MicroRNA Changes Up to 24 h following Induced Hypoglycemia in Type 2 Diabetes

Hypoglycemia, as a complication of type 2 diabetes (T2D), causes increased morbidity and mortality but the physiological response underlying hypoglycemia has not been fully elucidated. Small noncoding microRNA (miRNA) have multiple downstream biological effects. This pilot exploratory study was undertaken to determine if induced miRNA changes would persist and contribute to effects seen 24 h post-hypoglycemia. A parallel, prospective study design was employed, involving T2D (n = 23) and control (n = 23) subjects. The subjects underwent insulin-induced hypoglycemia (2 mmol/L; 36 mg/dL); blood samples were drawn at baseline, upon the induction of hypoglycemia, and 4 h and 24 h post-hypoglycemia, with a quantitative polymerase chain reaction analysis of miRNA undertaken. The baseline miRNAs did not differ. In the controls, 15 miRNAs were downregulated and one was upregulated (FDR < 0.05) from the induction of hypoglycemia to 4 h later while, in T2D, only four miRNAs were altered (downregulated), and these were common to both cohorts (miR-191-5p; miR-143-3p; let-7b-5p; let-7g-5p), correlated with elevated glucagon levels, and all were associated with energy balance. From the induction of hypoglycemia to 24 h, 14 miRNAs were downregulated and 5 were upregulated (FDR < 0.05) in the controls; 7 miRNAs were downregulated and 7 upregulated (FDR < 0.05) in T2D; a total of 6 miRNAs were common between cohorts, 5 were downregulated (miR-93-5p, let-7b-5p, miR-191-5p, miR-185-5p, and miR-652-3p), and 1 was upregulated (miR-369-3p). An ingenuity pathway analysis indicated that many of the altered miRNAs were associated with metabolic and coagulation pathways; however, of the inflammatory proteins expressed, only miR-143-3p at 24 h correlated positively with tumor necrosis factor-α (TNFa; p < 0.05 and r = 0.46) and negatively with toll-like receptor-4 (TLR4; p < 0.05 and r = 0.43). The MiRNA levels altered by hypoglycemia reflected changes in counter-regulatory glucagon and differed between cohorts, and their expression at 24 h suggests miRNAs may potentiate and prolong the physiological response. Trial registration: ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03102801","term_id":"NCT03102801"}}NCT03102801.     

Human Milk Oligosaccharide 2′-Fucosyllactose Modulates Local Viral Immune Defense by Supporting the Regulatory Functions of Intestinal Epithelial and Immune Cells

Human milk contains bioactive components that provide protection against viral infections in early life. In particular, intestinal epithelial cells (IEC) have key regulatory roles in the prevention of enteric viral infections. Here we established an in vitro model to study the modulation of host responses against enteric viruses mimicked by poly I:C (pIC). The effects of 2′-fucosyllactose (2′FL), abundantly present in human milk, were studied on IEC and/or innate immune cells, and the subsequent functional response of the adaptive immune cells. IEC were pre-incubated with 2′FL and stimulated with naked or Lyovec™-complexed pIC (LV-pIC). Additionally, monocyte-derived dendritic cells (moDC) alone or in co-culture with IEC were stimulated with LV-pIC. Then, conditioned-moDC were co-cultured with naïve CD4⁺ T helper (Th)-cells. IEC stimulation with naked or LV-pIC promoted pro-inflammatory IL-8, CCL20, GROα and CXCL10 cytokine secretion. However, only exposure to LV-pIC additionally induced IFNβ, IFNλ1 and CCL5 secretion. Pre-incubation with 2′FL further increased pIC induced CCL20 secretion and LV-pIC induced CXCL10 secretion. LV-pIC-exposed IEC/moDC and moDC cultures showed increased secretion of IL-8, GROα, IFNλ1 and CXCL10, and in the presence of 2′FL galectin-4 and -9 were increased. The LV-pIC-exposed moDC showed a more pronounced secretion of CCL20, CXCL10 and CCL5. The moDC from IEC/moDC cultures did not drive T-cell development in moDC/T-cell cultures, while moDC directly exposed to LV-pIC secreted Th1 driving IL-12p70 and promoted IFNγ secretion by Th-cells. Hereby, a novel intestinal model was established to study mucosal host-defense upon a viral trigger. IEC may support intestinal homeostasis, regulating local viral defense which may be modulated by 2′FL. These results provide insights regarding the protective capacity of human milk components in early life.     

Peroxisome Proliferator-Activated Receptor-α (PPARα) Expression in a Clinical Population of Pakistani Patients with Type 2 Diabetes and Dyslipidemia

Poor glycemic control and dyslipidemia are hallmarks of type 2 diabetes mellitus (T2DM), which predispose to cardiovascular diseases. Peroxisome proliferator-activated receptor-α (PPARα) has been associated with atherosclerosis, but its role in T2DM is less clear. Previously, we studied PPARα expression levels in diabetics with and without dyslipidemia (DD). In this study we described the association with fasting blood glucose, HbA1c levels and lipid levels of the study population. Patient demography and biochemical data were collected from hospitals in Islamabad, Pakistan, and RT-PCR data of PPARα expression were retrieved from our previous study from the same cohort. We performed t-tests and regression analysis to evaluate the relationships between PPARα expression and demographic and clinical variables. As expected, body mass index and HbA1c were elevated in T2DM and DD patients compared to controls. Blood lipids (total cholesterol, triglycerides, LDL and HDL) were significantly higher in the DD group compared to the other two groups. In the T2DM and DD groups, the PPARα expression was not associated with any of the physical and biochemical parameters measured in this study. Expression of the PPARα gene was independent of blood lipids and glycemic control in this study. Further research is necessary to better understand the biological parameters of PPARα expression.     

Temperature Rise Inside Shear Bands in a Simple Model Glass

One of the key factors, which hampers the application of metallic glasses as structural components, is the localization of deformation in narrow bands of a few tens up to one hundred nanometers thickness, the so-called shear bands. Processes, which occur inside shear bands are of central importance for the question whether a catastrophic failure of the material is unavoidable or can be circumvented or, at least, delayed. Via molecular dynamics simulations, this study addresses one of these processes, namely the local temperature rise due to viscous heat generation. The major contribution to energy dissipation is traced back to the plastic work performed by shear stress during steady deformation. Zones of largest strain contribute the most to this process and coincide with high-temperature domains (hottest spots) inside the sample. Magnitude of temperature rise can reach a few percent of the sample’s glass transition temperature. Consequences of these observations are discussed in the context of the current research in the field.     

Flow-Seq Evaluation of Translation Driven by a Set of Natural Escherichia coli 5′-UTR of Variable Length

Flow-seq is a method that combines fluorescently activated cell sorting and next-generation sequencing to deduce a large amount of data about translation efficiency from a single experiment. Here, we constructed a library of fluorescent protein-based reporters preceded by a set of 648 natural 5′-untranslated regions (5′-UTRs) of Escherichia coli genes. Usually, Flow-seq libraries are constructed using uniform-length sequence elements, in contrast to natural situations, where functional elements are of heterogenous lengths. Here, we demonstrated that a 5′-UTR library of variable length could be created and analyzed with Flow-seq. In line with previous Flow-seq experiments with randomized 5′-UTRs, we observed the influence of an RNA secondary structure and Shine–Dalgarno sequences on translation efficiency; however, the variability of these parameters for natural 5′-UTRs in our library was smaller in comparison with randomized libraries. In line with this, we only observed a 30-fold difference in translation efficiency between the best and worst bins sorted with this factor. The results correlated with those obtained with ribosome profiling.     

The Stress Response of the Holothurian Central Nervous System: A Transcriptomic Analysis

Injury to the central nervous system (CNS) results in permanent damage and lack of function in most vertebrate animals, due to their limited regenerative capacities. In contrast, echinoderms can fully regenerate their radial nerve cord (RNC) following transection, with little to no scarring. Investigators have associated the regenerative capacity of some organisms to the stress response and inflammation produced by the injury. Here, we explore the gene activation profile of the stressed holothurian CNS. To do this, we performed RNA sequencing on isolated RNC explants submitted to the stress of transection and enzyme dissection and compared them with explants kept in culture for 3 days following dissection. We describe stress-associated genes, including members of heat-shock families, ubiquitin-related pathways, transposons, and apoptosis that were differentially expressed. Surprisingly, the stress response does not induce apoptosis in this system. Other genes associated with stress in other animal models, such as hero proteins and those associated with the integrated stress response, were not found to be differentially expressed either. Our results provide a new viewpoint on the stress response in the nervous system of an organism with amazing regenerative capacities. This is the first step in deciphering the molecular processes that allow echinoderms to undergo fully functional CNS regeneration, and also provides a comparative view of the stress response in other organisms.     

Reaction of Corroles with Sarcosine and Paraformaldehyde: A New Facet of Corrole Chemistry

Details on the unexpected formation of two new (dimethylamino)methyl corrole isomers from the reaction of 5,10,15-tris(pentafluorophenyl)corrolatogallium(III) with sarcosine and paraformaldehyde are presented. Semi-empirical calculations on possible mechanism pathways seem to indicate that the new compounds are probably formed through a Mannich-type reaction. The extension of the protocol to the free-base 5,10,15-tris(pentafluorophenyl)corrole afforded an unexpected new seven-membered ring corrole derivative, confirming the peculiar behavior of corroles towards known reactions when compared to the well-behaved porphyrin counterparts.     

Evaluation of Cisplatin-Induced Pathology in the Larval Zebrafish Lateral Line

Cisplatin is an effective anticancer agent, but also causes permanent hearing loss by damaging hair cells—the sensory receptors essential for hearing. There is an urgent clinical need to protect cochlear hair cells in patients undergoing cisplatin chemotherapy. The zebrafish lateral line organ contains hair cells and has been frequently used in studies to screen for otoprotective compounds. However, these studies have employed a wide range of cisplatin dosages and exposure times. We therefore performed a comprehensive evaluation of cisplatin ototoxicity in the zebrafish lateral line with the goal of producing a standardized, clinically relevant protocol for future studies. To define the dose- and time-response patterns of cisplatin-induced hair-cell death, we treated 6-day-old larvae for 2 h in 50 µM–1 mM cisplatin and allowed them to recover. We observed delayed hair cell death, which peaked at 4–8 h post-exposure. Cisplatin also activated a robust inflammatory response, as determined by macrophage recruitment and phagocytosis of hair cells. However, selective depletion of macrophages did not affect hair cell loss. We also examined the effect of cisplatin treatment on fish behavior and found that cisplatin-induced lateral line injury measurably impaired rheotaxis. Finally, we examined the function of remaining hair cells that appeared resistant to cisplatin treatment. We observed significantly reduced uptake of the cationic dye FM1-43 in these cells relative to untreated controls, indicating that surviving hair cells may be functionally impaired. Cumulatively, these results indicate that relatively brief exposures to cisplatin can produce hair cell damage and delayed hair cell death. Our observations provide guidance on standardizing methods for the use of the zebrafish model in studies of cisplatin ototoxicity.     

CCBE1 Is Essential for Epicardial Function during Myocardium Development

The epicardium is a single cell layer of mesothelial cells that plays a critical role during heart development contributing to different cardiac cell types of the developing heart through epithelial-to-mesenchymal transition (EMT). Moreover, the epicardium is a source of secreted growth factors that promote myocardial growth. CCBE1 is a secreted extracellular matrix protein expressed by epicardial cells that is required for the formation of the primitive coronary plexus. However, the role of CCBE1 during epicardial development was still unknown. Here, using a Ccbe1 knockout (KO) mouse model, we observed that loss of CCBE1 leads to congenital heart defects including thinner and hyper-trabeculated ventricular myocardium. In addition, Ccbe1 mutant hearts displayed reduced proliferation of cardiomyocyte and epicardial cells. Epicardial outgrowth culture assay to assess epicardial-derived cells (EPDC) migration showed reduced invasion of the collagen gel by EPDCs in Ccbe1 KO epicardial explants. Ccbe1 KO hearts also displayed fewer nonmyocyte/nonendothelial cells intramyocardially with a reduced proliferation rate. Additionally, RNA-seq data and experimental validation by qRT-PCR showed a marked deregulation of EMT-related genes in developing Ccbe1 mutant hearts. Together, these findings indicate that the myocardium defects in Ccbe1 KO mice arise from disruption of epicardial development and function.