ARF1 maintains intestinal homeostasis by modulating gut microbiota and stem cell function.

AIMS: The small GTPase protein ARF1 has been shown to be involved in the lipolysis pathway and to selectively kill stem cells in Drosophila melanogaster. However, the role of ARF1 in mammalian intestinal homeostasis remains elusive. This study aimed to explore the role of ARF1 in intestinal epithelial cells (IECs) and reveal the possible mechanism. MATERIALS AND METHODS: IEC-specific ARF1 deletion mouse model was used to evaluate the role of ARF1 in intestine. Immunohistochemistry and immunofluorescence analyses were performed to detect specific cell type markers, and intestinal organoids were cultured to assess intestinal stem cell (ISC) proliferation and differentiation. Fluorescence in situ hybridization, 16S rRNA-seq analysis, and antibiotic treatments were conducted to elucidate the role of gut microbes in ARF1-mediated intestinal function and the underlying mechanism. Colitis was induced in control and ARF1-deficient mice by dextran sulfate sodium (DSS). RNA-seq was performed to elucidate the transcriptomic changes after ARF1 deletion. KEY FINDINGS: ARF1 was essential for ISC proliferation and differentiation. Loss of ARF1 increased susceptibility to DSS-induced colitis and gut microbial dysbiosis. Gut microbiota depletion by antibiotics could rescue the intestinal abnormalities to a certain extent. Furthermore, RNA-seq analysis revealed alterations in multiple metabolic pathways. SIGNIFICANCE: This work is the first to elucidate the essential role of ARF1 in regulating gut homeostasis, and provides novel insights into the pathogenesis of intestinal diseases and potential therapeutic targets.

An miRNA-mRNA integrative analysis in human placentas and mice: role of the Smad2/miR-155-5p axis in the development of fetal growth restriction.

Introduction: Functional disorder of the placenta is the principal cause of fetal growth restriction (FGR), usually cured with suitable clinical treatment and good nursing. However, some FGR mothers still give birth to small for gestational age (SGA) babies after treatment. The ineffectiveness of treatment in such a group of patients confused physicians of obstetrics and gynecology. Methods: In this study, we performed a microRNA-messenger RNA integrative analysis of gene expression profiles obtained from Gene Expression Omnibus. Differentially expressed genes were screened and checked using quantitative polymerase chain reaction. Target genes of significantly changed microRNA were screened and enriched for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Function of the obtained microRNA-messenger RNA was evaluated using HTR-8/SVneo trophoblast cells, human umbilical vein endothelial cells, and heterozygote male mice. Result: MiR-155-5p was upregulated (p = 0.001, fold-change = 2.275) in fetal-side placentals. Among the hub genes identified as key targets for miR-155-5p in fetal reprogramming, Smad2 was downregulated (p = 0.002, fold change = 0.426) and negatively correlated with miR-155-5p expression levels (r = -0.471, p < 1.0 E - 04) in fetal-side placental tissues. The miR-155-5p mimic blocks Smad2 expression and suppresses villous trophoblast cell and endothelial cell function (proliferation, migration, and invasion), indicating a close relationship with placental development. Luciferase assays further confirmed the targeting of miR-155-5p to Smad2. Furthermore, Smad2+/- heterozygote male mice were born small with low body weight (p = 0.0281) and fat composition (p = 0.013) in the fourth week post-natal. Discussion: We provide the first evidence of the role of the Smad2/miR-155-5p axis in the placental pathologies of FGR. Our findings elucidate the pathogenesis of FGR and provide new therapeutic targets.

Magnetic torque-driven living microrobots for increased tumor infiltration.

Biohybrid bacteria-based microrobots are increasingly recognized as promising externally controllable vehicles for targeted cancer therapy. Magnetic fields in particular have been used as a safe means to transfer energy and direct their motion. Thus far, the magnetic control strategies used in this context rely on poorly scalable magnetic field gradients, require active position feedback, or are ill-suited to diffuse distributions within the body. Here, we present a magnetic torque-driven control scheme for enhanced transport through biological barriers that complements the innate taxis toward tumor cores exhibited by a range of bacteria, shown for Magnetospirillum magneticum as a magnetically responsive model organism. This hybrid control strategy is readily scalable, independent of position feedback, and applicable to bacterial microrobots dispersed by the circulatory system. We observed a fourfold increase in translocation of magnetically responsive bacteria across a model of the vascular endothelium and found that the primary mechanism driving increased transport is torque-driven surface exploration at the cell interface. Using spheroids as a three-dimensional tumor model, fluorescently labeled bacteria colonized their core regions with up to 21-fold higher signal in samples exposed to rotating magnetic fields. In addition to enhanced transport, we demonstrated that our control scheme offers further advantages, including the possibility for closed-loop optimization based on inductive detection, as well as spatially selective actuation to reduce off-target effects. Last, after systemic intravenous injection in mice, we showed significantly increased bacterial tumor accumulation, supporting the feasibility of deploying this control scheme clinically for magnetically responsive biohybrid microrobots.

Disruption of the lipolysis pathway results in stem cell death through a sterile immunity-like pathway in adult Drosophila.

We previously showed that the Arf1-mediated lipolysis pathway sustains stem cells and cancer stem cells (CSCs); its ablation resulted in necrosis of stem cells and CSCs, which further triggers a systemic antitumor immune response. Here we show that knocking down Arf1 in intestinal stem cells (ISCs) causes metabolic stress, which promotes the expression and translocation of ISC-produced damage-associated molecular patterns (DAMPs; Pretaporter [Prtp] and calreticulin [Calr]). DAMPs regulate macroglobulin complement-related (Mcr) expression and secretion. The secreted Mcr influences the expression and localization of enterocyte (EC)-produced Draper (Drpr) and LRP1 receptors (pattern recognition receptors [PRRs]) to activate autophagy in ECs for ATP production. The secreted ATP possibly feeds back to kill ISCs by activating inflammasome-like pyroptosis. We identify an evolutionarily conserved pathway that sustains stem cells and CSCs, and its ablation results in an immunogenic cascade that promotes death of stem cells and CSCs as well as antitumor immunity.

Lmod3 promotes myoblast differentiation and proliferation via the AKT and ERK pathways.

Mutations in the Lmod3 gene have been identified as a genetic cause of nemaline myopathy. However, the mechanism underlying this disease and the function of Lmod3 remain largely unknown. In this study, we found that Lmod3 knockdown in C2C12 cells impaired myoblast differentiation, whereas enforced Lmod3 expression enhanced such differentiation. We also discovered that myoblast proliferation was promoted by Lmod3 overexpression but impeded by its knockdown. Additionally, knockdown of Lmod3 led to apoptosis in myoblasts. Concurrently, forced Lmod3 expression in C2C12 cells contributed to activation of the AKT and ERK pathways during myoblast differentiation and proliferation, respectively. Conversely, knockdown of Lmod3 in C2C12 cells produced the opposite results. Furthermore, administration of IGF-1, a booster of both AKT and ERK pathways, partially rescued the inhibitory effect of Lmod3 knockdown on both differentiation and proliferation of C2C12 cells. These results suggest that Lmod3 promotes differentiation and proliferation of myoblasts through the AKT and ERK pathways, respectively.

Undiagnosed type 2 diabetes during pregnancy is associated with increased perinatal mortality: a large population-based cohort study in Ontario, Canada.

AIM: To compare perinatal outcomes in women with undiagnosed diabetes with gestational diabetes alone, pre-existing diabetes and women without diabetes, and to identify risk factors which distinguish them from women with gestational diabetes alone. METHODS: This population-based cohort study included administrative data on all women who gave birth in Ontario, Canada, during 2002-2015. Maternal/neonatal outcomes were compared across groups using logistic regression, adjusting for confounders. A nested case control study compared women with undiagnosed type 2 diabetes with women with gestational diabetes alone to determine risk factors that would help identify these women. RESULTS: Among 995 990 women, 68 163 had gestational diabetes (6.8%) and, of those women with gestational diabetes,1772 had undiagnosed type 2 diabetes (2.6%). Those with undiagnosed type 2 diabetes were more likely to be older, from a lower income area, have parity > 3 and BMI ≥ 30 kg/m2 compared with gestational diabetes alone. Infants had a higher risk of perinatal mortality (OR 2.3 [1.6-3.4]), preterm birth (OR 2.6 [2.3-2.9]), congenital anomalies (OR 2.1 [1.7-2.5]), neonatal intensive care unit admission (OR 3.1 [2.8-3.5]) and neonatal hypoglycaemia (OR 406.0 [357-461]), which were similar to women with pre-existing diabetes. The strongest predictive risk factors included early gestational diabetes diagnosis, previous gestational diabetes and chronic hypertension. CONCLUSIONS: Women diagnosed with gestational diabetes who develop diabetes within 1 year postpartum are at higher risk of adverse pregnancy outcomes, including perinatal mortality. This highlights the need for earlier diagnosis, preferably pre-pregnancy, and more aggressive treatment and surveillance of suspected type 2 diabetes during pregnancy.

Effect of multiparity and ethnicity on the risk of development of diabetes: a large population-based cohort study.

AIMS: To investigate the relationship between increasing parity and diabetes in a large, population-based cohort, and to examine if this relationship is different among high-risk ethnic groups. METHODS: A population-based, retrospective cohort study was performed in 738 440 women aged 18-50 years, who delivered babies in Ontario between 1 April 2002 and 31 March 2011. Diabetes incidence postpartum was calculated for each parity and ethnic group. A multivariable analysis of the effect of parity and ethnicity on the incidence of diabetes was performed using a Cox proportional hazards model, adjusting for confounders. RESULTS: The diabetes incidence rate per 1000 person-years was 3.69 in women with 1 delivery, 4.12 in women with 3 deliveries and 7.62 in women with ≥5 deliveries. Women with ≥3 deliveries had a higher risk of developing diabetes compared with women with 1 delivery [adjusted hazard ratios 1.06 (95% CI 1.01-1.11) for 3 deliveries, 1.33 (95% CI 1.25-1.43) for 4 deliveries and 1.53 (95% CI 1.41-1.66) for ≥5 deliveries). A similar rise in risk could be seen in Chinese and South-Asian women, with the most influence in Chinese women [hazard ratio 4.59 (95% CI 2.36-8.92) for ≥5 deliveries]. CONCLUSIONS: There was a positive and graded relationship between increasing parity and risk of development of diabetes. The influence of parity was seen in all ethnicities. This association may be partly related to increasing weight gain and retention with increasing parity, or deterioration in ß-cell function. This merits further exploration.

Disruption of Gpr45 causes reduced hypothalamic POMC expression and obesity.

A rise in the occurrence of obesity has driven exploration of its underlying genetic basis and potential targets for intervention. GWAS studies have identified obesity susceptibility pathways involving several neuropeptides that control energy homeostasis, suggesting that variations in the genes that regulate food intake and energy expenditure may contribute to obesity. In this study, we identified 5 additional obesity loci, including a neuronal orphan GPCR called Gpr45, in a forward genetic screen of mutant mice generated by piggyBac insertional mutagenesis. Disruption of Gpr45 led to increased adiposity at the time of weaning and increases in body mass, fat content, glucose intolerance, and hepatic steatosis with advancing age. Mice with disruptions in Gpr45 also displayed a reduction in expression of the metabolic regulator POMC and less energy expenditure prior to the onset of obesity. Mechanistically, we determined that GPR45 regulates POMC expression via the JAK/STAT pathway in a cell-autonomous manner. Consistent with this finding, intraventricular administration of melanotan-2, an analog of the POMC derivative α-MSH, suppressed adult obesity in Gpr45 mutants. These results reveal that GPR45 is a regulator of POMC signaling and energy expenditure, which suggests that it may be a potential intervention target to combat obesity.

Lmod2 piggyBac mutant mice exhibit dilated cardiomyopathy.

BACKGROUND: Leiomodin proteins, Lmod1, Lmod2 and Lmod3, are key regulators of the thin filament length in muscles. While Lmod1 is specifically expressed in smooth muscles, both Lmod2 and Lmod3 are expressed in striated muscles including both cardiac and skeletal muscles. We and others have previously shown that Lmod3 mainly function in skeletal muscles and the mutant mice display disorganized sarcomere. Lmod2 protein has been found to act as an actin filament nucleator in both cell-free assays and in cultured rat and chicken cardiomyocytes. RESULTS: To better understand the function of Lmod2 in vivo, we have identified and characterized a piggyBac (PB) insertional mouse mutant. Our analysis revealed that the PB transposon inserts in the first exon of the Lmod2 gene and severely disrupts its expression. We found that Lmod2 (PB/PB) mice exhibit typical dilated cardiomyopathy (DCM) with ventricular arrhythmias and postnatal lethality. Electron microscope reveals that the Lmod2 (PB/PB) hearts carry disordered sarcomere, disarrayed thin filaments, and distorted intercalated discs (ICDs). Those ICDs display not only decreased convolutions, but also reduced electron-dense staining, indicating less ICDs component proteins in Lmod2 (PB/PB) hearts. Consistent with the phenotype, the expression of the ICD component genes, ß-catenin and Connexin43, are down-regulated. CONCLUSIONS: Taken together, our data reveal that Lmod2 is required in heart thin filaments for integrity of sarcomere and ICD and deficient mice exhibit DCM with ventricular arrhythmias and postnatal lethality. The Lmod2 (PB/PB) mutant offers a valuable resource for interrogation of pathogenesis and development of therapeutics for DCM.

The mechanism of increased biliary lipid secretion in mice with genetic inactivation of bile salt export pump.

Human bile salt export pump (BSEP) mutations underlie progressive familial intrahepatic cholestasis type 2 (PFIC2). In the PFIC2 animal model, Bsep(-/-) mice, biliary secretion of bile salts (BS) is decreased, but that of phospholipids (PL) and cholesterol (CH) is increased. Under physiological conditions, the biliary secretion of PL and CH is positively related ("coupled") to that of BS. We aimed to elucidate the mechanism of increased biliary lipid secretion in Bsep(-/-) mice. The secretion of the BS tauro-ß-muricholic acid (TßMCA) is relatively preserved in Bsep(-/-) mice. We infused Bsep(-/-) and Bsep(+/+) (control) mice with TßMCA in stepwise increasing dosages (150-600 nmol/min) and determined biliary bile flow, BS, PL, and CH secretion. mRNA and protein expression of relevant canalicular transporters was analyzed in livers from noninfused Bsep(-/-) and control mice. TßMCA infusion increased BS secretion in both Bsep(-/-) and control mice. The secreted PL or CH amount per BS, i.e., the "coupling," was continuously two- to threefold higher in Bsep(-/-) mice (P < 0.05). Hepatic mRNA expression of canalicular lipid transporters Mdr2, Abcg5, and Abcg8 was 45-55% higher in Bsep(-/-) mice (Abcg5; P < 0.05), as was canalicular Mdr2 and Abcg5 protein expression. Potential other explanations for the increased coupling of the biliary secretion of PL and CH to that of BS in Bsep(-/-) mice could be excluded. We conclude that the mechanism of increased biliary lipid secretion in Bsep(-/-) mice is based on increased expression of the responsible canalicular transporter proteins.