Systemic deletion of DMD exon 51 rescues clinically severe Duchenne muscular dystrophy in a pig model lacking DMD exon 52.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal musculature and myocardium. In DMD patients and in a corresponding pig model with a deletion of DMD exon 52 (DMDΔ52), expression of an internally shortened dystrophin can be achieved by skipping of DMD exon 51 to reframe the transcript. To predict the best possible outcome of this strategy, we generated DMDΔ51-52 pigs, additionally representing a model for Becker muscular dystrophy (BMD). DMDΔ51-52 skeletal muscle and myocardium samples stained positive for dystrophin and did not show the characteristic dystrophic alterations observed in DMDΔ52 pigs. Western blot analysis confirmed the presence of dystrophin in the skeletal muscle and myocardium of DMDΔ51-52 pigs and its absence in DMDΔ52 pigs. The proteome profile of skeletal muscle, which showed a large number of abundance alterations in DMDΔ52 vs. wild-type (WT) samples, was normalized in DMDΔ51-52 samples. Cardiac function at age 3.5 mo was significantly reduced in DMDΔ52 pigs (mean left ventricular ejection fraction 58.8% vs. 70.3% in WT) but completely rescued in DMDΔ51-52 pigs (72.3%), in line with normalization of the myocardial proteome profile. Our findings indicate that ubiquitous deletion of DMD exon 51 in DMDΔ52 pigs largely rescues the rapidly progressing, severe muscular dystrophy and the reduced cardiac function of this model. Long-term follow-up studies of DMDΔ51-52 pigs will show if they develop symptoms of the milder BMD.

Proteomics- and Metabolomics-Based Analysis of Metabolic Changes in a Swine Model of Pulmonary Hypertension.

Pulmonary vein stenosis (PVS) causes a rare type of pulmonary hypertension (PH) by impacting the flow and pressure within the pulmonary vasculature, resulting in endothelial dysfunction and metabolic changes. A prudent line of treatment in this type of PH would be targeted therapy to relieve the pressure and reverse the flow-related changes. We used a swine model in order to mimic PH after PVS using pulmonary vein banding (PVB) of the lower lobes for 12 weeks to mimic the hemodynamic profile associated with PH and investigated the molecular alterations that provide an impetus for the development of PH. Our current study aimed to employ unbiased proteomic and metabolomic analyses on both the upper and lower lobes of the swine lung to identify regions with metabolic alterations. We detected changes in the upper lobes for the PVB animals mainly pertaining to fatty acid metabolism, reactive oxygen species (ROS) signaling and extracellular matrix (ECM) remodeling and small, albeit, significant changes in the lower lobes for purine metabolism.

Multi-omics analysis of diabetic pig lungs reveals molecular derangements underlying pulmonary complications of diabetes mellitus.

Growing evidence shows that the lung is an organ prone to injury by diabetes mellitus. However, the molecular mechanisms of these pulmonary complications have not yet been characterized comprehensively. To systematically study the effects of insulin deficiency and hyperglycaemia on the lung, we combined proteomics and lipidomics with quantitative histomorphological analyses to compare lung tissue samples from a clinically relevant pig model for mutant INS gene-induced diabetes of youth (MIDY) with samples from wild-type littermate controls. Among others, the level of pulmonary surfactant-associated protein A (SFTPA1), a biomarker of lung injury, was moderately elevated. Furthermore, key proteins related to humoral immune response and extracellular matrix organization were significantly altered in abundance. Importantly, a lipoxygenase pathway was dysregulated as indicated by 2.5-fold reduction of polyunsaturated fatty acid lipoxygenase ALOX15 levels, associated with corresponding changes in the levels of lipids influenced by this enzyme. Our multi-omics study points to an involvement of reduced ALOX15 levels and an associated lack of eicosanoid switching as mechanisms contributing to a proinflammatory milieu in the lungs of subjects with diabetes mellitus.

Low-dose Bisphenol A exposure alters the functionality and cellular environment in a human cardiomyocyte model.

Early embryonic development represents a sensitive time-window during which the foetus might be vulnerable to the exposure of environmental contaminants, potentially leading to heart diseases also later in life. Bisphenol A (BPA), a synthetic chemical widely used in plastics manufacturing, has been associated with heart developmental defects, even in low concentrations. This study aims to investigate the effects of environmentally relevant doses of BPA on developing cardiomyocytes using a human induced pluripotent stem cell (hiPSC)-derived model. Firstly, a 2D in vitro differentiation system to obtain cardiomyocytes from hiPSCs (hiPSC-CMs) have been established and characterised to provide a suitable model for the early stages of cardiac development. Then, the effects of a repeated BPA exposure, starting from the undifferentiated stage throughout the differentiation process, were evaluated. The chemical significantly decreased the beat rate of hiPSC-CMs, extending the contraction and relaxation time in a dose-dependent manner. Quantitative proteomics analysis revealed a high abundance of basement membrane (BM) components (e.g., COL4A1, COL4A2, LAMC1, NID2) and a significant increase in TNNC1 and SERBP1 proteins in hiPSC-CMs treated with BPA. Network analysis of proteomics data supported altered extracellular matrix remodelling and provided a disease-gene association with well-known pathological conditions of the heart. Furthermore, upon hypoxia-reoxygenation challenge, hiPSC-CMs treated with BPA showed higher rate of apoptotic events. Taken together, our results revealed that a long-term treatment, even with low doses of BPA, interferes with hiPSC-CMs functionality and alters the surrounding cellular environment, providing new insights about diseases that might arise upon the toxin exposure. Our study contributes to the current understanding of BPA effects on developing human foetal cardiomyocytes, in correlation with human clinical observations and animal studies, and it provides a suitable model for New Approach Methodologies (NAMs) for environmental chemical hazard and risk assessment.

Structural and proteomic repercussions of growth hormone receptor deficiency on the pituitary gland: Lessons from a translational pig model.

Growth hormone receptor deficiency (GHRD) results in low serum insulin-like growth factor 1 (IGF1) and high, but non-functional serum growth hormone (GH) levels in human Laron syndrome (LS) patients and animal models. This study investigated the quantitative histomorphological and molecular alterations associated with GHRD. Pituitary glands from 6 months old growth hormone receptor deficient (GHR-KO) and control pigs were analyzed using a quantitative histomorphological approach in paraffin (9 GHR-KO [5 males, 4 females] vs. 11 controls [5 males, 6 females]), ultrathin sections tissue sections (3 male GHR-KO vs. 3 male controls) and label-free proteomics (4 GHR-KO vs. 4 control pigs [2 per sex]). GHR-KO pigs displayed reduced body weights (60% reduction in comparison to controls; p < .0001) and decreased pituitary volumes (54% reduction in comparison to controls; p < .0001). The volume proportion of the adenohypophysis did not differ in GHR-KO and control pituitaries (65% vs. 71%; p = .0506) and GHR-KO adenohypophyses displayed a reduced absolute volume but an unaltered volume density of somatotrophs in comparison to controls (21% vs. 18%; p = .3164). In GHR-KO pigs, somatotroph cells displayed a significantly reduced volume density of granules (23.5%) as compared to controls (67.7%; p < .0001). Holistic proteome analysis of adenohypophysis samples identified 4660 proteins, of which 592 were differentially abundant between the GHR-KO and control groups. In GHR-KO samples, the abundance of somatotropin precursor was decreased, whereas increased abundances of proteins involved in protein production, transport and endoplasmic reticulum (ER) stress were revealed. Increased protein production and secretion as well as significantly reduced proportion of GH-storing granules in somatotroph cells of the adenohypophysis without an increase in volume density of somatotroph cells in the adenohypophysis could explain elevated serum GH levels in GHR-KO pigs.

Proteomics and disease network associations evaluation of environmentally relevant Bisphenol A concentrations in a human 3D neural stem cell model.

Bisphenol A (BPA) exposure is associated with a plethora of neurodevelopmental abnormalities and brain disorders. Previous studies have demonstrated BPA-induced perturbations to critical neural stem cell (NSC) characteristics, such as proliferation and differentiation, although the underlying molecular mechanisms remain under debate. The present study evaluated the effects of a repeated-dose exposure of environmentally relevant BPA concentrations during the in vitro 3D neural induction of human induced pluripotent stem cells (hiPSCs), emulating a chronic exposure scenario. Firstly, we demonstrated that our model is suitable for NSC differentiation during the early stages of embryonic brain development. Our morphological image analysis showed that BPA exposure at 0.01, 0.1 and 1 µM decreased the average spheroid size by day 21 (D21) of the neural induction, while no effect on cell viability was detected. No alteration to the rate of the neural induction was observed based on the expression of key neural lineage and neuroectodermal transcripts. Quantitative proteomics at D21 revealed several differentially abundant proteins across all BPA-treated groups with important functions in NSC proliferation and maintenance (e.g., FABP7, GPC4, GAP43, Wnt-8B, TPPP3). Additionally, a network analysis demonstrated alterations to the glycolytic pathway, potentially implicating BPA-induced changes to glycolytic signalling in NSC proliferation impairments, as well as the pathophysiology of brain disorders including intellectual disability, autism spectrum disorders, and amyotrophic lateral sclerosis (ALS). This study enhances the current understanding of BPA-related NSC aberrations based mostly on acute, often high dose exposures of rodent in vivo and in vitro models and human GWAS data in a novel human 3D cell-based model with real-life scenario relevant prolonged and low-level exposures, offering further mechanistic insights into the ramifications of BPA exposure on the developing human brain and consequently, later life neurological disorders.

Maternal hyperglycemia induces alterations in hepatic amino acid, glucose and lipid metabolism of neonatal offspring: Multi-omics insights from a diabetic pig model.

OBJECTIVE: To gain mechanistic insights into adverse effects of maternal hyperglycemia on the liver of neonates, we performed a multi-omics analysis of liver tissue from piglets developed in genetically diabetic (mutant INS gene induced diabetes of youth; MIDY) or wild-type (WT) pigs. METHODS: Proteome, metabolome and lipidome profiles of liver and clinical parameters of serum samples from 3-day-old WT piglets (n = 9) born to MIDY mothers (PHG) were compared with those of WT piglets (n = 10) born to normoglycemic mothers (PNG). Furthermore, protein-protein interaction network analysis was used to reveal highly interacting proteins that participate in the same molecular mechanisms and to relate these mechanisms with human pathology. RESULTS: Hepatocytes of PHG displayed pronounced lipid droplet accumulation, although the abundances of central lipogenic enzymes such as fatty acid-synthase (FASN) were decreased. Additionally, circulating triglyceride (TG) levels were reduced as a trend. Serum levels of non-esterified free fatty acids (NEFA) were elevated in PHG, potentially stimulating hepatic gluconeogenesis. This is supported by elevated hepatic phosphoenolpyruvate carboxykinase (PCK1) and circulating alanine transaminase (ALT) levels. Even though targeted metabolomics showed strongly elevated phosphatidylcholine (PC) levels, the abundances of multiple key enzymes involved in major PC synthesis pathways - most prominently those from the Kennedy pathway - were paradoxically reduced in PHG liver. Conversely, enzymes involved in PC excretion and breakdown such as PC-specific translocase ATP-binding cassette 4 (ABCB4) and phospholipase A2 were increased in abundance. CONCLUSIONS: Our study indicates that maternal hyperglycemia without confounding obesity induces profound molecular changes in the liver of neonatal offspring. In particular, we found evidence for stimulated gluconeogenesis and hepatic lipid accumulation independent of de novo lipogenesis. Reduced levels of PC biosynthesis enzymes and increased levels of proteins involved in PC translocation or breakdown may represent counter-regulatory mechanisms to maternally elevated PC levels. Our comprehensive multi-omics dataset provides a valuable resource for future meta-analysis studies focusing on liver metabolism in newborns from diabetic mothers.

Pig models for Duchenne muscular dystrophy - from disease mechanisms to validation of new diagnostic and therapeutic concepts.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal muscles and heart. Animal models are essential for preclinical evaluation of novel diagnostic procedures and treatment strategies. Gene targeting/editing offers the possibility of developing tailored pig models for monogenic diseases. The first porcine DMD model was generated by deletion of DMD exon 52 (DMDΔ52) in cultured kidney cells, which were used for somatic cell nuclear transfer to produce DMDΔ52 offspring. The animals resembled clinical, biochemical, and pathological hallmarks of DMD, but died before sexual maturity, thus preventing their propagation by breeding. This limitation was overcome by the generation of female heterozygous DMDΔ52 carrier pigs, which allowed the establishment of a large breeding colony. In this overview, we summarize how porcine DMD models have been used for dissecting disease mechanisms, for validating multispectral optoacoustic tomography as an imaging modality for monitoring fibrosis, and for preclinical testing of a CRISPR/Cas9 based approach to restore an intact DMD reading frame. Particular advantages of porcine DMD models include their targeted design and the rapid disease progression with early cardiac involvement, facilitating translational studies in reasonable time frames.

Sensitized photo-oxidation of plant cytokinin-specific binding protein - Does the environment of the thioether group influence the oxidation reaction? From primary intermediates to stable products.

The reactions of protein oxidation play a significant role in many biological processes, especially in diseases development. Therefore, it is important to understand, how the protein molecule behaves in the presence of oxidants. In the present work, photo-oxidation of phytohormone-binding plant protein (VrPhBP) was investigated using light and 3-carboxybenzophenone (3CB) as a sensitizer (one electron oxidant). The protein interacts with the sensitizer in the ground state forming a weak binding complex leading to the presence of bound and free 3CB in solution. The early events and transient species (such as radicals and radical ions) formed during irradiation were characterised by transient spectroscopy showing the formation of the sulphur radical cation Met>S●+ (stabilized by (S∴N)+)and the tyrosyl radical TyrO● on VrPhBP. Thus the 3CB excited triplet state was quenched by the Met and Tyr residues and mostly by Met (based on the deconvoluted transient absorption spectra).The presence of a Tyr side chain in the vicinity of a Met residue results in intramolecular electron transfer from Tyr to the Met>S●+ radical cation, leading to regeneration of the thioether side chain and formation of TyrO●. The presence of other side chains close to Met, such as Arg or Lys can induce the stabilization of Met>S●+ via the formation of two-centered three-electron bonded species (S∴N)+. The transient species were additionally confirmed by stable product analysis. Based on SDS-PAGE, chromatography and mass spectrometry, the formation of methionine sulphoxide and Met-3CB adduct was identified together with di-Tyr cross links. On the basis of the experimental results the overall mechanism of VrPhBP photo-oxidation, from its early events to the formation of stable products, is described. In addition, a good correlation between the mechanisms of photooxidation of model compounds such as Met derivatives and peptides and those for real biological systems is emphasized.

Developmental Effects of (Pre-)Gestational Diabetes on Offspring: Systematic Screening Using Omics Approaches.

Worldwide, gestational diabetes affects 2-25% of pregnancies. Due to related disturbances of the maternal metabolism during the periconceptional period and pregnancy, children bear an increased risk for future diseases. It is well known that an aberrant intrauterine environment caused by elevated maternal glucose levels is related to elevated risks for increased birth weights and metabolic disorders in later life, such as obesity or type 2 diabetes. The complexity of disturbances induced by maternal diabetes, with multiple underlying mechanisms, makes early diagnosis or prevention a challenging task. Omics technologies allowing holistic quantification of several classes of molecules from biological fluids, cells, or tissues are powerful tools to systematically investigate the effects of maternal diabetes on the offspring in an unbiased manner. Differentially abundant molecules or distinct molecular profiles may serve as diagnostic biomarkers, which may also support the development of preventive and therapeutic strategies. In this review, we summarize key findings from state-of-the-art Omics studies addressing the impact of maternal diabetes on offspring health.

Differential Effects of Insulin-Deficient Diabetes Mellitus on Visceral vs. Subcutaneous Adipose Tissue-Multi-omics Insights From the Munich MIDY Pig Model.

Adipose tissue (AT) is no longer considered to be responsible for energy storage only but is now recognized as a major endocrine organ that is distributed across different parts of the body and is actively involved in regulatory processes controlling energy homeostasis. Moreover, AT plays a crucial role in the development of metabolic disease such as diabetes. Recent evidence has shown that adipokines have the ability to regulate blood glucose levels and improve metabolic homeostasis. While AT has been studied extensively in the context of type 2 diabetes, less is known about how different AT types are affected by absolute insulin deficiency in type 1 or permanent neonatal diabetes mellitus. Here, we analyzed visceral and subcutaneous AT in a diabetic, insulin-deficient pig model (MIDY) and wild-type (WT) littermate controls by RNA sequencing and quantitative proteomics. Multi-omics analysis indicates a depot-specific dysregulation of crucial metabolic pathways in MIDY AT samples. We identified key proteins involved in glucose uptake and downstream signaling, lipogenesis, lipolysis and ß-oxidation to be differentially regulated between visceral and subcutaneous AT in response to insulin deficiency. Proteins related to glycogenolysis, pyruvate metabolism, TCA cycle and lipogenesis were increased in subcutaneous AT, whereas ß-oxidation-related proteins were increased in visceral AT from MIDY pigs, pointing at a regionally different metabolic adaptation to master energy stress arising from diminished glucose utilization in MIDY AT. Chronic, absolute insulin deficiency and hyperglycemia revealed fat depot-specific signatures using multi-omics analysis. The generated datasets are a valuable resource for further comparative and translational studies in clinical diabetes research.

A scalable, clinically severe pig model for Duchenne muscular dystrophy.

Large-animal models for Duchenne muscular dystrophy (DMD) are crucial for the evaluation of diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 (DMDΔ52) exhibit molecular, clinical and pathological hallmarks of DMD, but die before sexual maturity and cannot be propagated by breeding. Therefore, we generated female DMD+/- carriers. A single founder animal had 11 litters with 29 DMDY/-, 34 DMD+/- as well as 36 male and 29 female wild-type offspring. Breeding with F1 and F2 DMD+/- carriers resulted in an additional 114 DMDY/- piglets. With intensive neonatal management, the majority survived for 3-4 months, providing statistically relevant cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance to human disease mechanisms. Importantly, DMDY/- pigs displayed progressive myocardial fibrosis and increased expression of connexin-43, associated with significantly reduced left ventricular ejection fraction, at 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability. Our breeding cohort of DMDΔ52 pigs and standardized tissue repositories provide important resources for studying DMD disease mechanisms and for testing novel treatment strategies.