Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P₂ and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca²⁺ acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P₂ complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca²⁺ concentrations is likely to be important for Ca²⁺-regulated secretion.     

Genome-Wide and Species-Wide In Silico Screening for Intragenic MicroRNAs in Human,Mouse and Chicken

MicroRNAs (miRNAs) are non-coding RNAs (ncRNAs) involved in regulation of gene expression. Intragenic miRNAs, especially those exhibiting a high degree of evolutionary conservation, have been shown to be coordinately regulated and/or expressed with their host genes, either with synergistic or antagonistic correlation patterns. However, the degree of cross-species conservation of miRNA/host gene co-location is not known and co-expression information is incomplete and fragmented among several studies. Using the genomic resources (miRBase and Ensembl) we performed a genome-wide in silico screening (GWISS) for miRNA/host gene pairs in three well-annotated vertebrate species: human, mouse, and chicken. Approximately half of currently annotated miRNA genes resided within host genes: 53.0% (849/1,600) in human, 48.8% (418/855) in mouse, and 42.0% (210/499) in chicken, which we present in a central publicly available Catalog of intragenic miRNAs (http://www.integratomics-time.com/miR-host/catalog). The miRNA genes resided within either protein-coding or ncRNA genes, which include long intergenic ncRNAs (lincRNAs) and small nucleolar RNAs (snoRNAs). Twenty-seven miRNA genes were found to be located within the same host genes in all three species and the data integration from literature and databases showed that most (26/27) have been found to be co-expressed. Particularly interesting are miRNA genes located within genes encoding for miRNA silencing machinery (DGCR8, DICER1, and SND1 in human and Cnot3, Gdcr8, Eif4e, Tnrc6b, and Xpo5 in mouse). We furthermore discuss a potential for phenotype misattribution of miRNA host gene polymorphism or gene modification studies due to possible collateral effects on miRNAs hosted within them. In conclusion, the catalog of intragenic miRNAs and identified 27 miRNA/host gene pairs with cross-species conserved co-location, co-expression, and potential co-regulation, provide excellent candidates for further functional annotation of intragenic miRNAs in health and disease.     

Impact of health research capacity strengthening in low- and middle-income countries: the case of WHO/TDR programmes

Background

Measuring the impact of capacity strengthening support is a priority for the international development community. Several frameworks exist for monitoring and evaluating funding results and modalities. Based on its long history of support, we report on the impact of individual and institutional capacity strengthening programmes conducted by the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and on the factors that influenced the outcome of its Research Capacity Strengthening (RCS) activities.

Methodology and Principal Findings

A mix of qualitative and quantitative methods (questionnaires and in-depth interviews) was applied to a selected group of 128 individual and 20 institutional capacity development grant recipients that completed their training/projects between 2000 and 2008. A semi-structured interview was also conducted on site with scientists from four institutions. Most of the grantees, both individual and institutional, reported beneficial results from the grant. However, glaring inequities stemming from gender imbalances and a language bias towards English were identified. The study showed that skills improvement through training contributed to better formulation of research proposals, but not necessarily to improved project implementation or communication of results. Appreciation of the institutional grants'' impact varied among recipient countries. The least developed countries saw the programmes as essential for supporting basic infrastructure and activities. Advanced developing countries perceived the research grants as complementary to available resources, and particularly suitable for junior researchers who were not yet able to compete for major international grants.

Conclusion

The study highlights the need for a more equitable process to improve the effectiveness of health research capacity strengthening activities. Support should be tailored to the existing research capacity in disease endemic countries and should focus on strengthening national health research systems, particularly in the least developing countries. The engagement of stakeholders at country level would facilitate the design of more specific and comprehensive strategies based on local needs.     

Comparison of biological effects of a sustained delivery system and nonencapsulated LH-RH antagonist SB-75 in rats.

Recently, we developed long-acting microcapsules and microgranules of the LH-RH antagonist SB-75. In this study, we compared the inhibitory effects of a single injection of encapsulated and nonencapsulated LH-RH antagonist SB-75 on gonadotropin and testosterone secretion. The resulting serum SB-75 levels were also measured by RIA. Microgranules containing 4% of this antagonist in poly(DL-lactide-co-glycolide) were administered IM at two different doses (30 and 60 mg/rat) to male rats. Other groups of rats were injected SC with equivalent doses of nonencapsulated SB-75 (1.25 and 2.5 mg/rat). The administration of microgranules at a dose of 60 mg/rat produced a significant elevation of serum SB-75 until day 76, and serum testosterone and LH levels were suppressed below the detection limit of the RIA for a period of 70 days. An equivalent dose of nonencapsulated SB-75 acetate (2.5 mg/rat) produced a significant elevation of SB-75 levels for 20 days and decreased testosterone to castration values and LH levels for merely 21 days. In rats treated with 30 mg microgranules of SB-75 or an equivalent dose of SB-75 acetate (1.25 mg/rat), serum testosterone and LH were suppressed to a similar extent, but for only 2 weeks. In another study, the effect of a single SC injection of 1.25 mg/rat of antagonist SB-75 on pituitary LH-RH receptors was determined, 7 and 60 days after administration. SB-75 produced a significant (p < 0.01) downregulation of membrane receptors for LH-RH 7 days after administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deletion of IL-33R (ST2) Abrogates Resistance to EAE in BALB/C Mice by Enhancing Polarization of APC to Inflammatory Phenotype

The administration of interleukin 33 and deletion of IL-33 receptor, ST2 molecule, affects the induction of autoimmunity in different experimental models of human autoimmune diseases. The aim of this study was to analyze the effect of ST2 deletion on the induction of experimental autoimmune encephalomyelitis (EAE) in resistant BALB/c mice. Mice were immunized with MOG_35–55 peptide or disease was induced by passive transfer of encephalitogenic singenic cells and EAE was clinically and histologically evaluated. Expression of intracellular inflammatory cytokines, markers of activation and chemokine receptors on lymphoid tissue and CNS infiltrating mononuclear cells was analyzed by flow cytometry. We report here that deletion of ST2^−/− molecule abrogates resistance of BALB/c mice to EAE induction based on clinical and histopathological findings. Brain and spinal cord infiltrates of ST2^−/− mice had significantly higher number of CD4⁺ T lymphocytes containing inflammatory cytokines compared to BALB/c WT mice. Adoptive transfer of ST2^−/− primed lymphocytes induced clinical signs of the disease in ST2^−/− as well as in WT mice. MOG_35–55 restimulated ST2^−/− CD4⁺ cells as well as ex vivo analyzed lymph node cells had higher expression of T-bet and IL-17, IFN-γ, TNF-α and GM-CSF in comparison with WT CD4⁺ cells. ST2^−/− mice had higher percentages of CD4⁺ cells expressing chemokine receptors important for migration to CNS in comparison with WT CD4⁺ cells. Draining lymph nodes of ST2^−/− mice contained higher percentage of CD11c⁺CD11b⁺CD8⁻ cells containing inflammatory cytokines IL-6 and IL-12 with higher expression of activation markers. Transfer of ST2^−/− but not WT dendritic cells induced EAE in MOG_35–55 immunized WT mice. Our results indicate that ST2 deficiency attenuates inherent resistance of BALB/c mice to EAE induction by enhancing differentiation of proinflammatory antigen presenting cells and consecutive differentiation of encephalitogenic T cells in the draining lymph node rather than affecting their action in the target tissue.     

Decrease in the osteocyte lacunar density accompanied by hypermineralized lacunar occlusion reveals failure and delay of remodeling in aged human bone

Aging decreases the human femur’s fatigue resistance, impact energy absorption, and the ability to withstand load. Changes in the osteocyte distribution and in their elemental composition might be involved in age‐related bone impairment. To address this question, we carried out a histomorphometric assessment of the osteocyte lacunar distribution in the periosteal and endosteal human femoral cortexes of 16 female and 16 male donors with regard to age‐ and sex‐related bone remodeling. Measurements of the bone mineral density distribution by quantitative backscattered electron imaging and energy dispersive X‐ray analysis were taken to evaluate the osteocyte lacunar mineral composition and characteristics. Age‐dependent decreases in the total osteocyte lacunar number were measured in all of the cases. This change signifies a risk for the bone’s safety. Cortical subdivision into periosteal and endosteal regions of interest emphasized that, in both sexes, primarily the endosteal cortex is affected by age‐dependent reduction in number of osteocyte lacunae, whereas the periosteal compartment showed a less pronounced osteocyte lacunar deficiency. In aged bone, osteocyte lacunae showed an increased amount of hypermineralized calcium phosphate occlusions in comparison with younger cases. With respect to Frost’s early delineation of micropetrosis, our microanalyses revealed that the osteocyte lacunae are subject to hypermineralization. Intralacunar hypermineralization accompanied by a decrease in total osteocyte lacunar density may contribute to failure or delayed bone repair in aging bone. A decreased osteocyte lacunar density may cause deteriorations in the canalicular fluid flow and reduce the detection of microdamage, which counteracts the bone’s structural integrity, while hypermineralized osteocyte lacunae may increase bone brittleness and render the bone fragile.     

Absence of KCNQ1-dependent K+ fluxes in proximal tubular cells of frog kidney

The present study was designed to investigate the functional significance of KCNQ1-mediated K+ secretory fluxes in proximal tubular cells of the frog kidney. To this end, we investigated the effects on rapid depolarization and slow repolarization of the peritubular membrane potential after luminal addition of L-phenylalanine or L-alanine plus/minus KCNQ1 channel blockers. Perfusing the lumen with 10 mmol/L L-phenylalanine plus/minus luminal 293B, a specific blocker of KCNQ1, did not modify the rapid depolarization and the rate of slow repolarization. Perfusing the lumen with 10 mmol/L L-alanine plus/minus luminal HMR-1556, a more potent KCNQ1 channel blocker, did not also alter the rapid depolarization and the rate of slow repolarization. Pretreatment (1 h) of the lumen with HMR-1556 also failed to modify rapid depolarization and rate of slow repolarization upon luminal 10 mmol/L L-alanine. Perfusing the lumen with 1 mmol/L L-alanine plus/minus luminal HMR-1556 did not change the rapid depolarization and the rate of slow repolarization. The pretreatment (1 h) with luminal HMR-1556 did not modify the rapid depolarization and the rate of slow repolarization upon luminal 1 mmol/L L-alanine. The pretreatment (1 h) of the lumen with HMR-1556 did not change transference number for K+ of peritubular cell membrane. Finally, luminal barium blunted the rapid depolarization upon application of luminal 1 mmol/L L-alanine. RT-PCR showed that KCNQ1 mRNA was not expressed in frog kidney. In conclusion, the KCNQ1-dependent K+ secretory fluxes are absent in proximal tubule of frog kidney.     

Pigment formation in the dermatophytes

Avstract The moist mycelium ofT. tonsurans was extracted with 3 % w/v sodium hydroxide to yield a purple to violet solution which was passed through a cation-exchange resin column, evaporated partially, dialyzed and lyophilized. The lyophilized powder was extracted using absolute ethanol, acetone and chloroform (Process A) and separately by defatting with petroleum ether and n-hexane followed by chloroform-methanol 2:1 (Process B).Separation was achieved for the extract of Process B on thin layer chromatography using Silica Gel G and butanol-ethanol-water as the solvent system. Ultra-violet and visible spectrophotometric absorption data are reported for the lyophilized extract ofT. tonsurans, hydrolyzates of the lyophilized extract, and for sodium hydroxide extracts ofT. megnini, T. mentagrophytes, T. rubrum, T. tonsurans andT. violaceum. The spectra were similar in all cases.A review of pigment substance in dermatophytes is made, which together with the experimental data secured withT. tonsurans and previous experimentation, suggest the possible effects of drying procedures, contusion, solvents, acidic additives and method of extraction on the removal of pigment from the mycelium of dermatophytes. It is suggested that all the above conditions influence the substances ultimately extracted and that some alteration of a basic pigment association in the organism occurs to release pigment fragments or fractions of varying complexity. Oxidation and polymerization may influence the end result. It is possible that the mechanism presented can account for the number of pigment substances reportedly occurring in the dermatophytes and that a similar pigment structure or complex exists in other Trichophyton species.Grateful thanks are extended to the Medical Research Council of Canada for financial support.