Weight loss interventions, including dietary changes, pharmacotherapy, or bariatric surgery, prevent many of the adverse consequences of obesity, and may also confer intervention-specific benefits beyond those seen with decreased weight alone. We compared the molecular effects of different interventions on liver metabolism to understand the mechanisms underlying these benefits. Male rats on a high-fat, high-sucrose diet underwent sleeve gastrectomy (SG) or intermittent fasting with caloric restriction (IF-CR), achieving equivalent weight loss. The interventions were compared to (AL)-fed controls. Analysis of liver and blood metabolome and transcriptome revealed distinct and sometimes contrasting metabolic effects between the two interventions. SG primarily influenced one-carbon metabolic pathways, whereas IF-CR increased lipogenesis and glycogen storage. These findings suggest that the unique metabolic pathways affected by SG and IF-CR contribute to their distinct clinical benefits, with bariatric surgery potentially influencing long-lasting changes through its effect on one-carbon metabolism.

Cystic kidney disease is a leading cause of morbidity in patients with tuberous sclerosis complex (TSC). We characterize the misregulated metabolic pathways using cell lines, a TSC mouse model, and human kidney sections. Our study reveals a substantial perturbation in the arginine biosynthesis pathway in TSC models with overexpression of argininosuccinate synthetase 1 (ASS1). The rise in ASS1 expression is dependent on the mechanistic target of rapamycin complex 1 (mTORC1) activity. Arginine depletion prevents mTORC1 hyperactivation and cell cycle progression and averts cystogenic signaling overexpression of c-Myc and P65. Accordingly, an arginine-depleted diet substantially reduces the TSC cystic load in mice, indicating the potential therapeutic effects of arginine deprivation for the treatment of TSC-associated kidney disease.

In Type 2 diabetes (T2D), elevated lipid levels have been suggested to contribute to insulin resistance and β-cell dysfunction. We previously reported that the expression of the PGE2 receptor EP3 is elevated in islets of T2D individuals and is preferentially stimulated by palmitate, leading to β-cell failure. The mouse EP3 receptor generates three isoforms by alternative splicing which differ in their C-terminal domain and are referred to as mEP3α, mEP3β, and mEP3γ. We bring evidence that the expression of the mEP3γ isoform is elevated in islets of diabetic db/db mice and is selectively upregulated by palmitate. Specific knockdown of the mEP3γ isoform restores the expression of β-cell-specific genes and rescues MIN6 cells from palmitate-induced dysfunction and apoptosis. This study indicates that palmitate stimulates the expression of the mEP3γ by a posttranscriptional mechanism, compared to the other spliced isoforms, and that the de novo synthesized ceramide plays an important role in FFA-induced mEP3γ expression in β-cells. Moreover, induced levels of mEP3γ mRNA by palmitate or ceramide depend on p38 MAPK activation. Our findings suggest that mEP3γ gene expression is regulated at the posttranscriptional level and defines the EP3 signaling axis as an important pathway mediating β-cell-impaired function and demise.

T-cell acute lymphoblastic leukemia (T-ALL) protein 1 (TAL1) is a central transcription factor in hematopoiesis. The timing and level of TAL1 expression orchestrate the differentiation to specialized blood cells and its overexpression is a common cause of T-ALL. Here, we studied the 2 protein isoforms of TAL1, short and long, which are generated by the use of alternative promoters as well as by alternative splicing. We analyzed the expression of each isoform by deleting an enhancer or insulator, or by opening chromatin at the enhancer location. Our results show that each enhancer promotes expression from a specific TAL1 promoter. Expression from a specific promoter gives rise to a unique 5' UTR with differential regulation of translation. Moreover, our study suggests that the enhancers regulate TAL1 exon 3 alternative splicing by inducing changes in the chromatin at the splice site, which we demonstrate is mediated by KMT2B. Furthermore, our results indicate that TAL1-short binds more strongly to TAL1 E-protein partners and functions as a stronger transcription factor than TAL1-long. Specifically TAL1-short has a unique transcription signature promoting apoptosis. Finally, when we expressed both isoforms in mice bone marrow, we found that while overexpression of both isoforms prevents lymphoid differentiation, expression of TAL-short alone leads to hematopoietic stem cell exhaustion. Furthermore, we found that TAL1-short promoted erythropoiesis and reduced cell survival in the CML cell line K562. While TAL1 and its partners are considered promising therapeutic targets in the treatment of T-ALL, our results show that TAL1-short could act as a tumor suppressor and suggest that altering TAL1 isoform's ratio could be a preferred therapeutic approach.

Microbial adaptation to changing environmental conditions is frequently mediated by hypermutable sequences. Here we demonstrate that such a hypermutable hotspot within a gene encoding a flagellar unit of generated spontaneous non-swarming mutants with increased stress resistance. These mutants, which survived conditions that eliminated wild-type cultures, could be carried by their swarming siblings when the colony spread, consequently increasing their numbers at the spreading edge. Of interest, the hypermutable nature of the aforementioned sequence enabled the non-swarming mutants to serve as "seeds" for a new generation of wild-type cells through reversion of the mutation. Using a mathematical model, we examined the survival dynamics of colonies under fluctuating environments. Our experimental and theoretical results suggest that the non-swarming, stress-resistant mutants can save the colony from extinction. Notably, we identified this hypermutable sequence in flagellar genes of additional species, suggesting that this phenomenon could be wide-spread and ecologically important.

Systemic light chain amyloidosis (AL) is a clonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin light chains (LC) as insoluble fibrils in organs. The lack of suitable models has hindered the investigation of the disease mechanisms. Our aim was to establish AL producing PC lines and to use them to investigate the biology of the amyloidogenic clone. We used lentiviral vectors to generate cell lines expressing LCs from patients suffering from AL amyloidosis. The AL LC producing cell lines showed a significant decrease in proliferation, cell cycle arrest, and an increase in apoptosis and autophagy as compared with the multiple myeloma (MM) LC producing cells. Using RNAsequencing the AL LC producing lines showed higher mitochondrial oxidative stress, decreased activity of the myc and cholesterol pathways. The neoplastic behavior of PCs is altered by the constitutive expression of amyloidogenic LC causing intracellular toxicity. This observation may explain the disparity in the malignant behavior of the amyloid clone compared to the myeloma clone. These findings should enable future in vitro studies and help delineate AL's unique cellular pathways, thus expediting the development of specific treatments for AL patients.

Thermal intolerance may limit activity in hostile environments. After heat illness, two physiologically distinct phenotypes evolve: heat tolerant (HT) and heat intolerant (HI). The recognition that heat illness alters gene expression justified revisiting the established physiological concept of HI. We used a DNA microarray to examine the global transcriptional response in peripheral blood mononuclear cells (PMBCs) from HI and HT phenotypes, categorized 2-mo postheat injury using a functional physiological heat-tolerance test (HTT, 40°C)-Recovery (R, 24°C) protocol. The impact of recurrent heat stress was studied in vitro using peripheral blood mononuclear cells (PBMCs) from controls (participants with no history of heat injury), HI, and HT (categorized by functional HTT) with a customized NanoString array. There were significant differences under basal conditions between the HI and HT. HI were more immunological alerted. Almost no shared genes were found between end-HTT and recovery phases, suggesting vast cellular plasticity. In HI, mitochondrial function was dysregulated, canonical pathways associated with exercise endurance-NRF2 and insulin were downregulated, whereas AMPK and peroxisome proliferator-activated receptor (PPAR) were upregulated. HT exhibited reciprocal responses, suggesting that energy dysregulation found in HI interfered with performance in the heat. The endoplasmic-reticulum stress response was also suppressed in HI. In vitro HTT (43°C) abolished differences between HI and HT PBMCs including the HSPs genes, whereas controls showed profound HSPs upregulation.

Universal Minicircle Sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind the single-stranded G-rich UMS sequence, conserved at the replication origins of minicircles in the kinetoplast DNA, the mitochondrial genome of kinetoplastids. Trypanosoma brucei UMSBP2 has been recently shown to colocalize with telomeres and to play an essential role in chromosome end protection. Here we report that TbUMSBP2 decondenses in vitro DNA molecules, which were condensed by core histones H2B, H4 or linker histone H1. DNA decondensation is mediated via protein-protein interactions between TbUMSBP2 and these histones, independently of its previously described DNA binding activity. Silencing of the TbUMSBP2 gene resulted in a significant decrease in the disassembly of nucleosomes in T. brucei chromatin, a phenotype that could be reverted, by supplementing the knockdown cells with TbUMSBP2. Transcriptome analysis revealed that silencing of TbUMSBP2 affects the expression of multiple genes in T. brucei, with a most significant effect on the upregulation of the subtelomeric variant surface glycoproteins (VSG) genes, which mediate the antigenic variation in African trypanosomes. These observations suggest that UMSBP2 is a chromatin remodeling protein that functions in the regulation of gene expression and plays a role in the control of antigenic variation in T. brucei.

Obstructive sleep apnea (OSA) is a highly prevalent condition, characterized by intermittent hypoxia (IH), sleep disruption, and altered autonomic nervous system function. OSA has been independently associated with dyslipidemia, insulin resistance, and metabolic syndrome. Brown adipose tissue (BAT) has been suggested as a modulator of systemic glucose tolerance through adaptive thermogenesis. Reductions in BAT mass have been associated with obesity and metabolic syndrome. No studies have systematically characterized the effects of chronic IH on BAT. Thus, we aimed to delineate IH effects on BAT and concomitant metabolic changes. C57BL/6J 8-week-old male mice were randomly assigned to IH during sleep (alternating 90 s cycles of 6.5% FO followed by 21% FO) or normoxia (room air, RA) for 10 weeks. Mice were subjected to glucose tolerance testing and F-FDG PET-MRI towards the end of the exposures followed by BAT tissues analyses for morphological and global transcriptomic changes. Animals exposed to IH were glucose intolerant despite lower total body weight and adiposity. BAT tissues in IH-exposed mice demonstrated characteristic changes associated with "browning"-smaller lipids, increased vascularity, and a trend towards higher protein levels of UCP1. Conversely, mitochondrial DNA content and protein levels of respiratory chain complex III were reduced. Pro-inflammatory macrophages were more abundant in IH-exposed BAT. Transcriptomic analysis revealed increases in fatty acid oxidation and oxidative stress pathways in IH-exposed BAT, along with a reduction in pathways related to myogenesis, hypoxia, and IL-4 anti-inflammatory response. Functionally, IH-exposed BAT demonstrated reduced absorption of glucose on PET scans and reduced phosphorylation of AKT in response to insulin. Current studies provide initial evidence for the presence of a maladaptive response of interscapular BAT in response to chronic IH mimicking OSA, resulting in a paradoxical divergence, namely, BAT browning but tissue-specific and systemic insulin resistance. We postulate that oxidative stress, mitochondrial dysfunction, and inflammation may underlie these dichotomous outcomes in BAT.

Injury to muscle brings about the activation of stem cells, which then generate new myocytes to replace damaged tissue. We demonstrate that this activation is accompanied by a dramatic change in the stem-cell methylation pattern that prepares them epigenetically for terminal myocyte differentiation. These de- and de novo methylation events occur at regulatory elements associated with genes involved in myogenesis and are necessary for activation and regeneration. Local injury of one muscle elicits an almost identical epigenetic change in satellite cells from other muscles in the body, in a process mediated by circulating factors. Furthermore, this same methylation state is also generated in muscle stem cells (MuSCs) of female animals following pregnancy, even in the absence of any injury. Unlike the activation-induced expression changes, which are transient, the induced methylation profile is stably maintained in resident MuSCs and thus represents a molecular memory of previous physiological events that is probably programmed to provide a mechanism for long-term adaptation.

Biological sex is a fundamental trait influencing development, reproduction, pathogenesis, and medical treatment outcomes. Modeling sex differences is challenging because of the masking effect of genetic variability and the hurdle of differentiating chromosomal versus hormonal effects. In this work we developed a cellular model to study sex differences in humans. Somatic cells from a mosaic Klinefelter syndrome patient were reprogrammed to generate isogenic induced pluripotent stem cell (iPSC) lines with different sex chromosome complements: 47,XXY/46,XX/46,XY/45,X0. Transcriptional analysis of the hiPSCs revealed novel and known genes and pathways that are sexually dimorphic in the pluripotent state and during early neural development. Female hiPSCs more closely resembled the naive pluripotent state than their male counterparts. Moreover, the system enabled differentiation between the contributions of X versus Y chromosome to these differences. Taken together, isogenic hiPSCs present a novel platform for studying sex differences in humans and bear potential to promote gender-specific medicine in the future.

GNE Myopathy is a rare, recessively inherited neuromuscular worldwide disorder, caused by a spectrum of bi-allelic mutations in the human GNE gene. GNE encodes a bi-functional enzyme responsible for the rate-limiting step of sialic acid biosynthesis pathway. However, the process in which GNE mutations lead to the development of a muscle pathology is not clear yet. Cellular and mouse models for GNE Myopathy established to date have not been informative. Further, additional GNE functions in muscle have been hypothesized. In these studies, we aimed to investigate gne functions using zebrafish genetic and transgenic models, and characterized them using macroscopic, microscopic, and molecular approaches. We first established transgenic zebrafish lineages expressing the human GNE cDNA carrying the M743T mutation, driven by the zebrafish gne promoter. These fish developed entirely normally. Then, we generated a gne knocked-out (KO) fish using the CRISPR/Cas9 methodology. These fish died 8-10 days post-fertilization (dpf), but a phenotype appeared less than 24 h before death and included progressive body axis curving, deflation of the swim bladder and decreasing movement and heart rate. However, muscle histology uncovered severe defects, already at 5 dpf, with compromised fiber organization. Sialic acid supplementation did not rescue the larvae from this phenotype nor prolonged their lifespan. To have deeper insights into the potential functions of gne in zebrafish, RNA sequencing was performed at 3 time points (3, 5, and 7 dpf). Genotype clustering was progressive, with only 5 genes differentially expressed in gne KO compared to gne WT siblings at 3 dpf. Enrichment analyses of the primary processes affected by the lack of gne also at 5 and 7 dpf point to the involvement of cell cycle and DNA damage/repair processes in the gne KO zebrafish. Thus, we have established a gne KO zebrafish lineage and obtained new insights into gne functions. This is the only model where GNE can be related to clear muscle defects, thus the only animal model relevant to GNE Myopathy to date. Further elucidation of gne precise mechanism-of-action in these processes could be relevant to GNE Myopathy and allow the identification of novel therapeutic targets.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been employed in the past decade as therapeutic agents in various diseases, including central nervous system (CNS) disorders. We currently aimed to use MSC-EVs as potential treatment for cerebral small vessel disease (CSVD), a complex disorder with a variety of manifestations. MSC-EVs were intranasally administrated to salt-sensitive hypertension prone SBH/y rats that were DOCA-salt loaded (SBH/y-DS), which we have previously shown is a model of CSVD. MSC-EVs accumulated within brain lesion sites of SBH/y-DS. An in vitro model of an inflammatory environment in the brain demonstrated anti-inflammatory properties of MSC-EVs. Following in vivo MSC-EV treatment, gene set enrichment analysis (GSEA) of SBH/y-DS cortices revealed downregulation of immune system response-related gene sets. In addition, MSC-EVs downregulated gene sets related to apoptosis, wound healing and coagulation, and upregulated gene sets associated with synaptic signaling and cognition. While no specific gene was markedly altered upon treatment, the synergistic effect of all gene alternations was sufficient to increase animal survival and improve the neurological state of affected SBH/y-DS rats. Our data suggest MSC-EVs act as microenvironment modulators, through various molecular pathways. We conclude that MSC-EVs may serve as beneficial therapeutic measure for multifactorial disorders, such as CSVD.

GNE myopathy is an adult onset neuromuscular disorder characterized by slowly progressive distal and proximal muscle weakness, caused by missense recessive mutations in the GNE gene. Although the encoded bifunctional enzyme is well known as the limiting factor in the biosynthesis of sialic acid, no clear mechanisms have been recognized to account for the muscle atrophic pathology, and novel functions for GNE have been hypothesized. Two major issues impair studies on this protein. First, the expression of the GNE protein is minimal in human and mice muscles and there is no reliable antibody to follow up endogenous expression. Second, no reliable animal model is available for the disease and cellular models from GNE myopathy patients' muscle cells (expressing the mutated protein) are less informative than expected. In order to broaden our knowledge on GNE functions in muscle, we have taken advantage of the CRISPR/Cas9 method for genome editing to first, add a tag to the endogenous Gne gene in mouse, allowing the determination of the spatiotemporal expression of the protein in the organism, using well established and reliable antibodies against the specific tag. In addition we have generated a Gne knock out murine muscle cell lineage to identify the events resulting from the total lack of the protein. A thorough multi-omics analysis of both cellular systems including transcriptomics, proteomics, phosphoproteomics and ubiquitination, unraveled novel pathways for Gne, in particular its involvement in cell cycle control and in the DNA damage/repair pathways. The elucidation of fundamental mechanisms of Gne in normal muscle may contribute to the identification of the disrupted functions in GNE myopathy, thus, to the definition of novel biomarkers and possible therapeutic targets for this disease.

To combat the various DNA lesions and their harmful effects, cells have evolved different strategies, collectively referred as DNA damage response (DDR). The DDR largely relies on intranuclear protein networks, which sense DNA lesions, recruit DNA repair enzymes, and coordinates several aspects of the cellular response, including a temporary cell cycle arrest. In addition, external cues mediated by the surface EGF receptor (EGFR) through downstream signaling pathways contribute to the cellular DNA repair capacity. However, cell cycle progression driven by EGFR activation should be reconciled with cell cycle arrest necessary for effective DNA repair. Here, we show that in damaged cells, the expression of Mig-6 (mitogen-inducible gene 6), a known regulator of EGFR signaling, is reduced resulting in heightened EGFR phosphorylation and downstream signaling. These changes in Mig-6 expression and EGFR signaling do not occur in cells deficient of Mre-11, a component of the MRN complex, playing a central role in double-strand break (DSB) repair or when cells are treated with the MRN inhibitor, mirin. RNAseq and functional analysis reveal that DNA damage induces a shift in cell response to EGFR triggering that potentiates DDR-induced p53 pathway and cell cycle arrest. These data demonstrate that the cellular response to EGFR triggering is skewed by components of the DDR, thus providing a plausible explanation for the paradox of the known role played by a growth factor such as EGFR in the DNA damage repair.