Allele-specific amplification of exon 7 in the survival motor neuron (SMN) genes for molecular diagnosis of spinal muscular atrophy

There are two highly homologous survival motor neuron (SMN) genes in humans but molecular defects in the SMN1 gene cause spinal muscular atrophy (SMA). More than 90% of SMA patients are shown to have a homozygous deletion of exon 7 in the SMN1 gene. Therefore, a simple test for exon 7 deletion would be very useful in the molecular diagnosis of SMA. However, limited methods are available, and most of these methods utilize expensive instruments and consumables. Here, we describe a simple allele-specific PCR test, which can be performed using standard equipment in DNA laboratories. The principle of the test is based on a single nucleotide difference (C versus T) between the exon 7 of SMN1 and SMN2 genes. Using allele-specific primers, two PCR amplifications are performed for each sample to amplify a 404-bp diagnostic fragment, and consequent electrophoresis of PCR products on agarose gel provides definitive information concerning the exon 7 deletion To rule out false negatives, a 500-bp fragment from the N-acetyltransferase gene was coamplified as an internal control in each test. We have, so far, analyzed 41 SMA samples with our method, and tested the validity of results using an independent restriction fragment length polymorphism (RFLP) method. Genotyping results obtained by both methods were in complete agreement for all of the samples analyzed. Our method can also be used to detect heterozygous deletion of exon 7 in SMN genes, if the relative intensities of the diagnostic and internal control bands are determined.     

Cyclase-associated protein is essential for the functioning of the endo-lysosomal system and provides a link to the actin cytoskeleton

Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase-associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunits of the highly conserved vacuolar H(+)-ATPase (V-ATPase) and co-localizes to some extent with the V-ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology of V-ATPase- and Nramp1-decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V-ATPase, had a more severe effect on CAP bsr than on wild-type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein-CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V-ATPase in CAP bsr was drastically altered upon hypo-osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper-osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo-lysosomal system in Dictyostelium.     

Microarray phenotyping places cyclase associated protein CAP at the crossroad of signaling pathways reorganizing the actin cytoskeleton in Dictyostelium

Large-scale gene expression analysis has been applied recently to uncover groups of genes that are co-regulated in particular processes. Here we undertake such an analysis on CAP, a protein that participates in the regulation of the actin cytoskeleton and in cAMP signaling in Dictyostelium. microarray analysis revealed that loss of CAP altered the expression of many cytoskeletal components. One of these, the Rho GDP-dissociation inhibitor RhoGDI1, was analyzed further. RhoGDI1 null cells expressed lower amounts of CAP, which failed to accumulate predominantly at the cell cortex. To further position CAP in the corresponding signal transduction pathways we studied CAP localization and cellular functioning in mutants that have defects in several signaling components. CAP showed correct localization and dynamics in all analyzed strains except in mutants with deficient cAMP dependent protein kinase A activity, where CAP preferentially accumulated in crown shaped structures. Ectopic expression of CAP improved the efficiency of phagocytosis in Gβ-deficient cells and restored the pinocytosis, morphology and actin distribution defects in a PI3 kinase double mutant (pi3k1/2 null). Our results show that CAP acts at multiple crossroads and links signaling pathways to the actin cytoskeleton either by physical interaction with cytoskeletal components or through regulation of their gene expression.     

Biological Control of Chickpea Collar Rot by Co-inoculation of Antagonistic Bacteria and Compatible Rhizobia

Two hundred and seven bacteria were isolated from composts and macrofauna and screened for plant growth promoting and antagonistic traits. Seven of the 207 isolates showed antagonistic activity against Sclerotium rolfsii in plate culture. Inhibition of S. rolfsii by the bacterial isolates ranged between 61 and 84%. Two of the seven isolates were Bacillus sp. and rest belonged to Pseudomonas sp. Two isolates, Pseudomonas sp. CDB 35 and Pseudomonas sp. BWB 21 was compatible with chickpea Rhizobium sp. IC 59 and IC 76 in plate culture conditions. Increase in plant biomass (dry weight) ranged between 18 and 30% on application of these bacteria by seed coating and seed priming methods. However, by seed-priming there was an increase in plant biomass by 5–7% compared to seed coating. Number of nodules and the nodule weight was similar by both seed coating and seed priming methods. Disease incidence was reduced up to 47% in treatments where captan (fungicide) or antagonistic Pseudomonas sp. CDB 35 was applied. Increase in shoot weight was 36% by seed coating with Rhizobium sp. IC 59 and Pseudomonas sp. CDB 35 when compared to captan application. Whereas by seed priming with IC 59 and CDB 35 increased shoot weight by 3 and 39% increase in nodulation was observed.     

Effect of bacteria isolated from composts and macrofauna on sorghum growth and mycorrhizal colonization

Summary Beneficial plant–microbe interactions in the rhizosphere are primary determinants of plant health and soil fertility. The effect of combined inoculation of plant growth-promoting bacteria, Bacillus circulans EB 35, Serratia marcescens EB 67 and Pseudomonas sp. CDB 35 and arbascular mycorrhizal fungi, Glomus spp. on sorghum growth and mycorrhizal colonization was investigated. Plant growth observations taken at 45 days after sowing (DAS) revealed that all the three strains applied along with arbascular mycorrhizae (AM) improved plant biomass from 17 to 20% and mycorrhizal colonization from 25 to 35%. Further studies at 90 DAS also showed improvement in plant growth parameters measured. It was apparent that all the three strains stimulated plant and root growth in combination with AM and infection of sorghum roots with mycorrhizae at 45 DAS was equal to or even greater than the AM + rock phosphate (RP) inoculation at 90 DAS. This shows the possible reduction of AM culturing period to 45 days compared to its 3-month culturing in the pot cultures.     

Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways

We evaluated the ability of neonatal porcine islets to engraft and restore glucose control in pancreatectomized rhesus macaques. Although porcine islets transplanted into nonimmunosuppressed macaques were rapidly rejected by a process consistent with cellular rejection, recipients treated with a CD28-CD154 costimulation blockade regimen achieved sustained insulin independence (median survival, >140 days) without evidence of porcine endogenous retrovirus dissemination. Thus, neonatal porcine islets represent a promising solution to the crucial supply problem in clinical islet transplantation.     

Expression of Ixodes scapularis antifreeze glycoprotein enhances cold tolerance in Drosophila melanogaster

Drosophila melanogaster experience cold shock injury and die when exposed to low non-freezing temperatures. In this study, we generated transgenic D. melanogaster that express putative Ixodes scapularis antifreeze glycoprotein (IAFGP) and show that the presence of IAFGP increases the ability of flies to survive in the cold. Male and female adult iafgp-expressing D. melanogaster exhibited higher survival rates compared with controls when placed at non-freezing temperatures. Increased hatching rates were evident in embryos expressing IAFGP when exposed to the cold. The TUNEL assay showed that flight muscles from iafgp-expressing female adult flies exhibited less apoptotic damage upon exposure to non-freezing temperatures in comparison to control flies. Collectively, these data suggest that expression of iafgp increases cold tolerance in flies by preventing apoptosis. This study defines a molecular basis for the role of an antifreeze protein in cryoprotection of flies.     

Influence of micronutrients on yeast growth and β-<Emphasis Type="SmallCaps">d</Emphasis>-fructofuranosidase production

β-d-fructofuranosidase is one of the most important enzymes of the food industry especially due to its application in preparation of soft centered candies, confectioneries and to produce fructose syrups. Although several bacteria and filamentous fungi are reported for its production, yeasts are the most preferred source for this enzyme. In the present study mineral nutrients were screened for their effect on yeast growth and the enzyme production using Plackett-Burman design. Of the 11 nutrients screened, three variables (KH₂PO₄, FeCl₃ and CoCl₂) were significantly effecting yeast growth while six variables, i.e. KH₂PO₄, CaCl₂, MnSO₄, Na₂MoO₄, ZnSO₄ and CoCl₂ had significant effect on enzyme production.     

Effect of Carbon Substrates on Rock Phosphate Solubilization by Bacteria from Composts and Macrofauna

Five of the 207 isolates from different composts, farm waste compost (FWC), rice straw compost (RSC), Gliricidia vermicompost (GVC), and macrofauna, showed rock phosphate (RP) solubilization in buffered medium in plate culture. When tested in RP broth medium, all five strains, Enterobacter cloacae EB 27, Serratia marcescens EB 67, Serratia sp. EB 75, Pseudomonas sp. CDB 35, and Pseudomonas sp. BWB 21, showed gluconic acid production and solubilized RP. Based on cellulose-degrading and P-solubilizing ability, two strains were selected for further studies. In the presence of different carbon sources, both strains showed a drop in pH and solubilized RP. P released was maximum with glucose (1212 and 522 μmol) and minimum with cellobiose (455 and 306 μmol) by S. marcescens EB 67 and Pseudomonas sp. CDB 35, respectively. Glucose dehydrogenase (GDH) activity was 63 and 77% with galactose and 35 and 46% with cellobiose when compared to glucose (100%) by EB 67 and CDB 35, respectively. Both strains solubilized RP in the presence of different crop residues. EB 67 and CDB 35 showed maximum cellulase activity (0.027 units) in the presence of rice straw and a mixture of rice straw and root. P solubilized from RP in the presence of pigeonpea root was 134 and 140 μmol with EB 67 and CDB 35. Significantly, these bacteria isolated from composts and macrofauna solubilized rock phosphate in the presence of various pure carbon substrates and crop residues and their importance in soil/rhizosphere conditions is discussed.     

Coordination of different ligands to copper(II) and cobalt(III) metal centers enhances Zika virus and dengue virus loads in both arthropod cells and human keratinocytes

Trace elements such as copper and cobalt have been associated with virus-host interactions. However, studies to show the effect of conjugation of copper(II) or cobalt(III) metal centers to thiosemicarbazone ligand(s) derived from either food additives or mosquito repellent such as 2-acetylethiazole or citral, respectively, on Zika virus (ZIKV) or dengue virus (serotype 2; DENV2) infections have not been explored. In this study, we show that four compounds comprising of thiosemicarbazone ligand derived from 2-acetylethiazole viz., (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide (acetylethTSC) (compound 1), a copper(II) complex with acetylethTSC as a ligand (compound 2), a thiosemicarbazone ligand-derived from citral (compound 3) and a cobalt(III) complex with a citral-thiosemicarbazone ligand (compound 4) increased DENV2 and ZIKV replication in both mosquito C6/36 cells and human keratinocytes (HaCaT cells). Treatment of both cell lines with compounds 2 or 4 showed increased dengue viral titers at all three tested doses. Enhanced dengue viral plaque formation was also noted at the tested dose of 100 μM, suggesting higher production of infectious viral particles. Treatment with the compounds 2 or 4 enhanced ZIKV and DENV2 RNA levels in HeLa cell line and primary cultures of mouse bone marrow derived dendritic cells. Also, pre- or post treatments with conjugated compounds 2 or 4 showed higher loads of ZIKV or DENV2 envelope (E) protein in HaCaT cells. No changes in loads of E-protein were found in ZIKV-infected C6/36 cells, when compounds were treated after infection. In addition, we tested bis(1,10-phenanthroline)copper(II) chloride ([Cu(phen)₂]Cl₂, (compound 5) and tris(1,10-phenanthroline)cobalt(III) chloride ([Co(phen)₃]Cl₃, (compound 6) that also showed enhanced DENV2 loads. Also, we found that copper(II) chloride dehydrate (CuCl₂·2H₂O) or cobalt(II) chloride hexahydrate (CoCl₂·6H₂O) alone had no effects as “free” cations. Taken together, these findings suggest that use of Cu(II) or Co(III) conjugation to organic compounds, in insect repellents and/or food additives could enhance DENV2/ZIKV loads in human cells and perhaps induce pathogenesis in infected individuals or individuals pre-exposed to such conjugated complexes.