Polymorphisms Associated With Metabolic Dysfunction-Associated Steatotic Liver Disease Influence the Progression of End-Stage Liver Disease.

Background And Aims: Chronic liver injury that results in cirrhosis and end-stage liver disease (ESLD) causes more than 1 million deaths annually worldwide. Although the impact of genetic factors on the severity of metabolic dysfunction-associated steatotic liver disease (MASLD) and alcohol-related liver disease (ALD) has been previously studied, their contribution to the development of ESLD remains largely unexplored.

Methods: We genotyped 6 MASLD-associated polymorphisms in healthy (n = 123), metabolic dysfunction-associated steatohepatitis (MASH) (n = 145), MASLD-associated ESLD (n = 72), and ALD-associated ESLD (n = 57) cohorts and performed multinomial logistic regression to determine the combined contribution of genetic, demographic, and clinical factors to the progression of ESLD.

Modeling mechanisms underlying differential inflammatory responses to COVID-19 in type 2 diabetes using a patient-derived microphysiological organ-on-a-chip system.

: COVID-19 pandemic has caused more than 6 million deaths worldwide. Co-morbid conditions such as Type 2 Diabetes (T2D) have increased mortality in COVID-19. With limited translatability of and small animal models to human disease, human organ-on-a-chip models are an attractive platform to model disease conditions and test potential therapeutics.

Evaluation of Human Hepatocyte Drug Metabolism Carrying High-Risk or Protection-Associated Liver Disease Genetic Variants.

Metabolic-dysfunction-associated steatotic liver disease (MASLD), which affects 30 million people in the US and is anticipated to reach over 100 million by 2030, places a significant financial strain on the healthcare system. There is presently no FDA-approved treatment for MASLD despite its public health significance and financial burden. Understanding the connection between point mutations, liver enzymes, and MASLD is important for comprehending drug toxicity in healthy or diseased individuals.

The Combination of a Human Biomimetic Liver Microphysiology System with BIOLOGXsym, a Quantitative Systems Toxicology (QST) Modeling Platform for Macromolecules, Provides Mechanistic Understanding of Tocilizumab- and GGF2-Induced Liver Injury.

Biologics address a range of unmet clinical needs, but the occurrence of biologics-induced liver injury remains a major challenge. Development of cimaglermin alfa (GGF2) was terminated due to transient elevations in serum aminotransferases and total bilirubin. Tocilizumab has been reported to induce transient aminotransferase elevations, requiring frequent monitoring.

A Quantitative Systems Pharmacology Platform Reveals NAFLD Pathophysiological States and Targeting Strategies.

Non-alcoholic fatty liver disease (NAFLD) has a high global prevalence with a heterogeneous and complex pathophysiology that presents barriers to traditional targeted therapeutic approaches. We describe an integrated quantitative systems pharmacology (QSP) platform that comprehensively and unbiasedly defines disease states, in contrast to just individual genes or pathways, that promote NAFLD progression. The QSP platform can be used to predict drugs that normalize these disease states and experimentally test predictions in a human liver acinus microphysiology system (LAMPS) that recapitulates key aspects of NAFLD.

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins.

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C.

Quantifying the progression of non-alcoholic fatty liver disease in human biomimetic liver microphysiology systems with fluorescent protein biosensors.

Metabolic syndrome is a complex disease that involves multiple organ systems including a critical role for the liver. Non-alcoholic fatty liver disease (NAFLD) is a key component of the metabolic syndrome and fatty liver is linked to a range of metabolic dysfunctions that occur in approximately 25% of the population. A panel of experts recently agreed that the acronym, NAFLD, did not properly characterize this heterogeneous disease given the associated metabolic abnormalities such as type 2 diabetes mellitus (T2D), obesity, and hypertension.

A 3D microfluidic liver model for high throughput compound toxicity screening in the OrganoPlate®.

We report the development, automation and validation of a 3D, microfluidic liver-on-a-chip for high throughput hepatotoxicity screening, the OrganoPlate LiverTox™. The model is comprised of aggregates of induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep) seeded in an extracellular matrix in the organ channel and co-cultured with endothelial cells and THP-1 monoblasts differentiated to macrophages seeded in the vascular channel of the 96 well Mimetas OrganoPlate 2-lane. A key component of high throughput screening is automation and we report a protocol to seed, dose, collect and replenish media and add assay reagents in the OrganoPlate 2-lane using a standard laboratory liquid handling robot.

Modeling the Effect of the Metastatic Microenvironment on Phenotypes Conferred by Estrogen Receptor Mutations Using a Human Liver Microphysiological System.

Reciprocal coevolution of tumors and their microenvironments underlies disease progression, yet intrinsic limitations of patient-derived xenografts and simpler cell-based models present challenges towards a deeper understanding of these intercellular communication networks. To help overcome these barriers and complement existing models, we have developed a human microphysiological system (MPS) model of the human liver acinus, a common metastatic site, and have applied this system to estrogen receptor (ER)+ breast cancer. In addition to their hallmark constitutive (but ER-dependent) growth phenotype, different ESR1 missense mutations, prominently observed during estrogen deprivation therapy, confer distinct estrogen-enhanced growth and drug resistant phenotypes not evident under cell autonomous conditions.

Clinically Observed Estrogen Receptor Alpha Mutations within the Ligand-Binding Domain Confer Distinguishable Phenotypes.

Objective: Twenty to fifty percent of estrogen receptor-positive (ER+) metastatic breast cancers express mutations within the ER ligand-binding domain. While most studies focused on the constitutive ER signaling activity commonly engendered by these mutations selected during estrogen deprivation therapy, our study was aimed at investigating distinctive phenotypes conferred by different mutations within this class.

Methods: We examined the two most prevalent mutations, D538G and Y537S, employing corroborative genome-edited and lentiviral-transduced ER+ T47D cell models.

Activation of the Caenorhabditis elegans Degenerin Channel by Shear Stress Requires the MEC-10 Subunit.

Mechanotransduction in Caenorhabditis elegans touch receptor neurons is mediated by an ion channel formed by MEC-4, MEC-10, and accessory proteins. To define the role of these subunits in the channel's response to mechanical force, we expressed degenerin channels comprising MEC-4 and MEC-10 in Xenopus oocytes and examined their response to laminar shear stress (LSS). Shear stress evoked a rapid increase in whole cell currents in oocytes expressing degenerin channels as well as channels with a MEC-4 degenerin mutation (MEC-4d), suggesting that C.

Deficient and Null Variants of SERPINA1 Are Proteotoxic in a Caenorhabditis elegans Model of α1-Antitrypsin Deficiency.

α1-antitrypsin deficiency (ATD) predisposes patients to both loss-of-function (emphysema) and gain-of-function (liver cirrhosis) phenotypes depending on the type of mutation. Although the Z mutation (ATZ) is the most prevalent cause of ATD, >120 mutant alleles have been identified. In general, these mutations are classified as deficient (<20% normal plasma levels) or null (<1% normal levels) alleles.

SERPINB12 Is a Slow-Binding Inhibitor of Granzyme A and Hepsin.

The clade B/intracellular serpins protect cells from peptidase-mediated injury by forming covalent complexes with their targets. SERPINB12 is expressed in most tissues, especially at cellular interfaces with the external environment. This wide tissue distribution pattern is similar to that of granzyme A (GZMA).

The aggregation-prone intracellular serpin SRP-2 fails to transit the ER in Caenorhabditis elegans.

Familial encephalopathy with neuroserpin inclusions bodies (FENIB) is a serpinopathy that induces a rare form of presenile dementia. Neuroserpin contains a classical signal peptide and like all extracellular serine proteinase inhibitors (serpins) is secreted via the endoplasmic reticulum (ER)-Golgi pathway. The disease phenotype is due to gain-of-function missense mutations that cause neuroserpin to misfold and aggregate within the ER.

A C. elegans model of human α1-antitrypsin deficiency links components of the RNAi pathway to misfolded protein turnover.

The accumulation of serpin oligomers and polymers within the endoplasmic reticulum (ER) causes cellular injury in patients with the classical form α1-antitrypsin deficiency (ATD). To better understand the cellular and molecular genetic aspects of this disorder, we generated transgenic C. elegans strains expressing either the wild-type (ATM) or Z mutant form (ATZ) of the human serpin fused to GFP.

A genome-wide RNAi screen identifies potential drug targets in a C. elegans model of α1-antitrypsin deficiency.

α1-Antitrypsin deficiency (ATD) is a common genetic disorder that can lead to end-stage liver and lung disease. Although liver transplantation remains the only therapy currently available, manipulation of the proteostasis network (PN) by small molecule therapeutics offers great promise. To accelerate the drug-discovery process for this disease, we first developed a semi-automated high-throughput/content-genome-wide RNAi screen to identify PN modifiers affecting the accumulation of the α1-antitrypsin Z mutant (ATZ) in a Caenorhabditis elegans model of ATD.

Isolation of serpin-interacting proteins in C. elegans using protein affinity purification.

Caenorhabditis elegans is a useful model organism for combining multiple imaging, genetic, and biochemical methodologies to gain more insight into the biological function of specific proteins. Combining both biochemical and genetic analyses can lead to a better understanding of how a given protein may function within the context of a network of other proteins or specific pathway. Here, we describe a protocol for the biochemical isolation of serpin-interacting proteins using affinity purification and proteomic analysis.

A pro-cathepsin L mutant is a luminal substrate for endoplasmic-reticulum-associated degradation in C. elegans.

Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C.

Loss of lysosomal ion channel transient receptor potential channel mucolipin-1 (TRPML1) leads to cathepsin B-dependent apoptosis.

Mucolipidosis type IV (MLIV) is a lysosomal storage disease caused by mutations in the gene MCOLN1, which codes for the transient receptor potential family ion channel TRPML1. MLIV has an early onset and is characterized by developmental delays, motor and cognitive deficiencies, gastric abnormalities, retinal degeneration, and corneal cloudiness. The degenerative aspects of MLIV have been attributed to cell death, whose mechanisms remain to be delineated in MLIV and in most other storage diseases.

Using C. elegans to identify the protease targets of serpins in vivo.

Most serpins inhibit serine and/or cysteine proteases, and their inhibitory activities are usually defined in vitro. However, the physiological protease targets of most serpins are unknown despite many years of research. This may be due to the rapid degradation of the inactive serpin:protease complexes and/or the conditions under which the serpin inhibits the protease.