Background & Aims Metabolic dysfunction-associated steatohepatitis (MASH) is a prevalent chronic liver disease characterized by steatosis, inflammation, and hepatocyte damage, with or without fibrosis. Robust preclinical models that represent the clinical features of MASH are critical for drug development. We aimed to examine the effects of two widely used Western-type diets and thermoneutral housing on the induction of MASH and liver fibrosis in mice. Methods Male mice were fed either a Gubra-Amylin (GAN) MASH or Western non-trans-fat diet and were housed at standard room temperature (21°C) for a period of 25, 33, or 46 weeks or at thermoneutrality (30°C) for 25 weeks. Liver histology, transcriptome profiling, and in situ imaging cytometry were applied to analyze the differences between the models. Results Liver inflammation and hepatocyte damage were increased by housing mice at thermoneutrality. Remarkably, mice housed at thermoneutrality developed liver fibrosis after 25 weeks of diet feeding, reaching a degree of extracellular matrix deposition equivalent to the levels that required 46 weeks of diet feeding at standard temperature. Thermoneutral housing enhanced the expression of genes related to extracellular matrix organization and inflammatory pathways, demonstrating higher similarity to human MASH. This model was also characterized by distinctive hepatic immune cell infiltration and greater cell-cell interactions, compared to housing at standard temperatures. Conclusion Housing diet-induced MASH mice at thermoneutrality accelerated fibrosis onset and induced increased disease severity. Phenotypically and transcriptionally, this mouse model showed greater similarity to human MASH. In addition, MASH mice at thermoneutrality developed a distinct and enhanced hepatic innate and adaptive immune response.

BACKGROUND:Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) is an effective therapy for reducing low-density lipoprotein (LDL) cholesterol (LDL-C) in adults with hyperlipidemia, including heterozygous familial hypercholesterolemia, thereby lowering cardiovascular risk. Current PCSK9 inhibitors are injectable therapies; no oral small-molecule PCSK9 inhibitor has yet been approved. METHODS:Laroprovstat (AZD0780) is a novel small-molecule identified through structure-based design that binds to the PCSK9 C-terminal domain. The effects of laroprovstat on LDL receptor expression and LDL-C levels were assessed in vitro and in mice expressing human PCSK9. Safety, tolerability, and pharmacokinetic and pharmacodynamic properties of laroprovstat were assessed in healthy participants with LDL-C ≥70 and ≤190 mg/dL after single ascending doses. Laroprovstat was also assessed in participants with LDL-C ≥100 and ≤190 mg/dL at doses of 1 mg or 30 mg versus placebo administered once daily for 28 days after a rosuvastatin 20 mg run-in treatment period. RESULTS:Laroprovstat does not inhibit the PCSK9-LDL receptor interaction but stabilizes the PCSK9 C-terminal domain, preventing lysosomal trafficking and degradation of LDL receptor. Laroprovstat increased LDL receptor expression and reduced LDL-C levels in mice expressing human PCSK9. Laroprovstat displayed dose-proportional pharmacokinetics and a half-life suitable for once-daily dosing (≈40 hours). There was no clinically meaningful change in exposure when dosed with a high-fat meal compared with the fasted state (area under the plasma concentration-time curveinf and Cmax geometric mean reduction of 1.15 [90% CI, 1.11-1.19] and 1.06 [90% CI, 1.00-1.13], respectively). After a rosuvastatin 20 mg 3-week run-in treatment period, laroprovstat 1 and 30 mg reduced LDL-C by 29% (95% CI, 18%-38%) and 51% (95% CI, 44%-58%) compared with baseline. Combined rosuvastatin and laroprovstat treatment resulted in a total approximate reduction in LDL-C of 70% and 80% for laroprovstat 1 and 30 mg, respectively. CONCLUSIONS:Laroprovstat was well tolerated with no safety findings of concern and may be dosed with or without food. In treatment-naïve participants with hypercholesterolemia, combined rosuvastatin 20 mg and laroprovstat 30 mg treatment led to an 80% LDL-C reduction, supporting further development of laroprovstat as the first oral small-molecule PCSK9 inhibitor in patients with hypercholesterolemia. REGISTRATION:URL: https://www.clinicaltrials.gov; Unique identifier: NCT05384262.

Population estimation using capture-recapture modeling typically requires that individuals are identifiable by unique marks. North Atlantic right whales (Eubalaena glacialis) can be identified by natural callosity patterns on their heads that are established nearly a year after birth, which has facilitated population monitoring using extensive aerial surveys. A well-maintained catalog of individual sightings has been used to annually estimate population size with a Jolly-Seber (J-S) model using a Bayesian state-space framework. Given that young animals cannot enter the catalog before an established callosity pattern, the terminal-year population estimate never includes new calves despite breeding area surveys that provide a nearly complete census of births. Here, I illustrate a simple modification to the J-S likelihood whereby the number of expected entrants is a function of known births and a parameter representing initial offspring mortality. A simulation study was used as a proof of concept and indicated increased accuracy and precision of population estimates. The birth-integrated J-S model had more accurate terminal-year estimates of right whale population size that remained consistent during subsequent model fitting to additional years of sightings data. While the bias corrections were fairly small (5%) given low per capita calving rates, the demonstrated improvement in accuracy will be helpful to the conservation and management processes for this endangered species. Integrated modeling approaches make better use of available data and can improve inferences on population dynamics.

The recent steep decline of the endangered North Atlantic right whale Eubalaena glacialis can be attributed to high mortality combined with low reproduction. While the former is a clear result of anthropogenic activity, the latter involves more complexity. Evidence suggests that both short-term fluctuations in prey availability and long-term decline in health are responsible for depressed right whale calving rates. To facilitate an assessment of extinction risk, we developed a multistate capture-recapture model that estimated the probability of calving using extensive sightings data from 1990-2019. The model estimated sub-lethal effects of severe injury on calving probability and modeled temporal variability in calving as related to indices of prey availability (Calanus spp. biomass) and an apparent regime shift. The average annual probability of calving for known-breeding females, given average prey conditions, decreased from 0.217 [95% CI: 0.162, 0.281] to 0.142 [95% CI: 0.067, 0.252] after the 2010 regime shift. The model indicated strong evidence of a relationship between calving probability and the prey index from the eastern Gulf of Maine, although this relationship effectively disappeared after 2010; moderate evidence for a relationship with prey from the southwest Gulf of St. Lawrence remained. Weak evidence of reduced calving probability due to severe injury resulted from low sample sizes, given increased mortality for individuals observed with severe injuries. The regime effect is hypothesized to be capturing a long-term decline in health due to a combination of decreasing habitat quality resulting from climate change and potentially chronic sublethal injuries (e.g. entanglements). Our reproduction model provides demographic parameter estimates that can be used in population projections for North Atlantic right whales, although uncertainty remains in the mechanisms responsible for recent declines in calving.

Background/Aims Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global epidemic. The disease has a strong genetic component, and a common missense variant (rs2642438) in the mitochondrial amidoxime-reducing component 1 (MARC1) gene confers protection against its onset and severity. However, there are contrasting results regarding the mechanisms that promote this protection. Methods We downregulated MARC1 in primary human hepatocytes (PHHs) using short interfering RNA (siRNA). We measured neutral lipid content by Oil-Red O staining and fatty acid oxidation by radiolabeled tracers. We also performed RNA-sequencing and proteomic analysis using LC-MS. Additionally, we analyzed data from 239,075 participants from the UK Biobank. Results Downregulation of MARC1 reduced neutral lipid content in PHHs homozygous for the wild type (p.A165, risk), but not for the mutant (p.T165, protective), allele. We found that this reduction was mediated by increased fatty acid utilization via β-oxidation. Consistent with these results, we found that the levels of 3-hydroxybutyrate, a by-product of β-oxidation, were higher in carriers of the rs2642438 minor allele among samples from the UK biobank, indicating higher β-oxidation in these individuals. Moreover, downregulation of the MARC1 p.A165 variant resulted in a more favorable phenotype by reducing ferroptosis and reactive oxygen species levels. Conclusions MARC1 downregulation in carriers of the risk allele results in lower hepatocyte neutral lipids content due to higher β-oxidation, while upregulating beneficial pathways involved in cell survival.

Abstract Understanding the causes of mortality for a declining species is essential for developing effective conservation and management strategies, particularly when anthropogenic activities are the primary threat. Using a competing hazards framework allows for robust estimation of the cause‐specific variation in risk that may exist across multiple dimensions, such as time and individual. Here, we estimated cause‐specific rates of severe injury and mortality for North Atlantic right whales (Eubalaena glacialis), a critically endangered species that is currently in peril due to human‐caused interactions. We developed a multistate capture–recapture model that leveraged 30 years of intensive survey effort yielding sightings of individuals with injury assessments and necropsies of carcass recoveries. We examined variation in the hazard rates of severe injury and mortality due to entanglements in fishing gear and vessel strikes as explained by temporal patterns and the age and reproductive status of the individual. We found strong evidence for increased rates of severe entanglement injuries after 2013 and for females with calves, with consequently higher marginal mortality. The model results also suggested that despite vessel strikes causing a lower average rate of severe injuries, the higher mortality rate conditional on injury results in significant total mortality risk, particularly for females resting from a recent calving event. Large uncertainty in the estimation of carcass recovery rate for vessel strike deaths permeated into the apportionment of mortality causes. The increased rates of North Atlantic right whale mortality in the last decade, particularly for reproducing females, has been responsible for the severe decline in the species. By apportioning the human‐caused threats using a quantitative approach with estimation of relevant uncertainty, this work can guide development of conservation and management strategies to facilitate species recovery. Our approach is relevant to other monitored populations where cause‐specific injuries from multiple threats can be observed in live and dead individuals.

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, encompasses steatosis and metabolic dysfunction-associated steatohepatitis (MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASLD research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male) and ranked them using an unbiased MASLD 'human proximity score' to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western diets align closely with human MASH; high cholesterol content, extended study duration and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with the selection of appropriate in vivo models to accelerate preclinical research.

Objective :Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern with no effective and specific drug treatment available. The rs2642438 minor allele in mitochondrial amidoxime-reducing component 1 (MARC1) results in an aminoacidic substitution (p.Ala165Thr) and associates with protection against MASLD. However, the mechanisms behind this protective effect are unknown. In this study, we examined the consequences of this aminoacidic substitution on protein stability and subcellular localization. Methods :We overexpressed the human MARC1 A165 (wild-type) or 165T (mutant) in vivo in mice and in vitro in human hepatoma cells (HepG2 and HuH-7), generated several mutants at position 165 by in situ mutagenesis and then examined protein levels. We also generated HepG2 cells stably overexpressing MARC1 A165 or 165T to test the effect of this substitution on MARC1 subcellular localization. Results :MARC1 165T overexpression resulted in lower protein levels than A165 both in vivo and in vitro. Similarly, any mutant at position 165 showed lower protein levels compared to the wild-type protein. We showed that the 165T mutant protein is polyubiquitinated and its degradation is accelerated through lysine-48 ubiquitin-mediated proteasomal degradation. We also showed that the 165T substitution does not affect the MARC1 subcellular localization. Conclusions :This study shows that alanine at position 165 in MARC1 is crucial for protein stability, and the threonine substitution at this position leads to a hypomorphic protein variant due to lower protein levels. Our result supports the notion that lowering hepatic MARC1 protein level may be a successful therapeutic strategy for treating MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is caused by metabolic triggers and genetic predisposition. Among the genetic MASLD risk variants identified today, the common PNPLA3 148M variant exerts the largest effect size of MASLD heritability. The PNPLA3 148M protein is causatively linked to the development of liver steatosis, inflammation and fibrosis in experimental studies and is therefore an appealing target for therapeutic approaches to treat this disease. Several PNPLA3 targeted approaches are currently being evaluated in clinical trials for the treatment of metabolic dysfunction-associated steatohepatitis (MASH), the most severe form of MASLD and promising proof of principle data with reduced liver fat content in homozygous PNPLA3 148M risk allele carriers has been reported from phase 1 trials following hepatic silencing of PNPLA3 . Thus, targeting PNPLA3, the strongest genetic determinant of MASH may hold promise as the first precision medicine for the treatment of this disease. A histological endpoint-based phase 2b study has been initiated and several more are expected to be initiated to evaluate treatment effects on histological MASH and liver fibrosis in participants being homozygous for the PNPLA3 148M risk allele variant. The scope of this mini-review is to briefly describe the PNPLA3 148M genetics, function and preclinical experimental evidence with therapeutic approaches targeting PNPLA3 as well as to summarise the PNPLA3 based therapies currently in clinical development.

The I148M variant of PNPLA3 is closely associated with hepatic steatosis. Recent evidence indicates that the I148M mutant functions as an inhibitor of PNPLA2/ATGL-mediated lipolysis, leaving the role of wild-type PNPLA3 undefined. Despite showing a triglyceride hydrolase activity in vitro, PNPLA3 has yet to be established as a lipase in vivo. Here, we show that PNPLA3 preferentially hydrolyzes polyunsaturated triglycerides, mobilizing polyunsaturated fatty acids for phospholipid desaturation and enhancing hepatic secretion of triglyceride-rich lipoproteins. Under lipogenic conditions, mice with liver-specific knockout or acute knockdown of PNPLA3 exhibit aggravated liver steatosis and reduced plasma VLDL-triglyceride levels. Similarly, I148M-knockin mice show decreased hepatic triglyceride secretion during lipogenic stimulation. Our results highlight a specific context whereby the wild-type PNPLA3 facilitates the balance between hepatic triglyceride storage and secretion, and suggest the potential contribution of a loss-of-function by the I148M variant to the development of fatty liver disease in humans.

Abstract Advances in tagging technologies are expanding opportunities to estimate survival of fish and wildlife populations. Yet, capture and handling effects could impact survival outcomes and bias inference about natural mortality processes. We developed a multistage time-to-event model that can partition the survival process into sequential phases that reflect the tagged animal experience, including handling and release mortality, post-release recovery mortality, and subsequently, natural mortality. We demonstrate performance of multistage survival models through simulation testing and through fish and bird telemetry case studies. Models are implemented in a Bayesian framework and can accommodate left, right, and interval censorship events. Our results indicate that accurate survival estimates can be achieved with reasonable sample sizes ( $$n\approx 100+)$$ n ≈ 100 + ) and that multimodel inference can inform hypotheses about the configuration and length of survival stages needed to adequately describe mortality processes for tracked specimens. While we focus on survival estimation for tagged fish and wildlife populations, multistage time-to-event models could be used to understand other phenomena of interest such as migration, reproduction, or disease events across a range of taxa including plants and insects.

Effective conservation of threatened populations requires identification of the processes limiting recovery. When multiple population processes are potentially limiting, they are sometimes analyzed independently, often using different datasets. Analytically, this is suboptimal, as processes are often correlated, which can lead to biased estimates of parameters and quantities of interest. Integrated population models (IPMs) can synthesize several data streams in the same probabilistic framework to circumvent these issues. Lack of prey was identified as one of the primary threats to recovery of critically endangered southern resident killer whales (SRKWs), Orcinus orca. Previous studies have correlated SRKW demographic rates with indices of Chinook salmon Oncorhynchus tshawytscha abundance, but these approaches have modeled reproduction and survival independently. We developed an IPM for SRKWs that models both processes simultaneously, as a function of Chinook salmon abundance along the west coast of North America. We used this model to evaluate the relationship between Chinook salmon abundance and demographic rates of SRKWs, with the goal of updating our understanding of a potential causal relationship between prey availability and SRKW population dynamics, and how these relationships may have changed over time. Results suggest that SRKW mortality rates are more strongly associated with Chinook salmon abundance than birth rates. Our analysis also suggests northern resident killer whale abundance could also be affecting SRKW carrying capacity, possibly through competition over shared prey resources. The IPM fit observed total abundance data well and predicted temporal changes in population demographics with reasonable accuracy, but small sample size may have opacified model selection. Our approach offers a valuable tool for predicting the response of the SRKW population to alternative management strategies involving the recovery of Chinook salmon stocks in the eastern Pacific.

BACKGROUND/AIMS:Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global epidemic. The disease has a strong genetic component, and a common missense variant (rs2642438) in the mitochondrial amidoxime-reducing component 1 (MARC1) gene confers protection against its onset and severity. However, there are contrasting results regarding the mechanisms that promote this protection. METHODS:We downregulated MARC1 in primary human hepatocytes (PHHs) using short interfering RNA (siRNA). We measured neutral lipid content by Oil-Red O staining and fatty acid oxidation by radiolabeled tracers. We also performed RNA-sequencing and proteomic analysis using LC-MS. Additionally, we analyzed data from 239,075 participants from the UK Biobank. RESULTS:Downregulation of MARC1 reduced neutral lipid content in PHHs homozygous for the wild type (p.A165, risk), but not for the mutant (p.T165, protective), allele. We found that this reduction was mediated by increased fatty acid utilization via β-oxidation. Consistent with these results, we found that the levels of 3-hydroxybutyrate, a by-product of β-oxidation, were higher in carriers of the rs2642438 minor allele among samples from the UK biobank, indicating higher β-oxidation in these individuals. Moreover, downregulation of the MARC1 p.A165 variant resulted in a more favorable phenotype by reducing ferroptosis and reactive oxygen species levels. CONCLUSION:MARC1 downregulation in carriers of the risk allele results in lower hepatocyte neutral lipids content due to higher β-oxidation, while upregulating beneficial pathways involved in cell survival.

The I148M variant of PNPLA3 is strongly linked to hepatic steatosis. Evidence suggests a gain-of-function role for the I148M mutant as an ATGL inhibitor, leaving the physiological relevance of wild-type PNPLA3 undefined. Here we show that PNPLA3 selectively degrades triglycerides (TGs) enriched in polyunsaturated fatty acids (PUFAs) independently of ATGL in cultured cells and mice. Lipidomics and metabolite tracing analyses demonstrated that PNPLA3 mobilizes PUFAs from intracellular TGs for phospholipid desaturation, supporting hepatic secretion of TG-rich lipoproteins. Consequently, mice with liver-specific knockout or acute knockdown of PNPLA3 both exhibited aggravated liver steatosis and concomitant decreases in plasma VLDL-TG, phenotypes that manifest only under lipogenic conditions. I148M-knockin mice similarly displayed impaired hepatic TG secretion during lipogenic stimulation. Our results highlight a specific context whereby PNPLA3 facilitates the balance between hepatic TG storage and secretion and suggest the potential contributions of I148M variant loss-of-function to the development of hepatic steatosis in humans. Summary Statement:We define the physiological role of wild type PNPLA3 in maintaining hepatic VLDL-TG secretion.

The identification of genetic variants associated with fatty liver disease (FLD) from genome-wide association studies started in 2008 when single nucleotide polymorphisms in PNPLA3, the gene encoding patatin-like phospholipase domain-containing 3, were found to be associated with altered hepatic fat content. Since then, several genetic variants associated with protection from, or an increased risk of, FLD have been identified. The identification of these variants has provided insight into the metabolic pathways that cause FLD and enabled the identification of potential therapeutic targets. In this mini-review, we will examine the therapeutic opportunities derived from genetically validated targets in FLD, including oligonucleotide-based therapies targeting PNPLA3 and HSD17B13 that are currently being evaluated in clinical trials for the treatment of NASH (non-alcoholic steatohepatitis).

The various debates around model selection paradigms are important, but in lieu of a consensus, there is a demonstrable need for a deeper appreciation of existing approaches, at least among the end-users of statistics and model selection tools. In the ecological literature, the Akaike information criterion (AIC) dominates model selection practices, and while it is a relatively straightforward concept, there exists what we perceive to be some common misunderstandings around its application. Two specific questions arise with surprising regularity among colleagues and students when interpreting and reporting AIC model tables. The first is related to the issue of 'pretending' variables, and specifically a muddled understanding of what this means. The second is related to p-values and what constitutes statistical support when using AIC. There exists a wealth of technical literature describing AIC and the relationship between p-values and AIC differences. Here, we complement this technical treatment and use simulation to develop some intuition around these important concepts. In doing so we aim to promote better statistical practices when it comes to using, interpreting and reporting models selected when using AIC.

Understanding the causes of mortality for a declining species is essential for developing effective conservation and management strategies, particularly when anthropogenic activities are the primary threat. Using a competing hazards framework allows for robust estimation of the cause-specific variation that may exist across multiple dimensions, such as time and individual. Here, we estimated cause-specific rates of severe injury and mortality for North Atlantic right whales ( Eubalaena glacialis ), a critically endangered species that is currently in peril due to human-caused interactions. We developed a multistate capture-recapture model that leveraged 30 years of intensive survey effort yielding sightings of individuals with injury assessments and necropsies of carcass recoveries. We examined variation in the hazard rates of severe injury and mortality due to entanglements in fishing gear and vessel strikes as explained by year and the age and reproductive status of the individual. We found strong evidence for increased rates of severe entanglement injuries after 2013 and for females with calves, with consequently higher marginal mortality. The model results also suggested that despite vessel strikes causing a lower average rate of severe injuries, the higher mortality rate conditional on injury results in significant total mortality risk, particularly for females resting from a recent calving event. Large uncertainty in the estimation of carcass recovery rate for vessel strike deaths permeated into the apportionment of mortality causes. The increased rates of North Atlantic right whale mortality in the last decade, particularly for reproducing females, puts the species at risk of severe decline. By apportioning the human-caused threats using a quantitative approach with estimation of relevant uncertainty, this work can guide development of conservation and management strategies to facilitate species recovery.