-
Nature Structural & Molecular Biology Jun 2022Proteins including FUS, hnRNPA2, and TDP-43 reversibly aggregate into amyloid-like fibrils through interactions of their low-complexity domains (LCDs). Mutations in LCDs...
Proteins including FUS, hnRNPA2, and TDP-43 reversibly aggregate into amyloid-like fibrils through interactions of their low-complexity domains (LCDs). Mutations in LCDs can promote irreversible amyloid aggregation and disease. We introduce a computational approach to identify mutations in LCDs of disease-associated proteins predicted to increase propensity for amyloid aggregation. We identify several disease-related mutations in the intermediate filament protein keratin-8 (KRT8). Atomic structures of wild-type and mutant KRT8 segments confirm the transition to a pleated strand capable of amyloid formation. Biochemical analysis reveals KRT8 forms amyloid aggregates, and the identified mutations promote aggregation. Aggregated KRT8 is found in Mallory-Denk bodies, observed in hepatocytes of livers with alcoholic steatohepatitis (ASH). We demonstrate that ethanol promotes KRT8 aggregation, and KRT8 amyloids co-crystallize with alcohol. Lastly, KRT8 aggregation can be seeded by liver extract from people with ASH, consistent with the amyloid nature of KRT8 aggregates and the classification of ASH as an amyloid-related condition.
Topics: Amyloid; Amyloidogenic Proteins; Hepatocytes; Humans; Liver; Mutation; Protein Domains
PubMed: 35637421
DOI: 10.1038/s41594-022-00774-y -
Scientific Reports Mar 2022Despite the increasing prevalence of Nonalcoholic steatohepatitis (NASH) worldwide, there is no effective treatment available for this disease. "Ballooned hepatocyte" is...
Despite the increasing prevalence of Nonalcoholic steatohepatitis (NASH) worldwide, there is no effective treatment available for this disease. "Ballooned hepatocyte" is a characteristic finding in NASH and is correlated with disease prognosis, but their mechanisms of action are poorly understood; furthermore, neither animal nor in vitro models of NASH have been able to adequately represent ballooned hepatocytes. Herein, we engineered cell sheets to develop a new in vitro model of ballooned hepatocytes. Primary human hepatocytes (PHH) and Hepatic stellate cells (HSC) were co-cultured to produce cell sheets, which were cultured in glucose and lipid containing medium, following which histological and functional analyses were performed. Histological findings showed hepatocyte ballooning, accumulation of fat droplets, abnormal cytokeratin arrangement, and the presence of Mallory-Denk bodies and abnormal organelles. These findings are similar to those of ballooned hepatocytes in human NASH. Functional analysis showed elevated levels of TGFβ-1, SHH, and p62, but not TNF-α, IL-8. Exposure of PHH/HSC sheets to a glucolipotoxicity environment induces ballooned hepatocyte without inflammation. Moreover, fibrosis is an important mechanism underlying ballooned hepatocytes and could be the basis for the development of a new in vitro NASH model with ballooned hepatocytes.
Topics: Animals; Hepatic Stellate Cells; Hepatocytes; Humans; Keratins; Kupffer Cells; Non-alcoholic Fatty Liver Disease
PubMed: 35351975
DOI: 10.1038/s41598-022-09428-x -
Redox Biology Jun 2022Autophagy is an evolutionarily conserved self-protecting mechanism implicated in cellular homeostasis. ATG4B plays a vital role in autophagy process via undertaking...
Autophagy is an evolutionarily conserved self-protecting mechanism implicated in cellular homeostasis. ATG4B plays a vital role in autophagy process via undertaking priming and delipidation of LC3. Chemical inhibitors and regulative modifications such as oxidation of ATG4B have been demonstrated to modulate autophagy function. Whether and how ATG4B could be regulated by metal ions is largely unknown. Copper is an essential trace metal served as static co-factors in redox reactions in physiology process. Excessive accumulation of copper in ATP7B mutant cells leads to pathology progression such as insoluble Mallory body (MB) in Wilson disease (WD). The clearance of MB via autophagy pathway was thought as a promising strategy for WD. Here, we discovered that copper ion instead of other ions could inhibit the activity of ATG4B followed by autophagy suppression. In addition, copper could induce ATG4B oligomers depending on cysteine oxidation which could be abolished in reduced condition. Copper also promotes the formation of insoluble ATG4B aggregates, as well as p62-and ubiquitin-positive aggregates, which is consistent with the components of MB caused by copper overload in WD cell model. Importantly, overexpression of ATG4B could partially reduce the formation of MB and rescue impaired autophagy. Taken together, our results uncovered for the first time a new damage mechanism mediated by copper and implied new insights of the crosstalk between the toxicity of copper and autophagy in the pathogenesis of WD.
Topics: Autophagy; Autophagy-Related Proteins; Copper; Cysteine Endopeptidases; Microtubule-Associated Proteins
PubMed: 35349929
DOI: 10.1016/j.redox.2022.102284 -
Hepatology (Baltimore, Md.) Oct 2022The NASH Clinical Research Network histologic scoring system, the gold-standard NASH histology assessment for clinical trials, has demonstrated intrarater and interrater...
BACKGROUND AND AIMS
The NASH Clinical Research Network histologic scoring system, the gold-standard NASH histology assessment for clinical trials, has demonstrated intrarater and interrater variability. An expert panel in a previous systematic Research and Development/University of California Los Angeles (RAND/UCLA) study determined that existing histologic scoring systems do not fully capture NASH disease activity and fibrosis, and standardized definitions of histologic features are needed. We evaluated the reliability of existing and alternate histologic measures and their correlations with a disease activity visual analog scale to propose optimal components for an expanded NAFLD activity score (NAS).
APPROACH AND RESULTS
Four liver pathologists who were involved in the prior RAND/UCLA study underwent standardized training and multiple discussions with the goal of improving agreement. They were blinded to clinical information and scored histologic measures twice, ≥2 weeks apart, for 40 liver biopsies representing the full spectrum of NAFLD. Index intraclass correlation coefficient (ICC) estimates demonstrated intrarater (0.80-0.85) and interrater (0.60-0.72) reliability. Hepatocyte ballooning items had similar interrater ICCs (0.68-0.79), including those extending scores from 0-2 to 0-4. Steatosis measures (interrater ICCs, 0.72-0.80) correlated poorly with disease activity. Correlations with disease activity were largest for hepatocyte ballooning and Mallory-Denk bodies (MDBs), with both used to develop the expanded NAS (intrarater ICC, 0.90; interrater ICC, 0.80). Fibrosis measures had ICCs of 0.70-0.87.
CONCLUSIONS
After extensive preparation among a group of experienced pathologists, we demonstrated improved reliability of multiple existing histologic NAFLD indices and fibrosis staging systems. Hepatocyte ballooning and MDBs most strongly correlated with disease activity and were used for the expanded NAS. Further validation including evaluation of responsiveness is required.
Topics: Biopsy; Fibrosis; Humans; Liver; Non-alcoholic Fatty Liver Disease; Reproducibility of Results; Severity of Illness Index
PubMed: 35332569
DOI: 10.1002/hep.32475 -
Journal of Applied Clinical Medical... May 2022To demonstrate the plan quality and delivery efficiency of volumetric-modulated arc therapy (VMAT) with the Halcyon Linac ring delivery system (RDS) in the treatment of...
PURPOSE
To demonstrate the plan quality and delivery efficiency of volumetric-modulated arc therapy (VMAT) with the Halcyon Linac ring delivery system (RDS) in the treatment of single-isocenter/two-lesion lung stereotactic body radiation therapy (SBRT).
MATERIALS/METHODS
Sixteen previously treated non-coplanar VMAT single-isocenter/two-lesion lung SBRT plans delivered with SBRT-dedicated C-arm TrueBeam Linac were selected. Prescribed dose was 50 Gy to each lesion over five fractions with treatment delivery every other day and AcurosXB algorithm as the final dose calculation algorithm. TrueBeam single-isocenter plans were reoptimized for Halcyon Linac with coplanar geometry. Both TrueBeam and Halcyon plans were normalized for identical combined target coverage and evaluated. Conformity indices (CIs), heterogeneity index (HI), gradient index (GI), gradient distance (GD), and D were compared. The normal lung V5Gy, V10Gy, V20Gy, mean lung dose (MLD), and dose to organs at risk (OAR) were evaluated. Treatment delivery parameters, including beam-on time, were recorded.
RESULTS
Halcyon plans were statistically similar to clinically delivered TrueBeam plans. No statistical differences in target conformity, dose heterogeneity, or intermediate-dose spillage were observed (all, p > 0.05). Halcyon plans, on average, demonstrated statistically insignificant reduced maximum dose to most adjacent OAR and normal lung. However, Halcyon yielded statistically significant lower maximal dose to the ribs (p = 0.041) and heart (p = 0.026), dose to 1 cc of ribs (p = 0.035) and dose to 5 cc of esophagus (p = 0.043). Plan complexity slightly increased as seen in the average increase of total monitor units, modulation factor, and beam-on time by 480, 0.48, and 2.78 min, respectively. However, the estimated overall treatment time was reduced by 2.22 min, on average. Mean dose delivery accuracy of clinical TrueBeam plans and the corresponding Halcyon plans was 98.9 ± 0.85% (range: 98.1%-100%) and 98.45 ± 0.99% (range: 97.9%-100%), respectively, demonstrating similar treatment delivery accuracy.
CONCLUSION
SBRT treatment of synchronous lung lesions via single-isocenter VMAT on Halcyon RDS is feasible and dosimetrically equivalent to clinically delivered TrueBeam plans. Halcyon provides excellent plan quality and shorter overall treatment time that may improve patient compliance, reduce intrafraction movement, improve clinic efficiency, and potentially offering lung SBRT treatments for underserved patients on a Halcyon only clinic.
Topics: Feasibility Studies; Humans; Lung; Lung Neoplasms; Radiosurgery; Radiotherapy Dosage; Radiotherapy Planning, Computer-Assisted; Radiotherapy, Intensity-Modulated
PubMed: 35128795
DOI: 10.1002/acm2.13555 -
BMC Musculoskeletal Disorders Jan 2022Intraoperative proximal femoral fractures (IPFF) are relevant complications during total hip arthroplasty. Fixation using cerclage wires (CW) represents a... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
Intraoperative proximal femoral fractures (IPFF) are relevant complications during total hip arthroplasty. Fixation using cerclage wires (CW) represents a minimally-invasive technique to address these fractures through the same surgical approach. The goal of treatment is to mobilise the patient as early as possible, which requires high primary stability. This study aimed to compare different cerclage wire configurations fixing IPFF with regard to biomechanical primary stability.
METHODS
Standardised IPFF (type II, Modified Mallory Classification) were created in human fresh frozen femora and were fixed either by two or three CW (1.6 mm, stainless steel). All cadaveric specimens (n = 42) were randomised to different groups (quasi-static, dynamic) or subgroups (2 CW, 3 CW) stratified by bone mineral density determined by Dual Energy X-ray Absorptiometry. Using a biomechanical testing setup, quasi-static and dynamic cyclic failure tests were carried out. Cyclic loading started from 200 N to 500 N at 1 Hz with increasing peak load by 250 N every 100 cycles until failure occurred or maximum load (5250 N) reached. The change of fracture gap size was optically captured.
RESULTS
No significant differences in failure load after quasi-static (p = 0.701) or dynamic cyclic loading (p = 0.132) were found between the experimental groups. In the quasi-static load testing, all constructs resisted 250% of the body weight (BW) of their corresponding body donor. In the dynamic cyclic load testing, all but one construct (treated by 3 CW) resisted 250% BW.
CONCLUSIONS
Based on this in vitro data, both two and three CW provided sufficient primary stability according to the predefined minimum failure load (250% BW) to resist. The authors recommend the treatment using two CW because it reduces the risk of vascular injury and shortens procedure time.
Topics: Arthroplasty, Replacement, Hip; Biomechanical Phenomena; Bone Wires; Femoral Fractures; Fracture Fixation, Internal; Humans
PubMed: 34996409
DOI: 10.1186/s12891-021-04956-5 -
Clinical, histological and molecular profiling of different stages of alcohol-related liver disease.Gut Sep 2022Alcohol-related liver disease (ALD) ranges from never-decompensated ALD (ndALD) to the life-threatening decompensated phenotype, known as alcohol-related hepatitis (AH).... (Observational Study)
Observational Study
OBJECTIVE
Alcohol-related liver disease (ALD) ranges from never-decompensated ALD (ndALD) to the life-threatening decompensated phenotype, known as alcohol-related hepatitis (AH). A multidimensional study of the clinical, histological and molecular features of these subtypes is lacking.
DESIGN
Two large cohorts of patients were recruited in an international, observational multicentre study: a retrospective cohort of patients with ndALD (n=110) and a prospective cohort of patients with AH (n=225). Clinical, analytical, immunohistochemistry and hepatic RNA microarray analysis of both disease phenotypes were performed.
RESULTS
Age and mean alcohol intake were similar in both groups. AH patients had greater aspartate amino transferase/alanine amino transferase ratio and lower gamma-glutamyl transferase levels than in ndALD patients. Patients with AH demonstrated profound liver failure and increased mortality. One-year mortality was 10% in ndALD and 50% in AH. Histologically, steatosis grade, ballooning and pericellular fibrosis were similar in both groups, while advanced fibrosis, Mallory-Denk bodies, bilirubinostasis, severe neutrophil infiltration and ductular reaction were more frequent among AH patients. Transcriptome analysis revealed a profound gene dysregulation within both phenotypes when compare to controls. While ndALD was characterised by deregulated expression of genes involved in matrisome and immune response, the development of AH resulted in a marked deregulation of genes involved in hepatocyte reprogramming and bile acid metabolism.
CONCLUSIONS
Despite comparable alcohol intake, AH patients presented with worse liver function compared with ndALD patients. Bilirubinostasis, severe fibrosis and ductular reaction were prominent features of AH. AH patients exhibited a more profound deregulation of gene expression compared with ndALD patients.
Topics: Fibrosis; Hepatitis, Alcoholic; Humans; Liver; Prospective Studies; Retrospective Studies
PubMed: 34992134
DOI: 10.1136/gutjnl-2021-324295 -
The FEBS Journal Feb 2023p62/Sequestosome-1 (SQSTM1) is a selective autophagy receptor that recruits and delivers intracellular substrates for bulk clearance through the autophagy lysosomal... (Review)
Review
p62/Sequestosome-1 (SQSTM1) is a selective autophagy receptor that recruits and delivers intracellular substrates for bulk clearance through the autophagy lysosomal pathway. Interestingly, p62 also serves as a signaling scaffold to participate in the regulation of multiple physiological processes, including oxidative stress response, metabolism, inflammation, and programmed cell death. Perturbation of p62 activity has been frequently found to be associated with the pathogenesis of many liver diseases. p62 has been identified as a critical component of protein aggregates in the forms of Mallory-Denk bodies (MDBs) or intracellular hyaline bodies (IHBs), which are known to be frequently detected in biopsy samples from alcoholic steatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), and hepatocellular carcinoma (HCC) patients. Importantly, abundance of these p62 inclusion bodies is increasingly recognized as a biomarker for NASH and HCC. Although the level of p62 bodies seems to predict the progression and prognosis of these liver diseases, understanding of the underlying mechanisms by which p62 regulates and contributes to the development and progression of these diseases remains incomplete. In this review, we will focus on the function and regulation of p62, and its pathophysiological roles in the liver, by critically reviewing the findings from preclinical models that recapitulate the pathogenesis and manifestation of these liver diseases in humans. In addition, we will also explore the suitability of p62 as a predictive biomarker and a potential therapeutic target for the treatment of liver diseases, including NASH and HCC, as well as recent development of small-molecule compounds for targeting the p62 signaling axis.
Topics: Humans; Carcinoma, Hepatocellular; Sequestosome-1 Protein; Liver Neoplasms; Non-alcoholic Fatty Liver Disease; Liver; Biomarkers; Autophagy
PubMed: 34882306
DOI: 10.1111/febs.16317 -
The Journal of Pediatrics Mar 2022
Topics: Humans; Mallory Bodies; Muscular Dystrophies; Scoliosis; Torticollis
PubMed: 34838582
DOI: 10.1016/j.jpeds.2021.11.048 -
Biomedicines Nov 2021Nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) is the most common liver disorder in developed countries. Although many new therapeutics for NASH are...
Nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) is the most common liver disorder in developed countries. Although many new therapeutics for NASH are present in the drug development pipeline, there are still no approved drugs. One of the reasons that makes NASH drug development challenging is the lack of appropriate animal NASH models that resolve issues arising from inter-species differences between humans and rodents. In the present study, we developed a choline-deficient, L-amino-acid-defined, high-fat-diet (CDAHFD)-induced human NASH model using human liver chimeric mice. We demonstrated human hepatocyte injury by an elevation of plasma human alanine aminotransferase 1 in mice fed CDAHFD. Histological analysis showed that CDAHFD feeding induced similar histological changes to human NASH patients, including ballooning, inflammation, apoptosis, regeneration of human hepatocytes, and pericellular and perisinusoidal fibrosis. The chimeric mice fed CDAHFD were treated with a peroxisome-proliferator-activated receptor α/δ agonist, Elafibranor. Elafibranor ameliorated steatosis, ballooning of hepatocytes, and preserved fibrosis progression. We developed a novel humanized NASH model that can elucidate pathophysiological mechanisms and predict therapeutic efficacy in human NASH. This model will be useful in exploring new drugs and biomarkers in the early stages of human NASH.
PubMed: 34829876
DOI: 10.3390/biomedicines9111647