Amplification of CRAC current by STIM1 and CRACM1 (Orai1)

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I(CRAC)). However, individual overexpression of either protein fails to significantly amplify I(CRAC). Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I(CRAC), suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.     

Candidemia by Species of the Candida parapsilosis Complex in Children’s Hospital: Prevalence, Biofilm Production and Antifungal Susceptibility

Opportunistic infections are an increasingly common problem in hospitals, and the yeast Candida parapsilosis has emerged as an important nosocomial pathogen. The aims of this study were to determine and compare (i) the prevalence rate among C. parapsilosis complex organisms isolated from blood in a public children’s hospital in São Paulo state, (ii) the ability of the complex C. parapsilosis species identified to produce biofilm and (iii) the antifungal susceptibility profiles. Forty-nine (49) specimens of isolated blood yeast were analyzed, previously identified as C. parapsilosis by conventional methods. After the molecular analysis, the isolates were characterized as C. parapsilosis sensu stricto (83.7 %), C. orthopsilosis (10.2 %) and C. metapsilosis (6.1 %). All species were able to form biofilm. The species with the highest biofilm production was C. parapsilosis sensu stricto, followed by C. orthopsilosis and further by C. metapsilosis. All of the strains have demonstrated similar susceptibility to fluconazole, caspofungin, voriconazole, cetoconazole and 5-flucytosine. Only one strain of C. parapsilosis was resistant to amphotericin B. Regarding itraconazole, 66.6 and 43.9 % isolates of C. metapsilosis and C. parapsilosis, respectively, have demonstrated to be susceptible dose-dependent, with one isolate of the latter species resistant to the drug. Candida parapsilosis sensu stricto has demonstrated to be the less susceptible, mainly to amphotericin B, caspofungin and “azoles” such as fluconazole. Therefore, C. metapsilosis and C. orthopsilosis are still involved in a restricted number of infections, but these data have become essential for there are very few studies of these species in Latin America.     

Phenome-Wide Association Studies on a Quantitative Trait: Application to TPMT Enzyme Activity and Thiopurine Therapy in Pharmacogenomics

Phenome-Wide Association Studies (PheWAS) investigate whether genetic polymorphisms associated with a phenotype are also associated with other diagnoses. In this study, we have developed new methods to perform a PheWAS based on ICD-10 codes and biological test results, and to use a quantitative trait as the selection criterion. We tested our approach on thiopurine S-methyltransferase (TPMT) activity in patients treated by thiopurine drugs. We developed 2 aggregation methods for the ICD-10 codes: an ICD-10 hierarchy and a mapping to existing ICD-9-CM based PheWAS codes. Eleven biological test results were also analyzed using discretization algorithms. We applied these methods in patients having a TPMT activity assessment from the clinical data warehouse of a French academic hospital between January 2000 and July 2013. Data after initiation of thiopurine treatment were analyzed and patient groups were compared according to their TPMT activity level. A total of 442 patient records were analyzed representing 10,252 ICD-10 codes and 72,711 biological test results. The results from the ICD-9-CM based PheWAS codes and ICD-10 hierarchy codes were concordant. Cross-validation with the biological test results allowed us to validate the ICD phenotypes. Iron-deficiency anemia and diabetes mellitus were associated with a very high TPMT activity (p = 0.0004 and p = 0.0015, respectively). We describe here an original method to perform PheWAS on a quantitative trait using both ICD-10 diagnosis codes and biological test results to identify associated phenotypes. In the field of pharmacogenomics, PheWAS allow for the identification of new subgroups of patients who require personalized clinical and therapeutic management.     

Nerve Cross-Bridging to Enhance Nerve Regeneration in a Rat Model of Delayed Nerve Repair

There are currently no available options to promote nerve regeneration through chronically denervated distal nerve stumps. Here we used a rat model of delayed nerve repair asking of prior insertion of side-to-side cross-bridges between a donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) nerve stump ameliorates poor nerve regeneration. First, numbers of retrogradely-labelled TIB neurons that grew axons into the nerve stump within three months, increased with the size of the perineurial windows opened in the TIB and CP nerves. Equal numbers of donor TIB axons regenerated into CP stumps either side of the cross-bridges, not being affected by target neurotrophic effects, or by removing the perineurium to insert 5-9 cross-bridges. Second, CP nerve stumps were coapted three months after inserting 0-9 cross-bridges and the number of 1) CP neurons that regenerated their axons within three months or 2) CP motor nerves that reinnervated the extensor digitorum longus (EDL) muscle within five months was determined by counting and motor unit number estimation (MUNE), respectively. We found that three but not more cross-bridges promoted the regeneration of axons and reinnervation of EDL muscle by all the CP motoneurons as compared to only 33% regenerating their axons when no cross-bridges were inserted. The same 3-fold increase in sensory nerve regeneration was found. In conclusion, side-to-side cross-bridges ameliorate poor regeneration after delayed nerve repair possibly by sustaining the growth-permissive state of denervated nerve stumps. Such autografts may be used in human repair surgery to improve outcomes after unavoidable delays.     

Effect of two different long-sprint training regimens on sprint performance and associated metabolic responses

The purpose of this study was to analyze 2 different long-sprint training programs (TPs) of equal total work load, completed either with short recovery (SR) or long recovery (LR) between sets and to compare the effects of 6 long-sprint training sessions (TSs) conducted over a 2-week period on a 300-m performance. Fourteen trained subjects performed 3 pretraining maximal sprints (50-, 100-, and 300-m), were paired according to their 300-m performance, and randomly allocated to an LR or SR group, which performed 6 TSs consisting of sets of 150, 200, or 250 m. The recovery in the LR group was double that of the SR group. During the third TS and the 300-m pretest and posttest, blood pH, bicarbonate concentration ([HCO??]), excess-base (EB), and lactate concentration were recorded. Compared with a similar TS performed with SR, the LR training tends to induce a greater alteration of the acid-base balance: pH: 7.09 ± 0.08 (LR) and 7.14 ± 0.05 (SR) (p = 0.10), [HCO??]: 7.8 ± 1.9 (LR) and 9.6 ± 2.7 (SR) (p = 0.04), and EB: -21.1 ± 3.8 (LR) and -17.7 ± 2.8 (SR) (p = 0.11). A significant improvement in the 300-m performance between pre-TP and post-TP (42.45 ± 2.64 vs. 41.52 ± 2.45, p = 0.01) and significant decreases in pH (p < 0.01), EB (p < 0.001) and increase in [La] (p < 0.001) have been observed post-TP compared with those pre-TP. Although sprint training with longer recovery induces higher metabolic disturbances, both sprint training regimens allow a similar 300-m performance improvement with no concomitant significant progress in the 50- and 100-m performance.     

Binding of the Heterogeneous Ribonucleoprotein K (hnRNP K) to the Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Enhances Viral LMP2A Expression

The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.     

Effects of variations in intragastric volume on blood pressure and splanchnic blood flow during intraduodenal glucose infusion in healthy older subjects

The postprandial reduction in blood pressure (BP) is triggered by the interaction of nutrients with the small intestine and associated with an increase in splanchnic blood flow. Gastric distension may attenuate the postprandial fall in BP. The aim of this study was to determine the effects of differences in intragastric volume, including distension at a low (100 ml) volume, on BP and superior mesenteric artery (SMA) blood flow responses to intraduodenal glucose in healthy older subjects. BP and heart rate (HR; automated device), SMA blood flow (Doppler ultrasound), mesenteric vascular resistance (MVR), and plasma norepinephrine of nine male subjects (65-75 yr old) were measured after an overnight fast on 4 separate days in random order. On each day, subjects were intubated with a nasoduodenal catheter, incorporating a duodenal infusion port, and orally with a second catheter, incorporating a barostat bag, positioned in the fundus. Each subject received a 60-min (t = 0-60 min) intraduodenal glucose infusion (3 kcal/min) and gastric distension at a volume of 1) 0 ml (V0), 2) 100 ml (V100), 3) 300 ml (V300), or 4) 500 ml (V500). Systolic BP fell (P < 0.05) during V0, but not during V100, V300, or V500. In contrast, HR (P < 0.01) and SMA blood flow (P < 0.001) increased and MVR decreased (P < 0.05) comparably on all 4 days. Plasma norepinephrine rose (P < 0.01) in response to intraduodenal glucose, with no difference between the four treatments. There was a relationship between the areas under the curve for the change in systolic BP from baseline with intragastric volume (r = 0.60, P < 0.001). In conclusion, low-volume (≤100 ml) gastric distension has the capacity to abolish the fall in BP induced by intraduodenal glucose in healthy older subjects without affecting SMA blood flow or MVR. These observations support the concept that nonnutrient gastric distension prior to a meal has potential therapeutic applications in the management of postprandial hypotension.