Effects of insulin on intracellular GLUT4 vesicles in adipocytes: Evidence for a secretory mode of regulation

The facilitative glucose transporter, GLUT4 undergoes insulin-dependent movement to the cell surface in adipocytes, The magnitude of the insulin effect is much greater for CLUT4 than other recycling proteins such as the CD-MPR, In the present study we have studied the colocalisation of these proteins in adipocytes in an effort to explain this selective insulin-dependent recruitment of CLUT4, Using immunofluorescence microscopy or immuno-EM on 3T3-L1 adipocytes we find that there is considerable colocalisation between these proteins particularly within the area of the TGN, However, the distribution of CD-MPR was not significantly effected by insulin. The insulin-dependent recruitment of GLUT4 was concomitant with a selective decrease in GLUT4 labelling of cytoplasmic vesicles whereas the amount of CLUT4 in the TGN region (approx. 50% of total GLUT4) was relatively unaffected. To explore the possibility that the cytoplasmic GLUT4(+) vesicles represent an intracellular insulin-responsive storage compartment we performed quantitative immuno-EM on whole mounts of intracellular vesicles isolated from basal and insulin-stimulated adipocytes. These studies revealed that: (1) GLUT4 and CD-MPR were concentrated in small (30-200 nm) vesicles at a labelling density of 1-20+ gold particles/vesicle; (2) there was significant overlap between both proteins in that 70% of the total GLUT4 pool colocalised with CD-MPR; (3) a significant amount of GLUT4 (approx, 50% of total) was found in a subpopulation of vesicles that contained as little as 5% of the total CD-MPR pool; (4) the GLUT4(+)/CD-MPR(-) vesicles were highly insulin-responsive, and (5) the total number of GLUT4(+) vesicles, but not CD-MPR(+) vesicles, decreased by approx. 30% in response to insulin treatment. These data are consistent with a model in which GLUT4 is selectively sorted into a vesicular compartment in adipocytes that is recruited to the plasma membrane by insulin stimulation

Laboratory-based ergometry for swimmers: a narrative review

INTRODUCTION: First widely available dry-land training machines for swimmers were introduced about 40 years ago. They were designed so that swimmers could perform resistance exercise whilst more-closely replicating the movements of swimming, than when using other gymnasium-based resistance training machines. This narrative review categorises and summarises what has been shown by the studies that have utilised laboratory-based ergometry for swimmers. EVIDENCE ACQUISITION: A systematic search was conducted in PubMed, Web of Science, ScienceDirect and Scopus (1970-2018) and relevant publications were included. Publications were grouped into 4 main areas of research: (i) physiological responses to exercise, (ii) functional evaluation of swimmers, (iii) monitoring of training, and (iv) muscular work output of swimmers. EVIDENCE SYNTHESIS: Significant differences were showed between swim bench exercise and real swimming, especially in regard to the muscles involved. The difficulties of accurate reproduction of the movements and coordinated dynamic actions of swimming have not been overcome. Nevertheless, the literature shows that the use of these devices has provided a valuablecontribution to swimming physiology, while overcoming difficulties presented by attempting to make physiological measurements in the water. CONCLUSIONS: In spite of its limitations, laboratory-based ergometry has allowed a valuable contribution to the understanding of the physiology, effects of training and efficiency of swimming

Inhibitors of trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease.

Chagas disease remains one of the most neglected diseases in the world despite being the most important parasitic disease in Latin America. The characteristic chronic manifestation of chagasic cardiomyopathy is the region's leading cause of heart-related illness, causing significant mortality and morbidity. Due to the limited available therapeutic options, new drugs are urgently needed to control the disease. Sirtuins, also called Silent information regulator 2 (Sir2) proteins have long been suggested as interesting targets to treat different diseases, including parasitic infections. Recent studies on Trypanosoma cruzi sirtuins have hinted at the possibility to exploit these enzymes as a possible drug targets. In the present work, the T. cruzi Sir2 related protein 1 (TcSir2rp1) is genetically validated as a drug target and biochemically characterized for its NAD+-dependent deacetylase activity and its inhibition by the classic sirtuin inhibitor nicotinamide, as well as by bisnaphthalimidopropyl (BNIP) derivatives, a class of parasite sirtuin inhibitors. BNIPs ability to inhibit TcSir2rp1, and anti-parasitic activity against T. cruzi amastigotes in vitro were investigated. The compound BNIP Spermidine (BNIPSpd) (9), was found to be the most potent inhibitor of TcSir2rp1. Moreover, this compound showed altered trypanocidal activity against TcSir2rp1 overexpressing epimastigotes and anti-parasitic activity similar to the reference drug benznidazole against the medically important amastigotes, while having the highest selectivity index amongst the compounds tested. Unfortunately, BNIPSpd failed to treat a mouse model of Chagas disease, possibly due to its pharmacokinetic profile. Medicinal chemistry modifications of the compound, as well as alternative formulations may improve activity and pharmacokinetics in the future. Additionally, an initial TcSIR2rp1 model in complex with p53 peptide substrate was obtained from low resolution X-ray data (3.5 Å) to gain insight into the potential specificity of the interaction with the BNIP compounds. In conclusion, the search for TcSir2rp1 specific inhibitors may represent a valuable strategy for drug discovery against T. cruzi

Fourth mRNA COVID-19 vaccination in immunocompromised patients with haematological malignancies (COBRA KAI): a cohort study

Background Patients with haematological malignancies have impaired antibody responses to SARS-CoV-2 vaccination. We aimed to investigate whether a fourth mRNA COVID-19 vaccination improved antibody quantity and quality. Methods In this cohort study, conducted at 5 sites in the Netherlands, we compared antibody concentrations 28 days after 4 mRNA vaccinations (3-dose primary series plus 1 booster vaccination) in SARS-CoV-2 naive, immunocompromised patients with haematological malignancies to those obtained by age-matched, healthy individuals who had received the standard primary 2-dose mRNA vaccination schedule followed by a first booster mRNA vaccination. Prior to and 4 weeks after each vaccination, peripheral blood samples and data on demographic parameters and medical history were collected. Concentrations of antibodies that bind spike 1 (S1) and nucleocapsid (N) protein of SARS-CoV-2 were quantified in binding antibody units (BAU) per mL according to the WHO International Standard for COVID-19 serological tests. Seroconversion was defined as an S1 IgG concentration > 10 BAU/mL and a previous SARS-CoV-2 infection as N IgG > 14.3 BAU/mL. Antibody neutralising activity was tested using lentiviral-based pseudoviruses expressing spike protein of SARS-CoV-2 wildtype (D614G), Omicron BA.1, and Omicron BA.4/5 variants. This study is registered with EudraCT, number 2021-001072-41. Findings Between March 24, 2021 and May 4, 2021, 723 patients with haematological diseases were enrolled, of which 414 fulfilled the inclusion criteria for the current analysis. Although S1 IgG concentrations in patients significantly improved after the fourth dose, they remained significantly lower compared to those obtained by 58 age-matched healthy individuals after their first booster (third) vaccination. The rise in neutralising antibody concentration was most prominent in patients with a recovering B cell compartment, although potent responses were also observed in patients with persistent immunodeficiencies. 19% of patients never seroconverted, despite 4 vaccinations. Patients who received their first 2 vaccinations when they were B cell depleted and the third and fourth vaccination during B cell recovery demonstrated similar antibody induction dynamics as patients with normal B cell numbers during the first 2 vaccinations. However, the neutralising capacity of these antibodies was significantly better than that of patients with normal B cell numbers after two vaccinations. Interpretation A fourth mRNA COVID-19 vaccination improved S1 IgG concentrations in the majority of patients with a haematological malignancy. Vaccination during B cell depletion may pave the way for better quality of antibody responses after B cell reconstitution

Regulation of epidermal growth factor receptor degradation by heterotrimeric G alpha s protein

Heterotrimeric G proteins have been implicated in the regulation of membrane trafficking, but the mechanisms involved are not well understood. Here, we report that overexpression of the stimulatory G protein subunit (Gas) promotes ligand-dependent degradation of epidermal growth factor (EGF) receptors and Texas Red EGF, and knock-down of Gas expression by RNA interference (RNAi) delays receptor degradation. We also show that Gas and its GTPase activating protein (GAP), RGS-PX1, interact with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a critical component of the endosomal sorting machinery. Gas coimmunoprecipitates with Hrs and binds Hrs in pull-down assays. By immunofluorescence, exogenously expressed Gas colocalizes with myc-Hrs and GFP-RGS-PX1 on early endosomes, and expression of either Hrs or RGS-PX1 increases the localization of Gas on endosomes. Furthermore, knock-down of both Hrs and Gas by double RNAi causes greater inhibition of EGF receptor degradation than knock-down of either protein alone, suggesting that Gas and Hrs have cooperative effects on regulating EGF receptor degradation. These observations define a novel regulatory role for Gas in EGF receptor degradation and provide mechanistic insights into the function of Gas in endocytic sorting

The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps

NETs (neutrophil extracellular traps) have been described as a fundamental innate immune defence mechanism. Duringformation of NETs, the nuclear membrane is disrupted by an asyet unknown mechanism. In the present study we investigated the role of human cathelicidin LL-37 in nuclear membrane disruption and formation of NETs. Immunofluorescence microscopy revealed that 5 μMLL-37 significantly facilitated NET formation by primary human blood-derived neutrophils alone, in the presence of the classical chemical NET inducer PMA or in the presence of Staphylococcus aureus. Parallel assays with a random LL-37 fragment library indicated that the NET induction is mediated by the hydrophobic character of the peptide. The translocalization of LL-37 towards the nucleus and the disruption of the nuclear membrane were visualized using confocal fluorescence microscopy. In conclusion, the present study demonstrates a novel role for LL-37 in the formation of NETs