholestasis through JAK/STAT3 signaling.

BACKGROUND AND AIMS: Bile acids trigger a hepatic inflammatory response, causing cholestatic liver injury. Runt-related transcription factor-1 (RUNX1), primarily known as a master modulator in hematopoiesis, plays a pivotal role in mediating inflammatory responses. However, RUNX1 in hepatocytes is poorly characterized, and its role in cholestasis is unclear. Herein, we aimed to investigate the role of hepatic RUNX1 and its underlying mechanisms in cholestasis. APPROACH AND RESULTS: Hepatic expression of RUNX1 was examined in cholestatic patients and mouse models. Mice with liver-specific ablation of Runx1 were generated. Bile duct ligation and 1% cholic acid diet were used to induce cholestasis in mice. Primary mouse hepatocytes and the human hepatoma PLC/RPF/5- ASBT cell line were used for mechanistic studies. Hepatic RUNX1 mRNA and protein levels were markedly increased in cholestatic patients and mice. Liver-specific deletion of Runx1 aggravated inflammation and liver injury in cholestatic mice induced by bile duct ligation or 1% cholic acid feeding. Mechanistic studies indicated that elevated bile acids stimulated RUNX1 expression by activating the RUNX1 -P2 promoter through JAK/STAT3 signaling. Increased RUNX1 is directly bound to the promotor region of inflammatory chemokines, including CCL2 and CXCL2 , and transcriptionally repressed their expression in hepatocytes, leading to attenuation of liver inflammatory response. Blocking the JAK signaling or STAT3 phosphorylation completely abolished RUNX1 repression of bile acid-induced CCL2 and CXCL2 in hepatocytes. CONCLUSIONS: This study has gained initial evidence establishing the functional role of hepatocyte RUNX1 in alleviating liver inflammation during cholestasis through JAK/STAT3 signaling. Modulating hepatic RUNX1 activity could be a new therapeutic target for cholestasis.

An experimental study on the visual identification of Fritillaria ussuriensis based on LAMP and nucleic acid colloidal gold technique.

Fritillaria ussuriensis Maxim is one of the traditional Chinese valuable herbs, which is the dried bulb of Fritillaria, a plant of the lily family. The identification of authenticity about F. ussuriensis is still technically challenging. In this study, visual identification was performed by ring-mediated isothermal amplification and nucleic acid colloidal gold techniques. Firstly, multiple sequence comparative analysis was performed by DNAMAN to find the differential sites of F. ussuriensis and its mixed pseudo-products, and the specific identification primers of F. ussuriensis were designed. Genomic DNA was extracted by the modified CTAB method, and the reaction system and reaction conditions were optimized to construct LAMP for the visual detection of F. ussuriensis, meanwhile, the genuine product was cloned and the extracted plasmid was sequenced. The specificity and sensitivity were detected, and also verified by nucleic acid colloidal gold method, and 20 commercially available samples were tested. The extracted DNA met the requirements of the experiment, and the genuine F. ussuriensis PCR product titrated on a test strip showed two bands on the T and C lines, while the counterfeit and negative control showed only one band on the C line, which matched the LAMP results. The specificity was 100 %, and the sensitivity of LAMP assay was up to 0.01 ng µL-1, while that of colloidal gold assay was 0.1 ng µL-1, thus the LAMP assay had high sensitivity. 14 out of 20 commercially available samples of F. ussuriensis were qualified, and 6 were unqualified, and the results of the two methods of identification were consistent. In this study, the combined detection method of LAMP and colloidal gold for nucleic acid was established to be specific, rapid, precise and visualized, which can provide a new technical idea for the detection of F. ussuriensis.

Transcriptome profiling of longissimus dorsi during different prenatal stages to identify genes involved in intramuscular fat deposition in lean and obese pig breeds.

BACKGROUND: There was significant difference in muscle development between fat-type and lean-type pig breeds. METHODS AND RESULTS: In current study, transcriptome analysis and bioinformatics analysis were used to compare the difference in longissimus dorsi (LD) muscle at three time-points (38 days post coitus (dpc), 58 dpc, and 78 dpc ) between Huainan (HN) and Large white (LW) pig breeds. A total of 24500 transcripts were obtained in 18 samples, and 2319, 2799, and 3713 differently expressed genes (DEGs) were identified between these two breeds at 38 dpc, 58 dpc, and 78 dpc, respectively. And the number and foldchange of DEGs were increased, the alternative splice also increased. The cluster analysis of DEGs indicated the embryonic development progress of LD muscle between these two breeds was different. There were 539 shared DEGs between HN and LW at three stages, and the top-shared DEGs were associated with muscle development and lipid deposition, such as KLF4, NR4A1, HSP70, ZBTB16 and so on. CONCLUSIONS: The results showed DEGs between Huainan (HN) and Large white (LW) pig breeds, and contributed to the understanding the muscle development difference between HN and LW, and provided basic materials for improvement of meat quality.

Dimethyl fumarate attenuates cholestatic liver injury by activating the NRF2 and FXR pathways and suppressing NLRP3/GSDMD signaling in mice.

The progression of cholestasis is characterized by excessive accumulation of bile acids (BAs) in the liver, which leads to oxidative stress (OS), inflammation and liver injury. There are currently limited treatments for cholestasis. Therefore, appropriate drugs for cholestasis treatment need to be developed. Dimethyl fumarate (DMF) has been widely used in the treatment of various diseases and exerts antioxidant and anti-inflammatory effects, but its effect on cholestatic liver disease remains unclarified. We fed mice 3,5-diethoxycarbonyl-1,4-dihydrocollidine or cholic acid to induce cholestatic liver injury and treated these mice with DMF to evaluate its protective ability. Alanine aminotransferase, aspartate aminotransferase, and total liver BAs were assessed as indicators of liver function. The levels of OS, liver inflammation, transporters and metabolic enzymes were also measured. DMF markedly altered the relative ALT and AST levels and enhanced the liver antioxidant capacity. DMF regulated the MST/NRF2 signaling pathway to protect against OS and reduced liver inflammation through the NLRP3/GSDMD signaling pathway. DMF also regulated the levels of BA transporters by promoting FXR protein expression. These findings provide new strategies for the treatment of cholestatic liver disorders.

GPX5-Enriched Exosomes Improve Sperm Quality and Fertilization Ability.

Semen preservation quality affects the artificial insemination success rate, and seminal exosomes are rich in various proteins that are transferable to sperm and conducive to sperm-function preservation during storage. However, the specific effects of these proteins remain unclear. In this study, the specific effects of these proteins on semen preservation quality and fertilization capacity were investigated through a proteomic analysis of seminal exosomes from boars with high conception rates (HCRs) and low conception rates (LCRs). The results revealed significant differences in the expression of 161 proteins between the two groups, with the GPX5 level being significantly higher in the HCR group (p < 0.05). The role of GPX5 was further investigated by constructing engineered exosomes enriched with GPX5 (Exo-GPX5), which could successfully transfer GPX5 to sperm. Compared to the control group, Exo-GPX5 could significantly improve sperm motility on storage days 4 and 5 and enhance the acrosome integrity on day 5 (p < 0.05). Additionally, Exo-GPX5 increased the total antioxidant capacity (T-AOC) of sperm, reduced the malondialdehyde (MDA) level, and decreased the expression of antioxidant proteins SOD1 and CAT (p < 0.05). In simulated fertilization experiments, Exo-GPX5-treated sperm exhibited higher capacitation ability and a significant increase in the acrosome reaction rate (p < 0.05). Overall, Exo-GPX5 can improve boar semen quality under 17 °C storage conditions and enhance sperm fertilization capacity.

Tumor necrosis factor α upregulates the bile acid efflux transporter OATP3A1 via multiple signaling pathways in cholestasis.

Cholestasis is a common condition in which the flow of bile from the liver to the intestines is inhibited. It has been shown that organic anion-transporting polypeptide 3A1 (OATP3A1) is upregulated in cholestasis to promote bile acid efflux transport. We have previously shown that the growth factor fibroblast growth factor 19 and inflammatory mediator tumor necrosis factor α (TNFα) increased OATP3A1 mRNA levels in hepatoma peritoneal lavage cell/PRF/5 cell lines. However, the mechanism underlying TNFα-stimulated OATP3A1 expression in cholestasis is unknown. To address this, we collected plasma samples from control and obstructive cholestasis patients and used ELISA to detect TNFα levels. We found that the TNFα levels of plasma and hepatic mRNA transcripts were significantly increased in obstructive cholestatic patients relative to control patients. A significant positive correlation was also observed between plasma TNFα and liver OATP3A1 mRNA transcripts in patients with obstructive cholestasis. Further mechanism analysis revealed that recombinant TNFα induced OATP3A1 expression and activated NF-κB and extracellular signal-regulated kinase (ERK) signaling pathways as well as expression of related transcription factors p65 and specificity protein 1 (SP1). Dual-luciferase reporter and chromatin immunoprecipitation assays showed that recombinant TNFα upregulated the binding activities of NF-κB p65 and SP1 to the OATP3A1 promoter in peritoneal lavage cell/PRF/5 cells. These effects were diminished following the application of NF-κB and ERK inhibitors BAY11-7082 and PD98059. We conclude that TNFα stimulates hepatic OATP3A1 expression in human obstructive cholestasis by activating NF-κB p65 and ERK-SP1 signaling. These results suggest that TNFα-activated NF-κB p65 and ERK-SP1 signaling may be a potential target to ameliorate cholestasis-associated liver injury.

A+T rich interaction domain protein 3a (Arid3a) impairs Mertk-mediated efferocytosis in cholestasis.

BACKGROUND & AIMS: Macrophages are key elements in the pathogenesis of cholestatic liver diseases. Arid3a plays a prominent role in the biologic properties of hematopoietic stem cells, B lymphocytes and tumor cells, but its ability to modulate macrophage function during cholestasis remains unknown. METHODS: Gene and protein expression and cellular localization were assessed by q-PCR, immunohistochemistry, immunofluorescence staining and flow cytometry. We generated myeloid-specific Arid3a knockout mice and established three cholestatic murine models. The transcriptome was analyzed by RNA-seq. A specific inhibitor of the Mertk receptor was used in vitro and in vivo. Promoter activity was determined by chromatin immunoprecipitation-seq against Arid3a and a luciferase reporter assay. RESULTS: In cholestatic murine models, myeloid-specific deletion of Arid3a alleviated cholestatic liver injury (accompanied by decreased accumulation of macrophages). Arid3a-deficient macrophages manifested a more reparative phenotype, which was eliminated by in vitro treatment with UNC2025, a specific inhibitor of the efferocytosis receptor Mertk. Efferocytosis of apoptotic cholangiocytes was enhanced in Arid3a-deficient macrophages via upregulation of Mertk. Arid3a negatively regulated Mertk transcription by directly binding to its promoter. Targeting Mertk in vivo effectively reversed the protective phenotype of Arid3a deficiency in macrophages. Arid3a was upregulated in hepatic macrophages and circulating monocytes in primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Mertk was correspondingly upregulated and negatively correlated with Arid3a expression in PBC and PSC. Mertk+ cells were located in close proximity to cholangiocytes, while Arid3a+ cells were scattered among immune cells with greater spatial distances to hyperplastic cholangiocytes in PBC and PSC. CONCLUSIONS: Arid3a promotes cholestatic liver injury by impairing Mertk-mediated efferocytosis of apoptotic cholangiocytes by macrophages during cholestasis. The Arid3a-Mertk axis is a promising novel therapeutic target for cholestatic liver diseases. IMPACT AND IMPLICATIONS: Macrophages play an important role in the pathogenesis of cholestatic liver diseases. This study reveals that macrophages with Arid3a upregulation manifest a pro-inflammatory phenotype and promote cholestatic liver injury by impairing Mertk-mediated efferocytosis of apoptotic cholangiocytes during cholestasis. Although we now offer a new paradigm to explain how efferocytosis is regulated in a myeloid cell autonomous manner, the regulatory effects of Arid3a on chronic liver diseases remain to be further elucidated.

Engineering triacylglycerol accumulation in duckweed (Lemna japonica).

Duckweeds are amongst the fastest growing of higher plants, making them attractive high-biomass targets for biofuel feedstock production. Their fronds have high rates of fatty acid synthesis to meet the demand for new membranes, but triacylglycerols (TAG) only accumulate to very low levels. Here we report on the engineering of Lemna japonica for the synthesis and accumulation of TAG in its fronds. This was achieved by expression of an estradiol-inducible cyan fluorescent protein-Arabidopsis WRINKLED1 fusion protein (CFP-AtWRI1), strong constitutive expression of a mouse diacylglycerol:acyl-CoA acyltransferase2 (MmDGAT), and a sesame oleosin variant (SiOLE(*)). Individual expression of each gene increased TAG accumulation by 1- to 7-fold relative to controls, while expression of pairs of these genes increased TAG by 7- to 45-fold. In uninduced transgenics containing all three genes, TAG accumulation increased by 45-fold to 3.6% of dry weight (DW) without severely impacting growth, and by 108-fold to 8.7% of DW after incubation on medium containing 100 µm estradiol for 4 days. TAG accumulation was accompanied by an increase in total fatty acids of up to three-fold to approximately 15% of DW. Lipid droplets from fronds of all transgenic lines were visible by confocal microscopy of BODIPY-stained fronds. At a conservative 12 tonnes (dry matter) per acre and 10% (DW) TAG, duckweed could produce 350 gallons of oil/acre/year, approximately seven-fold the yield of soybean, and similar to that of oil palm. These findings provide the foundation for optimizing TAG accumulation in duckweed and present a new opportunity for producing biofuels and lipidic bioproducts.

Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase.

Plant plastidial acyl-acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ6 to Δ9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ9 to Δ6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ6 position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

Autophagy benefits the in vitro and in vivo clearance of Talaromyces marneffei.

Talaromycosis, namely Talaromyces marneffei infection, is increasing gradually and has a high mortality rate even under antifungal therapy. Although autophagy acts differently on different pathogens, it is a promising therapeutic strategy. However, information on autophagy in macrophages and animals upon infection by T. marneffei is still limited. Therefore, several models were employed here to investigate the role of autophagy in host defense against T. marneffei, including RAW264.7 macrophages as in vitro models, different types of Caenorhabditis elegans and BALB/c mice as in vivo models. We applied the clinical T. marneffei isolate SUMS0152 in this study. T. marneffei-infected macrophages exhibit increased formation of autophagosomes. Further, macrophage autophagy promoted by rapamycin or Earle's balanced salt solution (EBSS) inhibited the viability of intracellular T. marneffei. In vivo, compared with uninfected Caenorhabditis elegans, the wild-type nematodes upregulated the expression of the autophagy-related gene lgg-1 and atg-18, and nematodes carrying GFP reporter were induced to form autophagosomes (GFP::LGG-1) after T. marneffei infection. Furthermore, the knockdown of lgg-1 significantly reduced the survival rate of T. marneffei-infected nematodes. Likewise, the autophagy activator rapamycin reduced the fungal burden and suppressed lung inflammation in a mouse model of infection. In conclusion, autophagy is essential for host defense against T. marneffei in vitro and in vivo. Therefore, autophagy may be an attractive target for developing new therapeutics to treat talaromycosis.

Semaphorin 7A interacts with nuclear factor NF-kappa-B p105 via integrin ß1 and mediates inflammation.

Semaphorin7a (SEMA7A), a membrane-anchored member of the semaphorin protein family, could be involved in a diverse range of immune responses via its receptor integrin ß1. Recently, we reported that the SEMA7AR148W mutation (a gain-of-function mutation, Sema7aR145W in mice) is a risk factor for progressive familial intrahepatic cholestasis and nonalcoholic fatty liver disease via upregulated membrane localization. In this study, we demonstrated that integrin ß1 is a membrane receptor for nuclear factor NF-kappa-B p105 (NF-κB p105) and a critical mediator of inflammation. Integrin ß1 could interact with the C-terminal domain of NF-κB p105 to promote p50 generation and stimulate the NF-κB p50/p65 signalling pathway, upregulate TNF-α and IL-1ß levels, and subsequently render hepatocytes more susceptible to inflammation. The induction of integrin ß1 depends on elevated Sema7a membrane localization. Moreover, we revealed elevated levels of Sema7aWT (SEMA7AWT) in hepatocellular carcinoma (HCC) patients and an HCC mouse model. In line with our findings, the NF-κB p50/p65 pathway could also be activated by high Sema7a expression and repressed by integrin ß1 silencing. In conclusion, our findings suggest that the Sema7aR145W (SEMA7AR148W) mutation and high Sema7aWT (SEMA7AWT) expression both activate the NF-κB p50/p65 pathway via integrin ß1 and play a crucial role in inflammatory responses. Video Abstract.

Melatonin attenuates cholestatic liver injury via inhibition of the inflammatory response.

Melatonin, an indole neurohormone secreted mainly by the pineal gland, has been found to be involved in a variety of liver diseases. However, the underlying mechanism by which melatonin ameliorates cholestatic liver injury is not fully understood. In this study, we investigated the mechanism by which melatonin attenuates cholestatic liver injury via inhibition of the inflammatory response. We measured the levels of serum melatonin in patients with obstructive cholestasis (n = 9), patients with primary biliary cholangitis (PBC) (n = 11), and control patients (n = 7). We performed experiments with C57BL/6 J mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and melatonin to verify the role of melatonin in the mouse model of cholestasis. Primary mouse hepatocytes were used for in vitro studies to study the mechanisms of action of melatonin in cholestasis. The levels of serum melatonin were markedly increased and negatively correlated with serum markers of liver injury in cholestatic patients. As expected, oral administration of melatonin significantly attenuated cholestasis-induced liver inflammation and fibrosis in 0.1% DDC diet-fed mice. Further mechanistic studies in cholestatic mice and primary hepatocytes revealed that melatonin reduced the conjugate BA-stimulated expression of cytokines (e.g. Ccl2, Tnfα, and Il6) through the ERK/Egr1 signalling pathway in these models. The levels of serum melatonin are significantly elevated in cholestatic patients. Melatonin treatment ameliorates cholestatic liver injury by suppressing the inflammatory response in vivo and in vitro. Therefore, melatonin is a promising novel therapeutic strategy for cholestasis.

Novel urinary protein panels for the non-invasive diagnosis of non-alcoholic fatty liver disease and fibrosis stages.

BACKGROUND & AIMS: There is an unmet clinical need for non-invasive tests to diagnose non-alcoholic fatty liver disease (NAFLD) and individual fibrosis stages. We aimed to test whether urine protein panels could be used to identify NAFLD, NAFLD with fibrosis (stage F ≥ 1) and NAFLD with significant fibrosis (stage F ≥ 2). METHODS: We collected urine samples from 100 patients with biopsy-confirmed NAFLD and 40 healthy volunteers, and proteomics and bioinformatics analyses were performed in this derivation cohort. Diagnostic models were developed for detecting NAFLD (UPNAFLD model), NAFLD with fibrosis (UPfibrosis model), or NAFLD with significant fibrosis (UPsignificant fibrosis model). Subsequently, the derivation cohort was divided into training and testing sets to evaluate the efficacy of these diagnostic models. Finally, in a separate independent validation cohort of 100 patients with biopsy-confirmed NAFLD and 45 healthy controls, urinary enzyme-linked immunosorbent assay analyses were undertaken to validate the accuracy of these new diagnostic models. RESULTS: The UPfibrosis model and the UPsignificant fibrosis model showed an AUROC of .863 (95% CI: .725-1.000) and 0.858 (95% CI: .712-1.000) in the training set; and .837 (95% CI: .711-.963) and .916 (95% CI: .825-1.000) in the testing set respectively. The UPNAFLD model showed an excellent diagnostic performance and the area under the receiver operator characteristic curve (AUROC) exceeded .90 in the derivation cohort. In the independent validation cohort, the AUROC for all three of the above diagnostic models exceeded .80. CONCLUSIONS: Our newly developed models constructed from urine protein biomarkers have good accuracy for non-invasively diagnosing liver fibrosis in NAFLD.

Association between body fat composition and disease duration, clinical activity, and intravenous corticosteroid-induced response in inflammatory bowel disease.

BACKGROUND: Body fat composition is believed to be associated with the progression, medical response, and prognosis of inflammatory bowel disease (IBD). Hence, we conducted this study to explore if fat metrics were associated with the disease activity of severe IBD and the response to intravenous corticosteroids (IVCS). METHODS: We included 69 patients with ulcerative colitis (UC) and 72 patients with Crohn's disease (CD) who had previously received IVCS during hospitalization. We quantified individual fat distribution using abdominal computed tomography slices. The correlations between fat parameters and disease activity were available with Spearman correlation analysis. The prediction model was developed using independent risk factors derived from multivariable logistic regression analysis. Model discrimination was evaluated leveraging the receiver operating characteristic curve. 1000 bootstrap resamples internally validated the model's prediction performance. RESULTS: Notable differences in age, nutritional status, serum cytomegalovirus replication, stool condition, and extraintestinal involvement between UC and CD patients were observed. UC subjects who responded to IVCS had higher subcutaneous adipose tissue index (SATI), visceral adipose tissue index (VATI), and mesorectal adipose tissue index (MATI) than non-responders. IVCS-responding CD individuals had lower VATI and mesenteric fat index (MFI) than non-responders. CD patients with a prolonged disease duration had a decreased SATI and an elevated MFI. VATI and MATI were reduced as UC clinically progressed, while more prominent clinical activity in CD correlated with increased VATI, MATI, and MFI. A high SATI indicated that patients with UC were more prone to be IVCS responders. For patients with CD, levels of VATI and MFI were negatively associated with effective IVCS treatment. The established models showed a discriminative accuracy of 0.834 [95% confidence interval (CI) 0.740-0.928] in the UC cohort and 0.871 (95% CI 0.793-0.949) in the CD cohort. Repeated samples supported the reliability of the developed models (AUCUC = 0.836, 95% CI 0.735-0.919; AUCCD = 0.876, 95% CI 0.785-0.946). CONCLUSION: Human fat indexes represent novel imaging biomarkers for identifying IBD patients who respond to IVCS, thus building accelerated therapy regimens and avoiding the adverse effects of ineffective IVCS.

Pyroptosis -related potential diagnostic biomarkers in steroid-induced osteonecrosis of the femoral head.

PURPOSE: Steroid-induced necrosis of the femoral head (SONFH) is a refractory orthopedic hip disease occurring in young and middle-aged people, with glucocorticoids being the most common cause. Previous experimental studies have shown that cell pyroptosis may be involved in the pathological process of SONFH, but its pathogenesis in SONFH is still unclear. This study aims to screen and validate potential pyroptosis-related genes in SONFH diagnosis by bioinformatics analysis to further elucidate the mechanism of pyroptosis in SONFH. METHODS: There were 33 pyroptosis-related genes obtained from the prior reviews. The mRNA expression was downloaded from GSE123568 dataset in the Gene Expression Omnibus (GEO) database, including 10 non-SONFH (following steroid administration) samples and 30 SONFH samples. The pyroptosis-related differentially expressed genes involved in SONFH were identified with "affy" and "limma" R package by intersecting the GSE123568 dataset with pyroptosis genes. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the pyroptosis-related differentially expressed genes involved in SONFH were conducted by "clusterProfiler" R package and visualized by "GOplot" R package. Then, the correlations between the expression levels of the pyroptosis-related differentially expressed genes involved in SONFH were confirmed with "corrplot" R package. Moreover, the protein-protein interaction (PPI) network was analysed by using GeneMANIA database. Next, The ROC curve of pyroptosis-related differentially expressed genes were analyzed by "pROC" R package. RESULTS: A total of 10 pyroptosis-related differentially expressed genes were identified between the peripheral blood samples of SONFH patients and non-SONFH patients based on the defined criteria, including 20 upregulated genes and 10 downregulated genes. The GO and KEGG pathway enrichment analyses revealed that these 10 pyroptosis-related differentially expressed genes involved in SONFH were particularly enriched in cysteine-type endopeptidase activity involved in apoptotic process, positive regulation of interleukin-1 beta secretion and NOD-like receptor signaling pathway. Correlation analysis revealed significant correlations among the 10 differentially expressed pyroptosis-related genes involved in SONFH. The PPI results demonstrated that the 10 pyroptosis-related differentially expressed genes interacted with each other. Compared to non-SONFH samples, these pyroptosis-related differentially expressed genes had good predictive diagnostic efficacy (AUC = 1.000, CI = 1.000-1.000) in the SONFH samples, and NLRP1 had the highest diagnostic value (AUC: 0.953) in the SONFH samples. CONCLUSIONS: There were 10 potential pyroptosis-related differentially expressed genes involved in SONFH were identified via bioinformatics analysis, which might serve as potential diagnostic biomarkers because they regulated pyroptosis. These results expand the understanding of SONFH associated with pyroptosis and provide new insights to further explore the mechanism of action and diagnosis of pyroptosis associated in SONFH.