Precirrhotic Primary Biliary Cholangitis with Portal Hypertension: Bile Duct Injury Correlate.

Background/Aims: : The histological characteristics and natural history of precirrhotic primary biliary cholangitis (PBC) with portal hypertension (PH) are unclear. Our aim was to clarify the prevalence, risk factors, and histological characteristics of precirrhotic PBC patients with PH. Methods: : This retrospective study compared the clinical features, histological characteristics, and response to ursodeoxycholic acid (UDCA) between the PH and non-PH groups of precirrhotic PBC patients. Results: : Out of 165 precirrhotic PBC patients, 40 (24.2%) also had PH. According to histological stage 1, 2 and 3 disease, 5.3% (1/19), 17.3% (17/98), and 45.8% (22/48) of patients also had PH, respectively. Precirrhotic PBC with PH was significantly positively correlated with bile duct loss, degree of cytokeratin 7 positivity, and degree of fibrosis in the portal area, but significantly negatively correlated with lymphoid follicular aggregation. Compared to the non-PH group, patients in the PH group showed a higher prevalence of obliterative portal venopathy, incomplete septal fibrosis, portal tract abnormalities and non-zonal sinusoidal dilatation (p<0.05). In addition, patients with PH were more likely to present with symptoms of jaundice, ascites, epigastric discomfort, a poorer response to UDCA, and more decompensation events (p<0.05). High alkaline phosphatase levels, low white blood cell counts, high Mayo scores, and high FIB-4 index values were risk factors for precirrhotic PBC with PH. Conclusions: : Approximately 24.2% of precirrhotic PBC patients have PH, which is histologically related to the injury of bile ducts. High alkaline phosphatase levels, low white blood cell counts, high Mayo scores, and high FIB-4 index values are associated with increased risk of precirrhotic PBC with PH.

ALKBH5-mediated m6A modification of IL-11 drives macrophage-to-myofibroblast transition and pathological cardiac fibrosis in mice.

Cardiac macrophage contributes to the development of cardiac fibrosis, but factors that regulate cardiac macrophages transition and activation during this process remains elusive. Here we show, by single-cell transcriptomics, lineage tracing and parabiosis, that cardiac macrophages from circulating monocytes preferentially commit to macrophage-to-myofibroblast transition (MMT) under angiotensin II (Ang II)-induced hypertension, with accompanying increased expression of the RNA N6-methyladenosine demethylases, ALKBH5. Meanwhile, macrophage-specific knockout of ALKBH5 inhibits Ang II-induced MMT, and subsequently ameliorates cardiac fibrosis and dysfunction. Mechanistically, RNA immunoprecipitation sequencing identifies interlukin-11 (IL-11) mRNA as a target for ALKBH5-mediated m6A demethylation, leading to increased IL-11 mRNA stability and protein levels. By contrast, overexpression of IL11 in circulating macrophages reverses the phenotype in ALKBH5-deficient mice and macrophage. Lastly, targeted delivery of ALKBH5 or IL-11 receptor α (IL11RA1) siRNA to monocytes/macrophages attenuates MMT and cardiac fibrosis under hypertensive stress. Our results thus suggest that the ALKBH5/IL-11/IL11RA1/MMT axis alters cardiac macrophage and contributes to hypertensive cardiac fibrosis and dysfunction in mice, and thereby identify potential targets for cardiac fibrosis therapy in patients.

Digital spatial profiling of segmental outflow regions in trabecular meshwork reveals a role for ADAM15.

In this study we used a spatial transcriptomics approach to identify genes specifically associated with either high or low outflow regions in the trabecular meshwork (TM) that could potentially affect aqueous humor outflow in vivo. High and low outflow regions were identified and isolated from organ cultured human anterior segments perfused with fluorescently-labeled 200 nm FluoSpheres. The NanoString GeoMx Digital Spatial Profiler (DSP) platform was then used to identified genes in the paraffin embedded tissue sections from within those regions. These transcriptome analyses revealed that 16 genes were statistically upregulated in high outflow regions and 57 genes were statistically downregulated in high outflow regions when compared to low outflow regions. Gene ontology enrichment analysis indicated that the top three biological categories of these differentially expressed genes were ECM/cell adhesion, signal transduction, and transcription. The ECM/cell adhesion genes that showed the largest differential expression (Log2FC ±1.5) were ADAM15, BGN, LDB3, and CRKL. ADAM15, which is a metalloproteinase that can bind integrins, was upregulated in high outflow regions, while the proteoglycan BGN and two genes associated with integrin signaling (LDB3, and CRKL) were downregulated. Immunolabeling studies supported the differential expression of ADAM15 and showed that it was specifically upregulated in high outflow regions along the inner wall of Schlemm's canal and in the juxtacanalicular (JCT) region of the TM. In addition to these genes, the studies showed that genes for decorin, a small leucine-rich proteoglycan, and the α8 integrin subunit were enriched in high outflow regions. These studies identify several novel genes that could be involved in segmental outflow, thus demonstrating that digital spatial profiling could be a useful approach for understanding segmental flow through the TM. Furthermore, this study suggests that changes in the expression of genes involved in regulating the activity and/or organization of the ECM and integrins in the TM are likely to be key players in segmental outflow.

Profiling IOP-responsive genes in anterior and posterior ocular tissues in the rat CEI glaucoma model.

Purpose: The rat Controlled Elevation of Intraocular pressure (CEI) model allows study of in vivo responses to defined intraocular pressures (IOP). In this study, we use Nanostring technology to investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals. Methods: Male and female rats (N=35) were subject to CEI for 8-hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5x IOP (CEI-50). Naïve animals, receiving no anesthesia or surgical interventions, served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA isolated, and samples were analyzed with a Nanostring panel containing 770 genes. Post-processing, raw count data were uploaded to Rosalind® for differential gene expression analyses. Results: For the TM, 45 IOP-related genes were significant in the "CEI-50 vs. CEI-20" and "CEI-50 vs. naïve" comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and TGFß pathways to be the most up- and down-regulated KEGG pathways, respectively. For ONH, 22 significantly regulated genes were identified in the "CEI-50 vs. naïve" comparison. Pathway analysis identified 'defense response' and 'immune response' as two significantly upregulated biological process pathways. Conclusions: This study demonstrates the ability to assay IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGFß pathway genes suggest that TM responses may prevent TGFß-induced extracellular matrix synthesis. For ONH, the initial response to elevated IOP may be protective, with astrocytes playing a key role in these gene responses.

Clinical characteristics and prognosis of non-APAP drug-induced acute liver failure: a large multicenter cohort study.

BACKGROUND: There is growing recognition of natural history, complications, and outcomes of patients who develop non-acetaminophen (APAP) drug-induced acute liver failure (ALF). To clarify high-risk factors and develop a nomogram model to predict transplant-free survival (TFS) in patients with non-APAP drug-induced ALF. METHODS: Patients with non-APAP drug-induced ALF from 5 participating centers were retrospectively analyzed. The primary endpoint was 21-day TFS. Total sample size was 482 patients. RESULTS: Regarding causative agents, the most common implicated drugs were herbal and dietary supplements (HDS) (57.0%). The hepatocellular type (R ≥ 5) was the main liver injury pattern (69.0%). International normalized ratio, hepatic encephalopathy grades, the use of vasopressor, N-acetylcysteine, or artificial liver support system were associated with TFS and incorporated to construct a nomogram model (drug-induced acute liver failure-5, DIALF-5). The AUROC of DIALF-5 for 7-day, 21-day, 60-day, and 90-day TFS in the internal cohort were 0.886, 0.915, 0.920, and 0.912, respectively. Moreover, the AUROC of DIALF-5 for 21-day TFS had the highest AUROC, which was significantly higher than 0.725 of MELD and 0.519 of KCC (p < 0.05), numerically higher than 0.905 of ALFSG-PI but without statistical difference (p > 0.05). These results were successfully validated in the external cohort (147 patients). CONCLUSIONS: Based on easily identifiable clinical data, the novel DIALF-5 model was developed to predict transplant-free survival in non-APAP drug-induced ALF, which was superior to KCC, MELD and had a similar prediction performance to ALFSG-PI but is more convenient, which can directly calculate TFS at multiple time points.

Development and validation of a noninvasive prediction model of autoimmune hepatitis in patients with liver diseases.

BACKGROUND AND AIMS: There is no golden standard for the diagnosis of autoimmune hepatitis which still dependent on liver biopsy currently. So, we developed a noninvasive prediction model to help optimize the diagnosis of autoimmune hepatitis. METHODS: From January 2017 to December 2019, 1739 patients who had undergone liver biopsy were seen in the second hospital of Nanjing, of which 128 were here for consultation. Clinical, laboratory, and histologic data were obtained retrospectively. Multivariable logistic regression analysis was employed to create a nomogram model that predicting the risk of autoimmune hepatitis. Internal and external validation was both performed to evaluate the model. RESULTS: A total of 1288 patients with liver biopsy were enrolled (1184 from the second hospital of Nanjing, the remaining 104 from other centers). After the univariate and multivariate logistic regression analysis, nine variables including ALT, IgG, ALP/AST, ALB, ANA, AMA, HBsAg, age, and gender were selected to establish the noninvasive prediction model. The nomogram model exhibits good prediction in diagnosing autoimmune hepatitis with AUROC of 0.967 (95% CI: 0.776-0.891) in internal validation and 0.835 (95% CI: 0.752-0.919) in external validation. CONCLUSIONS: ALT, IgG, ALP/AST, ALB, ANA, AMA, HBsAg, age, and gender are predictive factors for the diagnosis of autoimmune hepatitis in patients with unexplained liver diseases. The predictive nomogram model built by the nine predictors achieved good prediction for diagnosing autoimmune hepatitis.

Transjugular liver biopsy: enlarge the indications for liver biopsy with reliable diagnostic quality.

BACKGROUND: Complications and diagnostic efficiency for liver biopsy are main concerns for clinicians. This study aimed to assess the safety and efficacy of transjugular liver biopsy (TJLB) compared with percutaneous liver biopsy (PLB) when patients had equal level of liver function and number of passes, using propensity score matching (PSM). METHODS: The clinical and pathological data of patients who received TJLB or PLB between January 2012 and October 2022 were collected. Matching factors included age, gender, cirrhosis, portal hypertension, liver function, creatinine, number of passes, hemodialysis, history of anti-coagulation and anti-platelet, and comorbidities. Coagulation indexes were not considered as matching factors due to different indications of the two techniques. RESULTS: 2711 PLBs and 30 TJLBs were evaluated. By PSM, 75 patients (50 PLBs, 25 TJLBs) were matched. The complication rates for TJLB and PLB were 4.0% (1/25) and 10.0% (5/50) (P > 0.05). Two PLBs had hepatic hemorrhage, one of which required only close monitoring (Grade 1) and the other needed hemostasis and rehydration therapy (Grade 2). The other 3 cases presented with mild abdominal pain (Grade 1). And only one TJLB presented with mild pain. The median number of complete portal tracts were 6.0 and 10.0 for TJLBs and PLBs (P < 0.05). Moreover, the median length of sample for TJLBs and PLBs were 10.0 and 16.5 mm (P < 0.05). The diagnostic efficiency of hepatopathy of unknown etiology of TJLB versus PLB groups before and after matching were 96.4% vs. 94.1% and 95.7% vs. 93.2%, respectively (P > 0.05). CONCLUSION: TJLB is an effective invasive diagnostic procedure that expands indications for liver biopsy with reliable diagnostic quality.

AutoFibroNet: A deep learning and multi-photon microscopy-derived automated network for liver fibrosis quantification in MAFLD.

BACKGROUND: Liver fibrosis is the strongest histological risk factor for liver-related complications and mortality in metabolic dysfunction-associated fatty liver disease (MAFLD). Second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) is a powerful tool for label-free two-dimensional and three-dimensional tissue visualisation that shows promise in liver fibrosis assessment. AIM: To investigate combining multi-photon microscopy (MPM) and deep learning techniques to develop and validate a new automated quantitative histological classification tool, named AutoFibroNet (Automated Liver Fibrosis Grading Network), for accurately staging liver fibrosis in MAFLD. METHODS: AutoFibroNet was developed in a training cohort that consisted of 203 Chinese adults with biopsy-confirmed MAFLD. Three deep learning models (VGG16, ResNet34, and MobileNet V3) were used to train pre-processed images and test data sets. Multi-layer perceptrons were used to fuse data (deep learning features, clinical features, and manual features) to build a joint model. This model was then validated in two further independent cohorts. RESULTS: AutoFibroNet showed good discrimination in the training set. For F0, F1, F2 and F3-4 fibrosis stages, the area under the receiver operating characteristic curves (AUROC) of AutoFibroNet were 1.00, 0.99, 0.98 and 0.98. The AUROCs of F0, F1, F2 and F3-4 fibrosis stages for AutoFibroNet in the two validation cohorts were 0.99, 0.83, 0.80 and 0.90 and 1.00, 0.83, 0.80 and 0.94, respectively, showing a good discriminatory ability in different cohorts. CONCLUSION: AutoFibroNet is an automated quantitative tool that accurately identifies histological stages of liver fibrosis in Chinese individuals with MAFLD.

Prediction of treatment response to antipsychotic drugs for precision medicine approach to schizophrenia: randomized trials and multiomics analysis.

BACKGROUND: Choosing the appropriate antipsychotic drug (APD) treatment for patients with schizophrenia (SCZ) can be challenging, as the treatment response to APD is highly variable and difficult to predict due to the lack of effective biomarkers. Previous studies have indicated the association between treatment response and genetic and epigenetic factors, but no effective biomarkers have been identified. Hence, further research is imperative to enhance precision medicine in SCZ treatment. METHODS: Participants with SCZ were recruited from two randomized trials. The discovery cohort was recruited from the CAPOC trial (n = 2307) involved 6 weeks of treatment and equally randomized the participants to the Olanzapine, Risperidone, Quetiapine, Aripiprazole, Ziprasidone, and Haloperidol/Perphenazine (subsequently equally assigned to one or the other) groups. The external validation cohort was recruited from the CAPEC trial (n = 1379), which involved 8 weeks of treatment and equally randomized the participants to the Olanzapine, Risperidone, and Aripiprazole groups. Additionally, healthy controls (n = 275) from the local community were utilized as a genetic/epigenetic reference. The genetic and epigenetic (DNA methylation) risks of SCZ were assessed using the polygenic risk score (PRS) and polymethylation score, respectively. The study also examined the genetic-epigenetic interactions with treatment response through differential methylation analysis, methylation quantitative trait loci, colocalization, and promoter-anchored chromatin interaction. Machine learning was used to develop a prediction model for treatment response, which was evaluated for accuracy and clinical benefit using the area under curve (AUC) for classification, R2 for regression, and decision curve analysis. RESULTS: Six risk genes for SCZ (LINC01795, DDHD2, SBNO1, KCNG2, SEMA7A, and RUFY1) involved in cortical morphology were identified as having a genetic-epigenetic interaction associated with treatment response. The developed and externally validated prediction model, which incorporated clinical information, PRS, genetic risk score (GRS), and proxy methylation level (proxyDNAm), demonstrated positive benefits for a wide range of patients receiving different APDs, regardless of sex [discovery cohort: AUC = 0.874 (95% CI 0.867-0.881), R2 = 0.478; external validation cohort: AUC = 0.851 (95% CI 0.841-0.861), R2 = 0.507]. CONCLUSIONS: This study presents a promising precision medicine approach to evaluate treatment response, which has the potential to aid clinicians in making informed decisions about APD treatment for patients with SCZ. Trial registration Chinese Clinical Trial Registry ( https://www.chictr.org.cn/ ), 18. Aug 2009 retrospectively registered: CAPOC-ChiCTR-RNC-09000521 ( https://www.chictr.org.cn/showproj.aspx?proj=9014 ), CAPEC-ChiCTR-RNC-09000522 ( https://www.chictr.org.cn/showproj.aspx?proj=9013 ).

A2AR-mediated lymphangiogenesis via VEGFR2 signaling prevents salt-sensitive hypertension.

AIMS: Excess dietary sodium intake and retention lead to hypertension. Impaired dermal lymphangiogenesis and lymphatic dysfunction-mediated sodium and fluid imbalance are pathological mechanisms. The adenosine A2A receptor (A2AR) is expressed in lymphatic endothelial cells (LECs), while the roles and mechanisms of LEC-A2AR in skin lymphangiogenesis during salt-induced hypertension are not clear. METHODS AND RESULTS: The expression of LEC-A2AR correlated with lymphatic vessel density in both high-salt diet (HSD)-induced hypertensive mice and hypertensive patients. Lymphatic endothelial cell-specific A2AR knockout mice fed HSD exhibited 17 ± 2% increase in blood pressure and 17 ± 3% increase in Na+ content associated with decreased lymphatic density (-19 ± 2%) compared with HSD-WT mice. A2AR activation by agonist CGS21680 increased lymphatic capillary density and decreased blood pressure in HSD-WT mice. Furthermore, this A2AR agonist activated MSK1 directly to promote VEGFR2 activation and endocytosis independently of VEGF as assessed by phosphoprotein profiling and immunoprecipitation assays in LECs. VEGFR2 kinase activity inhibitor fruquintinib or VEGFR2 knockout in LECs but not VEGF-neutralizing antibody bevacizumab suppressed A2AR activation-mediated decrease in blood pressure. Immunostaining revealed phosphorylated VEGFR2 and MSK1 expression in the LECs were positively correlated with skin lymphatic vessel density and A2AR level in hypertensive patients. CONCLUSION: The study highlights a novel A2AR-mediated VEGF-independent activation of VEGFR2 signaling in dermal lymphangiogenesis and sodium balance, which might be a potential therapeutic target in salt-sensitive hypertension.

A method describing the microdissection of trabecular meshwork tissue from Brown Norway rat eyes.

Glaucoma is often associated with elevated intraocular pressure (IOP), generally due to obstruction of aqueous humor outflow within the trabecular meshwork (TM). Despite many decades of research, the molecular cause of this obstruction remains elusive. To study IOP regulation, several in vitro models, such as perfusion of anterior segments or mechanical stretching of TM cells, have identified several IOP-responsive genes and proteins. While these studies have proved informative, they do not fully recapitulate the in vivo environment where IOP is subject to additional factors, such as circadian rhythms. Thus, rodent animal models are now commonly used to study IOP-responsive genes in vivo. Several single-cell RNAseq studies have been performed where angle tissue, containing cornea, iris, ciliary body tissue in addition to TM, is dissected. However, it is advantageous to physically separate TM from other tissues because the ratio of TM cells is relatively low compared to the other cell types. In this report, we describe a new technique for rat TM microdissection. Evaluating tissue post-dissection by histology and immunostaining clearly shows successful removal of the TM. In addition, TaqMan PCR primers targeting biomarkers of trabecular meshwork (Myoc, Mgp, Chi3l1) or ciliary body (Myh11, Des) genes showed little contamination of TM tissue by the ciliary body. Finally, pitfalls encountered during TM microdissection are discussed to enable others to successfully perform this microsurgical technique in the rat eye.

Aberrant cytokeratin 7 expression by hepatocytes can predict the ductopenia grade in primary biliary cholangitis.

BACKGROUND: Aberrant cytokeratin 7 expression by hepatocytes (CK7+Hs) is the hallmark characteristic of cholestasis diseases, especially in ductopenia diseases such as primary biliary cholangitis (PBC). This study attempted to evaluate the differences and relationships between the clinical and histological features of aberrant cytokeratin 7 (CK7) expression by hepatocytes in PBC patients. METHODS: The clinicopathological data of patients diagnosed with PBC at the Second Hospital of Nanjing between January 2016 and September 2018 were analysed with SPSS 20.0. RESULTS: Eighty-nine PBC patients who underwent liver biopsy were enrolled in this study, and 15, 29 and 45 patients had aberrant CK7 expression by hepatocytes (CK7+Hs (2 +), CK7+Hs (1 +), and CK7-Hs, respectively). There were significant differences in TB, DB, ALP, TA, IgM, interface activity, and ductopenia grade between patients with CK7-Hs and CK7+Hs (2 +) (P < 0.05). The ductopenia grade was also significantly different between patients with CK7+Hs (2 +) and CK7+Hs (1 +) according to sex (P < 0.05). Upon merging the data of CK7+Hs (2 +) and CK7+Hs (1 +) into CK7+Hs, we found significant differences in AMA, AMA-M2, anti-gp210, TB, DB, ALP, TA, IgM, fibrosis, and ductopenia grade between CK7+Hs and CK7-Hs (P < 0.05). The odds ratios (ORs) (and 95% confidence intervals (CIs)) of CK7+Hs according to anti-gp210, ductopenia grade, and interface activity were 6.413 (95% CI 1.363-30.162), 4.145 (95% CI 1.898-9.052) and 3.247 (95% CI 1.556-6.775), respectively (P < 0.05). Spearman's rank correlation according to interface activity and ductopenia grade in patients with CK7+Hs (2 + , 1 + , 0) was r = 0.359 (P = 0.001) and r = 0.396 (P < 0.001), respectively. CONCLUSION: CK7+Hs serves as a cholestasis index of PBC and are associated with the ductopenia grade and interface activity. Aberrant cytokeratin 7 expression by hepatocytes can predict the ductopenia grade in primary biliary cholangitis.

The Effects of Mechanical Stretch on Integrins and Filopodial-Associated Proteins in Normal and Glaucomatous Trabecular Meshwork Cells.

The trabecular meshwork (TM) is the tissue responsible for regulating aqueous humor fluid egress from the anterior eye. If drainage is impaired, intraocular pressure (IOP) becomes elevated, which is a primary risk factor for primary open angle glaucoma. TM cells sense elevated IOP via changes in their biomechanical environment. Filopodia cellular protrusions and integrin transmembrane proteins may play roles in detecting IOP elevation, yet this has not been studied in detail in the TM. Here, we investigate integrins and filopodial proteins, such as myosin-X (Myo10), in response to mechanical stretch, an in vitro technique that produces mechanical alterations mimicking elevated IOP. Pull-down assays showed Myo10 binding to α5 but not the ß1 subunit, αvß3, and αvß5 integrins. Several of these integrins colocalized in nascent adhesions in the filopodial tip and shaft. Using conformation-specific antibodies, we found that ß1 integrin, but not α5 or αvß3 integrins, were activated following 1-h mechanical stretch. Cadherin -11 (CDH11), a cell adhesion molecule, did not bind to Myo10, but was associated with filopodia. Interestingly, CDH11 was downregulated on the TM cell surface following 1-h mechanical stretch. In glaucoma cells, CDH11 protein levels were increased. Finally, mechanical stretch caused a small, yet significant increase in Myo10 protein levels in glaucoma cells, but did not affect cellular communication of fluorescent vesicles via filopodia-like tunneling nanotubes. Together, these data suggest that TM cell adhesion proteins, ß1 integrin and CDH11, have relatively rapid responses to mechanical stretch, which suggests a central role in sensing changes in IOP elevation in situ.

Study on the mechanism of action of Scutellaria barbata on hepatocellular carcinoma based on network pharmacology and bioinformatics.

Background: Hepatocellular carcinoma is one of the most common cancers with the characteristics of invasion and high mortality. Current forms of prevention remain severe. Scutellaria barbata is widely used in traditional Chinese medicine treatment of various tumors. This study explored the mechanism of Scutellaria barbata in the treatment of hepatocellular carcinoma by network pharmacology and bioinformatics. Methods: The active ingredients of Scutellaria barbata and potential targets for the treatment of hepatocellular carcinoma were collected by network pharmacology. The protein interaction network was constructed to screen the core targets, and the association between the core targets and diseases was further verified by bioinformatics methods. Finally, the active ingredients corresponding to the targets closely related to the disease were screened for AMDE characteristics analysis. Molecular docking of drug-like ingredients with corresponding targets was performed. We used CCK-8 kit to determine the effect of active ingredients on cell proliferation. Results: 29 candidate active ingredients and 461 related targets of Scutellaria barbata were screened. A total of 8238 potential therapeutic targets for hepatocellular carcinoma were indentified. Finally, 373 potential targets for the treatment of HCC were obtained. The active ingredients: wogonin, Rhamnazin, eriodictyol, quercetin, baicalein, and luteolin, etc. The core targets were CDK1, CDK4, SRC, and E2F1. A total of 3056 GO enrichment entries were obtained, and 180 enrichment results were obtained by KEGG pathway analysis. Genes were mainly enriched in PI3K-Akt signaling pathway, IL-17 signaling pathway, TNF signaling pathway, apoptosis pathway, and hepatocellular carcinoma pathway. Molecular docking results showed that the screened compounds had strong binding ability with the corresponding target proteins. CCK8 assays showed that Rhamnazin and Luteolin suppressed the proliferation of HCC cells significantly compared with controls. Conclusion: This study revealed that the mechanism of Scutellaria barbata in the treatment of hepatocellular carcinoma may be that the active ingredients inhibit the expression of core genes and block the PI3K-AKT signaling pathway to inhibit the proliferation, and migration and induce apoptosis of cancer cells.

MYO5B-associated diseases: Novel liver-related variants and genotype-phenotype correlation.

BACKGROUND & AIMS: Biallelic pathogenic variants in MYO5B cause microvillus inclusion disease (MVID), or familial intrahepatic cholestasis (FIC). The reported FIC patients are scarce and so the genotype-phenotype correlation has not been fully characterised. This study aimed to report more MYO5B-associated FIC patients and correlate genotypes to phenotypes in more detail. METHODS: The phenotype and genetic data of 12 newly diagnosed MYO5B-associated (including 11 FIC) patients, as well as 118 previously reported patients with available genotypes, were summarised. Only patients with biallelic MYO5B variants were enrolled. Nonsense, frameshift, canonical splice sites, initiation codon loss, and single exon or multiexon deletion were defined as null MYO5B variants. RESULTS: Phenotypically, 50 were isolated MVID, 47 involved both liver and intestine (combined), and 33 were isolated FIC (9 persistent, 15 recurrent, 3 transient, and 6 un-sub-classified) patients. The severity of intestinal manifestation was positively correlated to an increased number of null variants (ρ = 0.299, P = .001). All FIC patients carried at least one non-null variant, and the severity of cholestasis was correlated to the presence of a null variant (ρ = 0.420, P = .029). The proportion of FIC patients (16/29, 55%) harbouring missense/in-frame variants affecting the non-motor regions of MYO5B was significantly higher than that of MVID (3/25, 12%, P = .001) and combined patients (3/31, 10%, P = .000). 10 of the 29 FIC patients harboured missense/in-frame variants at the IQ motifs comparing to none in the 56 MVID and combined patients (P = .000). CONCLUSIONS: The phenotype of MYO5B deficiency was associated with MYO5B genotypes, the nullity or the domain affected.

Diagnostic Performance of FibroTouch Ultrasound Attenuation Parameter and Liver Stiffness Measurement in Assessing Hepatic Steatosis and Fibrosis in Patients With Nonalcoholic Fatty Liver Disease.

INTRODUCTION: To evaluate the diagnostic performance of ultrasound attenuation parameter (UAP) and liver stiffness measurement (LSM) by FibroTouch for diagnosis of hepatic steatosis and fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). METHODS: We recruited 237 patients undergoing FibroTouch and liver biopsy within 2 weeks. The pathological findings of liver biopsy were scored by Nonalcoholic Steatohepatitis Clinical Research Network, and the diagnostic accuracy of UAP for steatosis and LSM for fibrosis was evaluated by area under the receiver operating characteristic curve (AUROC). The impacts of histological parameters on UAP and LSM were analyzed, and diagnostic performance of FibroTouch UAP and LSM was compared with other noninvasive biomarkers. RESULTS: The success rate of FibroTouch examination was 96.51%. The AUROC of UAP for diagnosis of steatosis ≥S1, ≥S2, and S3 was 0.88, 0.93, and 0.88, and the cutoff values were 244, 269, and 296 dB/m, respectively. The AUROC of LSM for the diagnosis of fibrosis stages ≥F2, ≥F3, and F4 was 0.71, 0.71, and 0.77, and the cutoff values were 9.4, 9.4, and 11 kPa, respectively. Multiple regression analysis showed that LSM was positively correlated with degree of fibrosis and NAFLD activity score. UAP was positively correlated with liver steatosis. The diagnostic performance of UAP for steatosis was significantly superior to that of the hepatic steatosis index. DISCUSSION: FibroTouch has a low failure rate with moderate to high diagnostic performance for discriminating the steatosis degree and fibrosis stage and is suitable for clinical evaluation and monitoring of patients with NAFLD.

Senescent T Cell Induces Brown Adipose Tissue "Whitening" Via Secreting IFN-γ.

Aging-associated chronic inflammation is a key contributing factor to a cluster of chronic metabolic disorders, such as cardiovascular disease, obesity, and type 2 diabetes. Immune cells particularly T cells accumulate in adipose tissue with advancing age, and there exists a cross talk between T cell and preadipocyte, contributing to age-related adipose tissue remodeling. Here, we compared the difference in morphology and function of adipose tissue between young (3-month-old) and old (18-month-old) mice and showed the phenomenon of brown adipose tissue (BAT) "whitening" in old mice. Flow cytometry analysis suggested an increased proportion of T cells in BAT of old mice comparing with the young and exhibited senescent characteristics. We take advantage of coculture system to demonstrate directly that senescent T cells inhibited brown adipocyte differentiation of preadipocytes in adipose tissue. Mechanistically, both in vitro and in vivo studies suggested that senescent T cells produced and released a higher level of IFN-γ, which plays a critical role in inhibition of preadipocyte-to-brown adipocyte differentiation. Taken together, the data indicate that senescent T cell-derived IFN-γ is a key regulator in brown adipocyte differentiation.

p.Cys223Tyr mutation causing Crigler-Najjar syndrome type II.

Crigler-Najjar syndrome (CNs) is a rare hereditary unconjugated hyperbilirubinemia caused by mutations in the bilirubin Uridine (UDP) glucuronosyltransferase family 1 member A1 (UGT1A1, ENSG00000241635) gene. Two patients were clinically diagnosed with Crigler-Najjar Syndrome types II (CNs-II) can be clinically diagnosed which were based on the level of total bilirubin, efficacy of phenobarbital treatment, normal liver architecture and exclusion of hemolysis. Diagnosis was also confirmed by UGT1A1 gene mutations, which by sequencing the coding region for UGT1A1 gene mutations, which were the homozygous mutations c.668G > A/p.Cys223Tyr and which caused less than 10% of activity of the enzyme. No data have been reported about this mutate in the population. These patients have a good prognosis and require no active intervention, indicating that an early accurate diagnosis is necessary for disease management and genetic counseling.

Transcriptomic analyses of humans and mice provide insights into depression.

Accumulating studies have been conducted to identify risk genes and relevant biological mechanisms underlying major depressive disorder (MDD). In particular, transcriptomic analyses in brain regions engaged in cognitive and emotional processes, e.g., the dorsolateral prefrontal cortex (DLPFC), have provided essential insights. Based on three independent DLPFC RNA-seq datasets of 79 MDD patients and 75 healthy controls, we performed differential expression analyses using two alternative approaches for cross-validation. We also conducted transcriptomic analyses in mice undergoing chronic variable stress (CVS) and chronic social defeat stress (CSDS). We identified 12 differentially expressed genes (DEGs) through both analytical methods in MDD patients, the majority of which were also dysregulated in stressed mice. Notably, the mRNA level of the immediate early gene FOS ( Fos proto-oncogene) was significantly decreased in both MDD patients and CVS-exposed mice, and CSDS-susceptible mice exhibited a greater reduction in Fos expression compared to resilient mice. These findings suggest the potential key roles of this gene in the pathogenesis of MDD related to stress exposure. Altered transcriptomes in the DLPFC of MDD patients might be, at least partially, the result of stress exposure, supporting that stress is a primary risk factor for MDD.

Analysis of interleukin-20 receptor complexes in trabecular meshwork cells and effects of cytokine signaling in anterior segment perfusion culture.

Purpose: Inflammatory responses may be involved in the glaucomatous process. Our previous studies mapped a T104M mutation in interleukin-20 receptor beta (IL-20RB) in a family with primary open angle glaucoma (POAG). IL-20RB can heterodimerize with IL-20RA to propagate signals from IL-20 family cytokines, IL-19, IL-20, and IL-24 (the type I receptor complex), or it can heterodimerize with IL-22RA to propagate signals from IL-20 and IL-24 (type II receptor complex). In this study, we investigated IL-20 heterodimeric receptor complexes in the trabecular meshwork (TM) compared to dermal fibroblast cell cultures, and examined the phosphorylation of signal transducer and activator of transcription (STAT)-1, -3, and -5 following exposure to IL-20 family cytokines. Additionally, we determined the effects of IL-20 family cytokines on outflow rates in anterior segment perfusion culture, an in vitro model of intraocular pressure (IOP) regulation. Methods: Primary human TM (HTM) cells were grown from dissected TM tissue, and IL-20 receptor expression was investigated with PCR. A Duolink assay was performed to investigate in situ IL-20 receptor protein interactions in HTM or dermal fibroblasts, and Imaris software was used to quantitate the association of the heterodimeric complexes. Phosphorylation of STAT-1, -3, and -5 were evaluated in HTM or dermal fibroblasts using Western immunoblotting after exposure to IL-10, IL-19, IL-20, IL-22, or IL-24. Anterior segment perfusion culture was performed in human cadaver and porcine eyes treated with IL-20, IL-19, or IL-24. Results: All of the IL-20 receptors, IL-20RA, IL-20RB, and IL-22RA1 were expressed in HTM cells. Two isoforms of IL-20RA were expressed: The V1 variant, which is the longest, is the predominant isoform, while the V3 isoform, which lacks exon 3, was also expressed. The Duolink assay demonstrated that the type I (IL-20RA-IL-20RB) and type II (IL-22RA1-IL-20RB) receptors were expressed in HTM cells and dermal fibroblasts. However, in the HTM cells, the type I receptor was present at significantly higher levels, while the type II receptor was preferentially used in the dermal fibroblasts. The HTM cells and the dermal fibroblasts predominantly phosphorylate the Ser727 site in STAT-3. The dermal fibroblasts had higher induction of phosphorylated STAT-1 compared to the HTM cells, while neither cell type had phosphorylated STAT-5 in the cell lysates. The outflow rates in the human anterior segment cultures were increased 2.3-fold by IL-20. However, IL-19 and IL-24 showed differential responses. For IL-19 and IL-24, 50% of the eyes responded with a 1.7- or 1.5-fold increase, respectively, while the other half did not respond. Similarly, perfused porcine anterior segments showed "responders" and "non-responders": IL-20 responders (2.3-fold increase in outflow, n=12) and non-responders (n=11); IL-19 responders (2.1-fold increase, n=7) and non-responders (n=5); and IL-24 responders (1.8-fold increase, n=12) and non-responders (n=5). Conclusions: Type I and type II IL-20 receptor complexes are expressed in human TM cells with predominant expression of the type I receptor (IL-20RA and IL-20RB), which propagates signals from all three IL-20 family cytokines. However, there was a variable response in the outflow rates following perfusion of cytokines in two different species. This may explain why some people are more susceptible to developing elevated IOP in response to inflammation.