Differential Subcellular Localization of the Splice Variants of the Zinc Transporter ZnT5 Is Dictated by the Different C-Terminal Regions

Zinc is emerging as an important intracellular signaling molecule, as well as fulfilling essential structural and catalytic functions through incorporation in a myriad of zinc metalloproteins so it is important to elucidate the molecular mechanisms of zinc homeostasis, including the subcellular localizations of zinc transporters.Two splice variants of the human SLC30A5 Zn transporter gene (ZnT5) have been reported in the literature. These variants differ at their N- and C-terminal regions, corresponding with the use of different 5' and 3' exons. We demonstrate that full length human ZnT5 variant B is a genuine transcript in human intestinal cells and confirm expression of both variant A and variant B in a range of untreated human tissues by splice variant-specific RT-PCR. Using N- or C-terminal GFP or FLAG fusions of both isoforms of ZnT5 we identify that the differential subcellular localization to the Golgi apparatus and ER respectively is a function of their alternative C-terminal sequences. These different C-terminal regions result from the incorporation into the mature transcript of either the whole of exon 14 (variant B) or only the 5' region of exon 14 plus exons 15-17 (variant A).We thus propose that exons 15 to 17 include a signal that results in trafficking of ZnT5 to the Golgi apparatus and that the 3' end of exon 14 includes a signal that leads to retention in the ER

On-chip Single Nanoparticle Detection and Sizing by Mode Splitting in an Ultra-high-Q Microresonator

The ability to detect and size individual nanoparticles with high resolution is crucial to understanding behaviours of single particles and effectively using their strong size-dependent properties to develop innovative products. We report real-time, in-situ detection and sizing of single nanoparticles, down to 30 nm in radius, using mode-splitting in a monolithic ultra-high-Q whispering-gallery-mode (WGM) microtoroid resonator. Particle binding splits a WGM into two spectrally shifted resonance modes, forming a self-referenced detection scheme. This technique provides superior noise suppression and enables extracting accurate size information in a single-shot measurement. Our method requires neither labelling of the particles nor apriori information on their presence in the medium, providing an effective platform to study nanoparticles at single particle resolution.Comment: 23 pages, 8 figure

The Cyclophilin-Binding Agent Sanglifehrin A Is a Dendritic Cell Chemokine and Migration Inhibitor

Sanglifehrin A (SFA) is a cyclophilin-binding immunosuppressant but the immunobiology of action is poorly understood. We and others have reported that SFA inhibits IL-12 production and antigen uptake in dendritic cells (DC) and exhibits lower activity against lymphocytes. Here we show that SFA suppresses DC chemokine production and migration. Gene expression analysis and subsequent protein level confirmation revealed that SFA suppressed CCL5, CCL17, CCL19, CXCL9 and CXCL10 expression in human monocyte-derived DC (moDC). A systems biology analysis, Onto Express, confirmed that SFA interferes with chemokine-chemokine receptor gene expression with the highest impact. Direct comparison with the related agent cyclosporine A (CsA) and dexamethasone indicated that SFA uniquely suppresses moDC chemokine expression. Competitive experiments with a 100-fold molar excess of CsA and with N-Methyl-Val-4-cyclosporin, representing a nonimmunosuppressive derivative of CsA indicated chemokine suppression through a cyclophilin-A independent pathway. Functional assays confirmed reduced migration of CD4+ Tcells and moDCs to supernatant of SFA-exposed moDCs. Vice versa, SFA-exposed moDC exhibited reduced migration against CCL19. Moreover, SFA suppressed expression of the ectoenzyme CD38 that was reported to regulate DC migration and cytokine production. These results identify SFA as a DC chemokine and migration inhibitor and provide novel insight into the immunobiology of SFA

An Assay to Monitor HIV-1 Protease Activity for the Identification of Novel Inhibitors in T-Cells

The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life

Neurod1 Suppresses Hair Cell Differentiation in Ear Ganglia and Regulates Hair Cell Subtype Development in the Cochlea

Background: At least five bHLH genes regulate cell fate determination and differentiation of sensory neurons, hair cells and supporting cells in the mammalian inner ear. Cross-regulation of Atoh1 and Neurog1 results in hair cell changes in Neurog1 null mice although the nature and mechanism of the cross-regulation has not yet been determined. Neurod1, regulated by both Neurog1 and Atoh1, could be the mediator of this cross-regulation. Methodology/Principal Findings: We used Tg(Pax2-Cre) to conditionally delete Neurod1 in the inner ear. Our data demonstrate for the first time that the absence of Neurod1 results in formation of hair cells within the inner ear sensory ganglia. Three cell types, neural crest derived Schwann cells and mesenchyme derived fibroblasts (neither expresses Neurod1) and inner ear derived neurons (which express Neurod1) constitute inner ear ganglia. The most parsimonious explanation is that Neurod1 suppresses the alternative fate of sensory neurons to develop as hair cells. In the absence of Neurod1, Atoh1 is expressed and differentiates cells within the ganglion into hair cells. We followed up on this effect in ganglia by demonstrating that Neurod1 also regulates differentiation of subtypes of hair cells in the organ of Corti. We show that in Neurod1 conditional null mice there is a premature expression of several genes in the apex of the developing cochlea and outer hair cells are transformed into inner hair cells. Conclusions/Significance: Our data suggest that the long noted cross-regulation of Atoh1 expression by Neurog1 migh

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Improvements on Restricted Insecticide Application Protocol for Control of Human and Animal African Trypanosomiasis in Eastern Uganda

African trypanosomes constrain livestock and human health in Sub-Saharan Africa, and aggravate poverty and hunger of these otherwise largely livestock-keeping communities. To solve this, there is need to develop and use effective and cheap tsetse control methods. To this end, we aimed at determining the smallest proportion of a cattle herd that needs to be sprayed on the legs, bellies and ears (RAP) for effective Human and Animal African Trypanosomiasis (HAT/AAT) control.; Cattle in 20 villages were ear-tagged and injected with two doses of diminazene diaceturate (DA) forty days apart, and randomly allocated to one of five treatment regimens namely; no treatment, 25%, 50%, 75% monthly RAP and every 3 month Albendazole drench. Cattle trypanosome re-infection rate was determined by molecular techniques. ArcMap V10.3 was used to map apparent tsetse density (FTD) from trap catches. The effect of graded RAP on incidence risk ratios and trypanosome prevalence was determined using Poisson and logistic random effect models in R and STATA V12.1 respectively. Incidence was estimated at 9.8/100 years in RAP regimens, significantly lower compared to 25.7/100 years in the non-RAP regimens (incidence rate ratio: 0.37; 95% CI: 0.22-0.65; P>0.001). Likewise, trypanosome prevalence after one year of follow up was significantly lower in RAP animals than in non-RAP animals (4% vs 15%, OR: 0.20, 95% CI: 0.08-0.44; P>0.001). Contrary to our expectation, level of protection did not increase with increasing proportion of animals treated.; Reduction in RAP coverage did not significantly affect efficacy of treatment. This is envisaged to improve RAP adaptability to low income livestock keepers but needs further evaluation in different tsetse challenge, HAT/AAT transmission rates and management systems before adopting it for routine tsetse control programs