The Human Cardiac "Age-OME": Age-Specific Changes in Myocardial Molecular Expression.

Ageing is one of the most significant risk factors for heart disease; however, it is still not clear how the human heart changes with age. Taking advantage of a unique set of pre-mortem, cryopreserved, non-diseased human hearts, we performed omics analyses (transcriptomics, proteomics, metabolomics, and lipidomics), coupled with biologically informed computational modelling in younger (≤ 25 years old) and older hearts (≥ 50 years old) to describe the molecular landscape of human cardiac ageing. In older hearts, we observed a downregulation of proteins involved in calcium signalling and the contractile apparatus.

Left ventricular myocardial molecular profile of human diabetic ischaemic cardiomyopathy.

Ischaemic cardiomyopathy is the most common cause of heart failure and often coexists with diabetes mellitus, which worsens patient symptom burden and outcomes. Yet, their combined effects are seldom investigated and are poorly understood. To uncover the influencing molecular signature defining ischaemic cardiomyopathy with diabetes, we performed multi-omic analyses of ischaemic and non-ischaemic cardiomyopathy with and without diabetes against healthy age-matched donors.

The Efficacy of Risk Factor Modification Compared to NAD Repletion in Diastolic Heart Failure.

Heart failure (HF) with left ventricular diastolic dysfunction is a growing global concern. This study evaluated myocardial oxidized nicotinamide adenine dinucleotide (NAD) levels in human systolic and diastolic HF and in a murine model of HF with preserved ejection fraction, exploring NAD repletion as therapy. We quantified myocardial NAD and nicotinamide phosphoribosyltransferase levels, assessing restoration with nicotinamide riboside (NR).

Deletion of miPEP in adipocytes protects against obesity and insulin resistance by boosting muscle metabolism.

Unlabelled: Mitochondria facilitate thousands of biochemical reactions, covering a broad spectrum of anabolic and catabolic processes. Here we demonstrate that the adipocyte mitochondrial proteome is markedly altered across multiple models of insulin resistance and reveal a consistent decrease in the level of the mitochondrial processing peptidase miPEP.

Objective: To determine the role of miPEP in insulin resistance.

Hepatic lipid droplet-associated proteome changes distinguish dietary-induced fatty liver from glucose tolerance in male mice.

Fatty liver is characterized by the expansion of lipid droplets (LDs) and is associated with the development of many metabolic diseases. We assessed the morphology of hepatic LDs and performed quantitative proteomics in lean, glucose-tolerant mice compared with high-fat diet (HFD) fed mice that displayed hepatic steatosis and glucose intolerance as well as high-starch diet (HStD) fed mice who exhibited similar levels of hepatic steatosis but remained glucose tolerant. Both HFD- and HStD-fed mice had more and larger LDs than Chow-fed animals.

The major TMEM106B dementia risk allele affects TMEM106B protein levels, fibril formation, and myelin lipid homeostasis in the ageing human hippocampus.

Background: The risk for dementia increases exponentially from the seventh decade of life. Identifying and understanding the biochemical changes that sensitize the ageing brain to neurodegeneration will provide new opportunities for dementia prevention and treatment. This study aimed to determine how ageing and major genetic risk factors for dementia affect the hippocampal proteome and lipidome of neurologically-normal humans over the age of 65.

Dietary restriction induces a sexually dimorphic type I interferon response in mice with gene-environment interactions.

Intermittent fasting (IF) is an established intervention to treat the growing obesity epidemic. However, the interaction between dietary interventions and sex remains a significant knowledge gap. In this study, we use unbiased proteome analysis to identify diet-sex interactions.

The Small-Protein Enrichment Assay (SPEA) for Analysis of Low Abundance Peptide Hormones in Plasma.

The analysis of low abundance peptide hormones such as insulin in blood plasma is difficult with unbiased mass spectrometry-based proteomics, as they are overshadowed by very abundant proteins such as albumin and IgG. The small-protein enrichment assay (SPEA) can greatly increase detection and discovery of these factors through specific enrichment, which enables fast and efficient analysis of many small-protein factors using a single untargeted LC-MS/MS acquisition. SPEA uses an alcohol-acid-based dissociation and precipitation step, prior to denaturing SEC to remove the large highly abundant plasma proteins leaving only a small-protein fraction.

Anabolic Factors and Myokines Improve Differentiation of Human Embryonic Stem Cell Derived Skeletal Muscle Cells.

Skeletal muscle weakness is linked to many adverse health outcomes. Current research to identify new drugs has often been inconclusive due to lack of adequate cellular models. We previously developed a scalable monolayer system to differentiate human embryonic stem cells (hESCs) into mature skeletal muscle cells (SkMCs) within 26 days without cell sorting or genetic manipulation.

Annotated Protein Database Using Known Cleavage Sites for Rapid Detection of Secreted Proteins.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of secreted proteins has contributed to our understanding of human disease and physiology but is limited by its need for accurate protein database annotation. Common assumptions used in proteomics of perfect protease specificity are inaccurate for secreted proteins, which are cleaved by numerous endogenous proteases. Here, we describe the generation of an optimized protein database that divides proteins into their individual biological chains and peptides to allow fast identification of semi-tryptic peptides from secreted proteins using fully tryptic searches.

Proteomics analysis of adipose depots after intermittent fasting reveals visceral fat preservation mechanisms.

Intermittent fasting is a beneficial dietary treatment for obesity. But the response of each distinct adipose depot is currently poorly defined. Here we explore the response of key adipose depots to every-other-day fasting (EODF) in mice using proteomics.

Elucidating the mechanisms by which disulfiram protects against obesity and metabolic syndrome.

There is an unmet need and urgency to find safe and effective anti-obesity interventions. Our recent study in mice fed on obesogenic diet found that treatment with the alcohol aversive drug disulfiram reduced feeding efficiency and led to a decrease in body weight and an increase in energy expenditure. The intervention with disulfiram improved glucose tolerance and insulin sensitivity, and mitigated metabolic dysfunctions in various organs through poorly defined mechanisms.

Disulfiram Treatment Normalizes Body Weight in Obese Mice.

Obesity is a top public health concern, and a molecule that safely treats obesity is urgently needed. Disulfiram (known commercially as Antabuse), an FDA-approved treatment for chronic alcohol addiction, exhibits anti-inflammatory properties and helps protect against certain types of cancer. Here, we show that in mice disulfiram treatment prevented body weight gain and abrogated the adverse impact of an obesogenic diet on insulin responsiveness while mitigating liver steatosis and pancreatic islet hypertrophy.

Multi-omics Analysis of the Intermittent Fasting Response in Mice Identifies an Unexpected Role for HNF4α.

Every-other-day fasting (EODF) is an effective intervention for the treatment of metabolic disease, including improvements in liver health. But how the liver proteome is reprogrammed by EODF is currently unknown. Here, we use EODF in mice and multi-omics analysis to identify regulated pathways.

Human induced pluripotent stem cell-derived GABAergic interneuron transplants attenuate neuropathic pain.

Neuropathic pain causes severe suffering, and most patients are resistant to current therapies. A core element of neuropathic pain is the loss of inhibitory tone in the spinal cord. Previous studies have shown that foetal GABAergic neuron precursors can provide relief from pain.

Small-protein Enrichment Assay Enables the Rapid, Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma.

Unbiased and sensitive quantification of low abundance small proteins in human plasma ( hormones, immune factors, metabolic regulators) remains an unmet need. These small protein factors are typically analyzed individually and using antibodies that can lack specificity. Mass spectrometry (MS)-based proteomics has the potential to address these problems, however the analysis of plasma by MS is plagued by the extremely large dynamic range of this body fluid, with protein abundances spanning at least 13 orders of magnitude.

Proteomic Analysis of Human Plasma during Intermittent Fasting.

Intermittent fasting (IF) increases lifespan and decreases metabolic disease phenotypes and cancer risk in model organisms, but the health benefits of IF in humans are less clear. Human plasma derived from clinical trials is one of the most difficult sample sets to analyze using mass spectrometry-based proteomics due to the extensive sample preparation required and the need to process many samples to achieve statistical significance. Here, we describe an optimized and accessible device (Spin96) to accommodate up to 96 StageTips, a widely used sample preparation medium enabling efficient and consistent processing of samples prior to LC-MS/MS.

Chromatin Modifiers SET-25 and SET-32 Are Required for Establishment but Not Long-Term Maintenance of Transgenerational Epigenetic Inheritance.

Some epigenetic modifications are inherited from one generation to the next, providing a potential mechanism for the inheritance of environmentally acquired traits. Transgenerational inheritance of RNAi phenotypes in Caenorhabditis elegans provides an excellent model to study this phenomenon, and although studies have implicated both chromatin modifications and small RNA pathways in heritable silencing, their relative contributions remain unclear. Here, we demonstrate that the putative histone methyltransferases SET-25 and SET-32 are required for establishment of a transgenerational silencing signal but not for long-term maintenance of this signal between subsequent generations, suggesting that transgenerational epigenetic inheritance is a multi-step process with distinct genetic requirements for establishment and maintenance of heritable silencing.

Efficient analysis of mammalian polysomes in cells and tissues using Ribo Mega-SEC.

We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue.

Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation.

Mitochondrial oxidative stress, mitochondrial dysfunction, or both have been implicated in insulin resistance. However, disentangling the individual roles of these processes in insulin resistance has been difficult because they often occur in tandem, and tools that selectively increase oxidant production without impairing mitochondrial respiration have been lacking. Using the dimer/monomer status of peroxiredoxin isoforms as an indicator of compartmental hydrogen peroxide burden, we provide evidence that oxidative stress is localized to mitochondria in insulin-resistant 3T3-L1 adipocytes and adipose tissue from mice.