Characterization of Zinc Influx Transporters (ZIPs) in pancreatic beta cells: roles in regulating cytosolic zinc homeostasis and insulin secretion

Zinc plays an essential role in the regulation of pancreatic beta cell function, affecting important processes including proinsulin biosynthesis, glucose-stimulated insulin secretion, and cell viability. Mutations in zinc efflux transport protein ZnT8, have been linked with both type 1 and type 2 diabetes, further supporting an important role for zinc in glucose homeostasis. However, very little is known about how cytosolic zinc is controlled by zinc influx proteins (ZIPs). In the current study, we have examined the beta cell and islet ZIP transcriptome and show consistent high expression of ZIP6 (Slc39a6) and ZIP7 (Slc39a7) genes across human, mouse islets and MIN6 beta cells. Modulation of ZIP6 and ZIP7 expression significantly altered cytosolic zinc influx in pancreatic beta cells, indicating an important role for ZIP6 and ZIP7 in regulating cellular zinc homeostasis. Functionally, this deregulated cytosolic zinc homeostasis led to impaired insulin exocytosis and insulin secretion. In parallel studies, we identified both ZIP6 and ZIP7 as potential interacting proteins with GLP-1R by a membrane yeast-two-hybrid (MYTH) assay. Knock-down of ZIP6 but not ZIP7 in MIN6 beta cells impaired the protective effects of GLP-1 on fatty acid-induced cell death possibly via reduced p-ERK pathway. Thus, our data suggests that ZIP6 and ZIP7 function as two important zinc influx transporters to regulate cytosolic zinc concentrations and insulin secretion in beta cells. In particular, ZIP6 is also capable of directly interacting with GLP-1R to facilitate the protective effect of GLP-1 on beta cell survival

Molecular prevalence of bovine noroviruses and nebovirusesin newborn calves in Iran

AbstractIn this study, bovine enteric caliciviruses (BECs) were detected in 49.4% of a total of 253 stool specimens for diarrheiccalves collected from 42 industrial dairy farms from March 2010 to February 2012. Genogroup III norovirus (NoVsGIII)were more prevalent (39.5%) than neboviruses (NBs) (15%), and coinfections were observed in 5.1% of the samples tested.Sequence analysis of the partial polymerase gene from 13 NoVsGIII samples indicated the circulation of both genotype 1and genotype 2 strains. Among the six NB strains sequenced, five were related to the Bo/Nebraska/80/US strain, while onewas related to the Bo/Newbury1/76/UK strain

Higher Expression Level and Lower Toxicity of Genetically Spliced Rotavirus NSP4 in Comparison to the Full-Length Protein in E. coli

Background: Rotavirus group A (RVA) is recognized as a major cause of severe gastroenteritis in children and new-born animals. Nonstructural protein 4 (NSP4) is responsible for the enterotoxic activity of these viruses in the villus epithelial cells. Amino acids 114-135 of NSP4 are known to form the diarrhea-inducing region of this viral enterotoxin. Therefore, developing an NSP4 lacking the enterotoxin domain could result in the introduction of a new subunit vaccine against rotaviruses in both humans and animals. Objectives: The aim of this study is the evaluation of rotavirus ANSP4 expression in E. coli expression system before and after removal of the diarrhea-inducing domain, which is the first step towards further immunological studies of the resulting protein. Materials and Methods: Splicing by overlap extension (SOEing) PCR was used to remove the diarrhea-inducing sequence from the NSP4 cDNA. Both the full-length (FL-NSP4) and the spliced (S-NSP4) cDNA amplicons were cloned into pET-32c and pGEX-6P-2. Expression levels of the recombinant proteins were evaluated in E. coli BL21 (DE3) by Western blot analysis. In addition, the toxicity of pET plasmids bearing the S-NSP4 and FL-NSP4 fragments was investigated by plasmid stability test. Results: For FL-NSP4, protein expression was detected for the strain containing the pGEX:FL-NSP4 plasmid, but not for the strain carrying pET:FL-NSP4. Hourly sampling up to 3 h showed that the protein production decreased by time. In contrast, expression of S-NSP4 was detected for pET:S-NSP4 strain, but not for pGEX:S-NSP4. Plasmid stability test showed that pET:S-NSP4 recombinant plasmid was almost stable, while pET:FL-NSP4 was unstable. Conclusions: This is the first report of production of rotavirus NSP4 lacking the diarrhea-inducing domain (S-NSP4). SNSP4 shows less toxicity in this expression system and potentially could be a promising goal for rotavirus immunological and vaccine studies in the future. Keywords: Diarrhea; Enterotoxin; Expression; NSP4; Rotavirus; Splicing by overlap extension PC

The molecular epidemiology of bovine rotaviruses circulating in Iran: a two-year study

International audienceBovine group A rotavirus (bovine RVA) is recognized as a major cause of severe gastroenteritis in newborn calves. The purpose of this study was to estimate the prevalence and identify the genotypes of circulating bovine RVA in newborn diarrheic calves. Two hundred fifty-three stool samples of diarrheic calves up to 1 month old were collected from 42 industrial dairy farms in two Iranian provinces during March 2010 to February 2012. All collected samples were screened for the presence of bovine RVA by RT-PCR, and the G and P genotypes were determined by semi-nested multiplex RT-PCR assay. The results of RT-PCR indicated that 49.4 % (125 out of 253) of the samples were positive for bovine RVA. The G and P genotyping of a subset of positive samples (n = 85) by semi-nested multiplex RT-PCR revealed that G6 (55.3 %) and G10 (43.5 %) and P[5] (51.8 %) and P[11] (27 %) were the most prevalent G and P genotypes, respectively. G6P[5] was the dominant genotype (35.3 %), followed by G10P[5], G10P[11] and G6P[11], with prevalence rates of 16.5 %, 15.3 % and 10.6 %, respectively. Sequence analysis of 20 VP7 and four VP4 genes showed highest nucleotide sequence identity with the corresponding genes of strains RVA/Cow-tc/GBR/UK/1973/G6P7[5] and RVA/Cow-tc/USA/B223/XXXX/G10P[11]. The results of this study reveal the diversity of G and P genotypes in bovine RVA samples from diarrheic Iranian calves and expands our knowledge of bovine RVA infections in the Middle East. These results also highlight the importance of producing of an effective rotavirus vaccine and its inclusion in the national cattle immunization program

Critical metals in porphyry copper deposits

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25-30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function

Molecular prevalence of bovine noroviruses and neboviruses in newborn calves in Iran

International audienceIn this study, bovine enteric caliciviruses (BECs) were detected in 49.4% of a total of 253 stool specimens for diarrheic calves collected from 42 industrial dairy farms from March 2010 to February 2012. Genogroup III norovirus (NoVsGIII) were more prevalent (39.5%) than neboviruses (NBs) (15%), and coinfections were observed in 5.1% of the samples tested. Sequence analysis of the partial polymerase gene from 13 NoVsGIII samples indicated the circulation of both genotype 1 and genotype 2 strains. Among the six NB strains sequenced, five were related to the Bo/Nebraska/80/US strain, while one was related to the Bo/Newbury1/76/UK strai

Analyses of Ca⁺² flux, and respiration capacities of the human H1 ES-DBCs.

<p>(A) Measurement of glucose-stimulated cytosolic Ca⁺² flux in the ES-DBCs, Non-Treated cell and MIN-6 beta-cell population. (B) Mitochondrial respiration (the potential of mitochondria to reserve energy) in ES-DBCs, Non-Treated and MIN-6 cells using the seahorse technique. (n = 4)-two technical replicates per batch, data are presented as Mean±SD. (*<i>p< 0</i>.<i>05</i>, **<i>p< 0</i>.<i>01</i>, p***<<i>0</i>.<i>001</i>, paired two-tailed <i>t</i>-test, n = 4).</p

Short protocol outline.

<p>(A) Schematic overview of the 25 to 30-day protocol to generate human H1 ES-derived beta-like cells (DBCs). Below, images of the differentiated H1 cells and the control cells (Non-Treated ES cell) at each stage are shown. The arrow symbol identifies tube-like structure in the differentiated cells in the stage 2. The star symbol identifies detached dead cells as spheres in the Non-Treated cells in stage 4. Scale bar = 100μm for all cell images. The <i>red font</i> indicates modifications to molecules or timing in comparison to the protocol described by Rezania et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164457#pone.0164457.ref009" target="_blank">9</a>]. (B) Expression analyses of <i>SOX17</i>, <i>FOXA2</i> and <i>Gooscoid</i> as Definitive Endoderm (DE), <i>Sox1</i> as ectoderm, and <i>Brachyury</i> as mesoderm-specific markers in the H1 ES cells differentiated on MEF, Mouse Embryonic Fibroblast; as EB (Embryoid Bodies) or on Geltrex, analyzed by quantitative RT-PCR. (* <i>p< 0</i>.<i>05</i>, **<i>p< 0</i>.<i>01</i>, p***<<i>0</i>.<i>001</i>, significant differences between the treated and control cells in each condition, unpaired two-tailed <i>t</i>-test, n = 3).</p

The efficiency of Definitive Endoderm (DE) and Gut Tube Endoderm formation at the stage 1 and 2 of the differentiation protocol.

<p>(A) Flow cytometry, and immunofluorescence staining for DE-specific markers in the differentiated H1 ES cells. (B) Quantitative RT-PCR results for Gut Tube Endoderm-specific markers are shown in (B), showing genes up-regulated in the stage 1, and (C) maintained highly expressed genes in the stage 2. Scale bar = 40μm. (*<i>p< 0</i>.<i>05</i>, **<i>p< 0</i>.<i>01</i>, p***<<i>0</i>.<i>001</i>, unpaired two-tailed <i>t</i>-test, n = 3).</p

Characterization of the differentiated H1 ES cells at the Pancreatic Progenitor (PP) and the Endocrine Progenitor (EN) stages.

<p>(A) From left to right, flow cytometry for PDX1/FOXA2, immunofluorescence staining for PDX1, and qRT-PCR analysis for the PP-specific genes in the differentiated cells at stage 3. (B) Flow cytometry for NGN3/NKX6.1, immunofluorescence staining for NGN3, qRT-PCR analysis for the EP-specific genes and below, immunofluorescence staining for PDX1/NKX6.1 in the differentiated cells at stage 4. (C) Immunofluorescence staining for ARX/PAX4, and qRT-PCR analysis for ARX and PAX4 in differentiated cells at the stage 4. Scale bar = 40μm. (*<i>p< 0</i>.<i>05</i>, **<i>p< 0</i>.<i>01</i>, p***<<i>0</i>.<i>001</i>, unpaired two-tailed <i>t</i>-test, n = 3).</p