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The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapies before moving into the human clinical setting. In this study, we report the generation of iPS cells from equine fibroblasts using a piggyBac (PB) transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally regulated fashion. The established iPS cell lines express hallmark pluripotency markers, display a stable karyotype even during long-term culture, and readily form complex teratomas containing all three embryonic germ layer derived tissues upon in vivo grafting into immunocompromised mice. Our EiPS cell lines hold the promise to enable the development of a whole new range of stem cell-based regenerative therapies in veterinary medicine, as well as aid the development of preclinical models for human applications. EiPS cell could also potentially be used to revive recently extinct or currently threatened equine species. [ABSTRACT FROM AUTHOR]

To test whether chronic enhanced blood flow alters insulin-stimulated glucose uptake. we measured skeletal muscle glucose uptake in chow-fed and high-fat-fed mice injected with adenovirus containing modified angiopoietin-1. COMP-Ang1. via euglycemic-hyperinsulinemic clamp. Blood flow rates and platelet endothelial cell adhesion molecule-1 positive endothelial cells in the hindlimb skeletal muscle were elevated in COMP-Ang1 compared with control LacZ. Whole body glucose uptake and whole body glycogen/lipid synthesis were elevated in COMP-Ang1 compared with LacZ in chow diet. High fat diet significantly reduced whole body glucose uptake and whole body glycolysis in LacZ mice. whereas high-fat-fed COMP-Ang1 showed a level of whole body glucose uptake that was comparable with chow-fed LacZ and showed increased glucose uptake compared with high-fat-fed LacZ. Glucose uptake and glycolysis in gastrocnemius muscle of chow-fed COMP-Ang1 mere increased compared with chow-fed LacZ. High-fat diet-induced whole body insulin resistance in the LacZ was mostly due to ∼40% decrease in insulin-stimulated glucose uptake in skeletal muscle. In contrast, COMP-Ang1 prevented diet-induced skeletal muscle insulin resistance compared with high-fat-fed LacZ. Akt phosphorylation in skeletal muscle was increased in COMP-Ang1 compared with LacZ in both chow-fed and high-fat-fed groups. These results suggest that increased blood flow by COMP-Ang1 increases insulin-stimulated glucose uptake and prevents high fat diet induced insulin resistance in skeletal muscle. [ABSTRACT FROM AUTHOR]

Abstract: Radiation therapy is a widely used cancer treatment, but it is unable to completely block cancer metastasis. The lymphatic vasculature serves as the primary route for metastatic spread, but little is known about how lymphatic endothelial cells respond to radiation. Here, we show that lymphatic endothelial cells in the small intestine and peri-tumor areas are highly resistant to radiation injury, while blood vessel endothelial cells in the small intestine are relatively sensitive. Our results suggest the need for alternative therapeutic modalities that can block lymphatic endothelial cell survival, and thus disrupt the integrity of lymphatic vessels in peri-tumor areas. [Copyright &y& Elsevier]

Lower oxidative capacity in skeletal muscles (SKMs) is a prevailing cause of metabolic diseases. Exercise not only enhances the fatty acid oxidation (FAO) capacity of SKMs but also increases lactate levels. Given that lactate may contribute to tricarboxylic acid cycle (TCA) flux and impact monocarboxylate transporter 1 in the SKMs, we hypothesize that lactate can influence glucose and fatty acid (FA) metabolism. To test this hypothesis, we investigated the mechanism underlying lactate-driven FAO regulation in the SKM of mice with diet-induced obesity (DIO). Lactate was administered to DIO mice immediately after exercise for over 3 wk. We found that increased lactate levels enhanced energy expenditure mediated by fat metabolism during exercise recovery and decreased triglyceride levels in DIO mice SKMs. To determine the lactate-specific effects without exercise, we administered lactate to mice on a high-fat diet (HFD) for 8 wk. Similar to our exercise conditions, lactate increased FAO, TCA cycle activity, and mitochondrial respiration in the SKMs of HFD-fed mice. In addition, under sufficient FA conditions, lactate increased uncoupling protein-3 abundance via the NADH-NAD+ shuttle. Conversely, ATP synthase abundance decreased in the SKMs of HFD mice. Taken together, our results suggest that lactate amplifies the adaptive increase in FAO capacity mediated by the TCA cycle and mitochondrial respiration in SKMs under sufficient FA abundance. NEW & NOTEWORTHY: Lactate administration post-exercise promotes triglyceride content loss in skeletal muscles (SKMs) and reduced body weight. Lactate enhances fatty acid oxidation in the SKMs of high-fat diet (HFD)-fed mice due to enhanced mitochondrial oxygen consumption. In addition, lactate restores the malate-aspartate shuttle, which is reduced by a HFD, and activates the tricarboxylic acid cycle (TCA) cycle in SKMs. Interestingly, supraphysiological lactate facilitates uncoupling protein-3 expression through NADH/NAD+, which is enhanced under high-fat levels in SKMs. [ABSTRACT FROM AUTHOR]

• Increasing body adiposity by diet lowers cerebral cortex volume in mice. • Higher body adiposity associates with lower cortical volume in humans. • Microglia and excitatory neurons are dysregulated in the obese mouse cortex. • Neuron-adverse dysregulation is dominated by reduced neuritogenesis and signalling. • Microglial changes in gene expression are similar in obese mice and Alzheimer's disease. Obesity is a major modifiable risk factor for Alzheimer's disease (AD), characterized by progressive atrophy of the cerebral cortex. The neurobiology of obesity contributions to AD is poorly understood. Here we show with in vivo MRI that diet-induced obesity decreases cortical volume in mice, and that higher body adiposity associates with lower cortical volume in humans. Single-nuclei transcriptomics of the mouse cortex reveals that dietary obesity promotes an array of neuron-adverse transcriptional dysregulations, which are mediated by an interplay of excitatory neurons and glial cells, and which involve microglial activation and lowered neuronal capacity for neuritogenesis and maintenance of membrane potential. The transcriptional dysregulations of microglia, more than of other cell types, are like those in AD, as assessed with single-nuclei cortical transcriptomics in a mouse model of AD and two sets of human donors with the disease. Serial two-photon tomography of microglia demonstrates microgliosis throughout the mouse cortex. The spatial pattern of adiposity-cortical volume associations in human cohorts interrogated together with in silico bulk and single-nucleus transcriptomic data from the human cortex implicated microglia (along with other glial cells and subtypes of excitatory neurons), and it correlated positively with the spatial profile of cortical atrophy in patients with mild cognitive impairment and AD. Thus, multi-cell neuron-adverse dysregulations likely contribute to the loss of cortical tissue in obesity. The dysregulations of microglia may be pivotal to the obesity-related risk of AD. [ABSTRACT FROM AUTHOR]

Forkhead box O1 (FOXO1) regulates muscle growth, but the metabolic role of FOXO1 in skeletal muscle and its mechanisms remain unclear. To explore the metabolic role of FOXO1 in skeletal muscle, we generated skeletal muscle–specific Foxo1 inducible knockout (mFOXO1 iKO) mice and fed them a high-fat diet to induce obesity. We measured insulin sensitivity, fatty acid oxidation, mitochondrial function, and exercise capacity in obese mFOXO1 iKO mice and assessed the correlation between FOXO1 and mitochondria-related protein in the skeletal muscle of patients with diabetes. Obese mFOXO1 iKO mice exhibited improved mitochondrial respiratory capacity, which was followed by attenuated insulin resistance, enhanced fatty acid oxidation, and improved skeletal muscle exercise capacity. Transcriptional inhibition of FOXO1 in peroxisome proliferator–activated receptor δ (PPARδ) expression was confirmed in skeletal muscle, and deletion of PPARδ abolished the beneficial effects of FOXO1 deficiency. FOXO1 protein levels were higher in the skeletal muscle of patients with diabetes and negatively correlated with PPARδ and electron transport chain protein levels. These findings highlight FOXO1 as a new repressor in PPARδ gene expression in skeletal muscle and suggest that FOXO1 links insulin resistance and mitochondrial dysfunction in skeletal muscle via PPARδ. Article Highlights: Skeletal muscle FOXO1 is upregulated in metabolically unhealthy conditions, such as obesity and aging. We generated skeletal muscle–specific inducible Foxo1 -deficient mice to clarify the metabolic role of FOXO1 in skeletal muscle. FOXO1 deficiency improved mitochondrial dysfunction by increasing peroxisome proliferator–activated receptor δ (PPARδ) transcription, leading to improved insulin resistance and lipid metabolism in the skeletal muscle of obese mice. This study shows that FOXO1 is a new repressor in PPARδ gene expression in skeletal muscle and links insulin resistance and mitochondrial dysfunction in skeletal muscle via PPARδ. [ABSTRACT FROM AUTHOR]

Obesity is strongly and independently associated with an increased risk of severe illness and death from coronavirus disease 2019 (COVID-19). The pathophysiological changes that result from elevated body weight lead to metabolic dysfunction, chronic inflammation, impaired immunological responses, and multisystem disorders, which increase vulnerability to severe illness from COVID-19. While vaccination strategies are under way across the world, the second and third waves of the pandemic, along with the emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains, continue to threaten the stability of medical systems worldwide. Furthermore, evidence from previous pandemics suggests that vaccines are less effective in obese individuals than in their healthy-weight counterparts over the long term. Therefore, a consideration of lifestyle changes that can boost metabolic health and immunity is critical to reduce the risk of complications and severe illness from viral infection. In this review, we discuss the potential mechanisms linking excess body weight with COVID-19 morbidity. We also present evidence that intermittent fasting (IF), a dietary program that has gained popularity in recent years, may be an effective strategy to improve metabolic health and immunity and thus reduce the impact of obesity on COVID-19 morbidity and mortality. Obesity is a strong risk factor for severe illness and mortality from coronavirus disease 2019 (COVID-19) infection. Mechanisms linking obesity with severe COVID-19 include diabetes-associated hyperglycemia, inflammation, weakened immune function, and metabolic dysfunction. Obese populations have a known history of poor response to vaccination and it is unknown whether this will also affect their vaccine-induced immunity to COVID-19. Therefore, it is important to implement dietary and lifestyle changes that potentially boost metabolic and immune health to mitigate the impacts of COVID-19. Intermittent fasting (IF) is associated with weight loss, improved glucose homeostasis, metabolic health restoration, and strengthened immune responses. Amid COVID-19 lockdowns, which are associated with more-sedentary lifestyles, the incorporation of IF may be a practical way to curb unhealthy eating habits, maximize healthy lifestyles, and improve mood and emotional well-being. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons, leading to muscle weakness, paralysis, and eventual death. Research from the past few decades has appreciated that ALS is not only a disease of the motor neurons but also a disease that involves systemic metabolic dysfunction. This review will examine the foundational research of understanding metabolic dysfunction in ALS and provide an overview of past and current studies in ALS patients and animal models, spanning from full systems to various metabolic organs. While ALS-affected muscle tissue exhibits elevated energy demand and a fuel preference switch from glycolysis to fatty acid oxidation, adipose tissue in ALS undergoes increased lipolysis. Dysfunctions in the liver and pancreas contribute to impaired glucose homeostasis and insulin secretion. The central nervous system (CNS) displays abnormal glucose regulation, mitochondrial dysfunction, and increased oxidative stress. Importantly, the hypothalamus, a brain region that controls whole-body metabolism, undergoes atrophy associated with pathological aggregates of TDP-43. This review will also cover past and present treatment options that target metabolic dysfunction in ALS and provide insights into the future of metabolism research in ALS. [ABSTRACT FROM AUTHOR]

Multipotent mesenchymal stromal cells (MSCs) are more and more frequently used to treat orthopedic injuries in horses. However, these cells are limited in their expandability and differentiation capacity. Recently, the first equine-induced pluripotent stem cell (iPSC) lines were reported by us []. In vitro differentiation of iPSCs into MSC-like cells is an attractive alternative to using MSCs derived from other sources, as a much larger quantity of patient-specific cells with broad differentiation potential could be generated. However, the differentiation capacity of iPSCs to MSCs and the potential for use in tissue engineering have yet to be explored. In this study, equine iPSCs were induced to differentiate into an MSC-like population. Upon induction, the iPSCs changed morphology toward spindle-shaped cells similar to MSCs. The ensuing iPSC-MSCs exhibited downregulation of pluripotency-associated genes and an upregulation of MSC-associated genes. In addition, the cells expressed the same surface markers as MSCs derived from equine umbilical cord blood. We then assessed the multilineage differentiation potential of iPSC-MSCs. Although chondrogenesis was not achieved after induction with transforming growth factor-beta 3 (TGFβ3) and/or bone morphogenic protein 4 (BMP-4) in 3D pellet culture, mineralization characteristic of osteogenesis and lipid droplet accumulation characteristic of adipogenesis were observed after chemical induction. We demonstrate a protocol for the derivation of MSC-like progenitor populations from equine iPS cells. [ABSTRACT FROM AUTHOR]

The circadian clock regulates metabolism in anticipation of regular changes in the environment. It is found throughout the body, including in key metabolic organs such as the liver, adipose tissues, and intestine, where the timing of the clock is set largely by nutrient signaling. However, the circadian clocks of these tissues during the fasted state have not been completely characterized. Moreover, the sufficiency of a functioning host clock to produce diurnal rhythms in the composition of the microbiome in fasted animals has not been explored. To this end, mice were fasted 24 h prior to collection of key metabolic tissues and fecal samples for the analysis of circadian clock gene expression and microbiome composition. Rhythm characteristics were determined using CircaCompare software. We identify tissue‐specific changes to circadian clock rhythms upon fasting, particularly in the brown adipose tissue, and for the first time demonstrate the rhythmicity of the microbiome in fasted animals. [ABSTRACT FROM AUTHOR]

Senescent cells accumulate in various tissues over time and contribute to tissue dysfunction and aging-associated phenotypes. Accumulating evidence suggests that cellular senescence can be inhibited through pharmacological intervention, as well as through treatment with soluble factors derived from embryonic stem cells (ESCs). In an attempt to investigate the anti-senescence factors secreted by ESCs, we analyzed mouse ESC-derived extracellular microRNAs in conditioned medium via microRNA array analysis. We selected mmu-miR-291a-3p as a putative anti-senescence factor via bioinformatics analysis. We validated its inhibitory effects on replicative, Adriamycin-induced, and ionizing radiation-induced senescence in human dermal fibroblasts. Treatment of senescent cells with mmu-miR-291a-3p decreased senescence-associated β-galactosidase activity, enhanced proliferative potential, and reduced mRNA and protein expression of TGF-β receptor 2, p53, and p21. mmu-miR-291a-3p in conditioned medium was enclosed in ESC-derived exosomes and exosomes purified from ESC conditioned medium inhibited cellular senescence. The inhibitory effects of mmu-miR-291a-3p were mediated through the TGF-β receptor 2 signaling pathway. Hsa-miR-371a-3p and hsa-miR-520e, the human homologs of mmu-miR-291a-3p, showed similar anti-senescence activity. Furthermore, mmu-miR-291a-3p accelerated the excisional skin wound healing process in aged mice. Our results indicate that the ESC-derived mmu-miR-291a-3p is a novel candidate agent that can be utilized for cell-free therapeutic intervention against aging and aging-related diseases. [ABSTRACT FROM AUTHOR]

Female reproductive capacity declines dramatically in the fourth decade of life as a result of an age-related decrease in oocyte quality and quantity. The primary causes of reproductive aging and the molecular factors responsible for decreased oocyte quality remain elusive. Here, we show that aging of the female germ line is accompanied by mitochondrial dysfunction associated with decreased oxidative phosphorylation and reduced Adenosine tri-phosphate ( ATP) level. Diminished expression of the enzymes responsible for CoQ production, Pdss2 and Coq6, was observed in oocytes of older females in both mouse and human. The age-related decline in oocyte quality and quantity could be reversed by the administration of CoQ10. Oocyte-specific disruption of Pdss2 recapitulated many of the mitochondrial and reproductive phenotypes observed in the old females including reduced ATP production and increased meiotic spindle abnormalities, resulting in infertility. Ovarian reserve in the oocyte-specific Pdss2-deficient animals was diminished, leading to premature ovarian failure which could be prevented by maternal dietary administration of CoQ10. We conclude that impaired mitochondrial performance created by suboptimal CoQ10 availability can drive age-associated oocyte deficits causing infertility. [ABSTRACT FROM AUTHOR]

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition. [ABSTRACT FROM AUTHOR]

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators. Alternative splicing represents a widely acting mode of gene regulation, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming. [ABSTRACT FROM AUTHOR]

Summary: Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior. [ABSTRACT FROM AUTHOR]

Background: Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI) for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells. Methods: According to this system, a “docking-site” was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an “incoming” vector containing the gene of interest was specifically inserted in the docking-site using PhiC31. Results: Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate format. Conclusions: The novel PhiC31-IMSI system described in this study represents a powerful tool that can facilitate the characterization of cancer-related genes. [ABSTRACT FROM AUTHOR]

Summary: Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs. [ABSTRACT FROM AUTHOR]

Summary: Two vascular growth factor families, VEGF and the angiopoietins, play critical and coordinate roles in tumor progression and metastasis. A single inhibitor targeting both VEGF and angiopoietins is not available. Here, we developed a chimeric decoy receptor, namely double anti-angiogenic protein (DAAP), which can simultaneously bind VEGF-A and angiopoietins, blocking their actions. Compared to VEGF-Trap or Tie2-Fc, which block either VEGF-A or angiopoietins alone, we believe DAAP is a highly effective molecule for regressing tumor angiogenesis and metastasis in implanted and spontaneous solid tumors; it can also effectively reduce ascites formation and vascular leakage in an ovarian carcinoma model. Thus, simultaneous blockade of VEGF-A and angiopoietins with DAAP is an effective therapeutic strategy for blocking tumor angiogenesis, metastasis, and vascular leakage. [ABSTRACT FROM AUTHOR]

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral, lentiviral, adenoviral and plasmid transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision, we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies. [ABSTRACT FROM AUTHOR]

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology. [Display omitted] • VEGFa/KDR suppresses mucous differentiation of club cells during regeneration • VEGFa/KDR signaling executes its epithelial function through MEK/ERK kinases • Sox9 mediates mucous metaplasia of the airway epithelium upon VEGFa/KDR inhibition • VEGFa/KDR/MEK blockage is linked to mucous metaplasia in asthma and cystic fibrosis Jiang et al. demonstrate that the VEGFa/KDR pathway protects the airway epithelium against mucous metaplasia during regeneration. Suppressed VEGF signaling leads to increased levels of SOX9, which promotes goblet cell differentiation. These findings introduce critical players in airway mucous metaplasia that is commonly seen in asthma and CF. [ABSTRACT FROM AUTHOR]

Intermittent fasting (IF) is an effective dietary intervention to counteract obesity-associated metabolic abnormalities. Previously, we and others have highlighted white adipose tissue (WAT) browning as the main underlying mechanism of IF-mediated metabolic benefits. However, whether IF retains its efficacy in different models, such as genetically obese/diabetic animals, is unknown. Here, leptin-deficient ob/ob mice were subjected to 16 weeks of isocaloric IF, and comprehensive metabolic phenotyping was conducted to assess the metabolic effects of IF. Unlike our previous study, isocaloric IF-subjected ob/ob animals failed to exhibit reduced body weight gain, lower fat mass, or decreased liver lipid accumulation. Moreover, isocaloric IF did not result in increased thermogenesis nor induce WAT browning in ob/ob mice. These findings indicate that isocaloric IF may not be an effective approach for regulating body weight in ob/ob animals, posing the possible limitations of IF to treat obesity. However, despite the lack of improvement in insulin sensitivity, isocaloric IF-subjected ob/ob animals displayed improved glucose tolerance as well as higher postprandial insulin level, with elevated incretin expression, suggesting that isocaloric IF is effective in improving nutrient-stimulated insulin secretion. Together, this study uncovers the insulinotropic effect of isocaloric IF, independent of adipose thermogenesis, which is potentially complementary for the treatment of type 2 diabetes. [ABSTRACT FROM AUTHOR]