Testing effects of signal transduction pathways on cadherin junctional complex assembly using quantitative image analysis

Cadherin adhesion molecules function in numerous cell biological processes that influence embryo development, normal cell physiology, and pathophysiology of many disease processes. Cadherins nucleate the assembly of the adherens junction, a cell-to-cell adhesion plaque that is prominent in simple epithelial cells and found in many cell types. Numerous cell biological approaches have been used to study this interesting class of molecules. Here, we outline methodology used in our studies of junctional complexes to examine effects of signaling molecules on assembly mechanisms. This is a quantitative method that allows the investigator to test the combined effect of two different signaling processes to determine whether these two signals act in concert within the same pathway. We discuss how this method could be generalized to other studies to examine consequences of various experimental manipulations on the assembly of cellular structures.     

Phylogenetic and Molecular Clock Analysis of Dengue Serotype 1 and 3 from New Delhi,India

Dengue fever is the most prevalent arboviral disease in the tropical and sub-tropical regions of the world. The present report describes molecular detection and serotyping of dengue viruses in acute phase blood samples collected from New Delhi, India. Phylogenetic and molecular clock analysis of dengue virus serotype 1 and 3 strains were also investigated. Dengue virus infection was detected in 68.87% out of 604 samples tested by RT-PCR between 2011 & 2014. Dengue serotype 1 was detected in 25.48% samples, dengue serotype 2 in 79.56% samples and dengue serotype 3 in 11.29% samples. Dengue serotype 4 was not detected. Co-infection by more than one dengue serotype was detected in 18.26% samples. Envelope gene of 29 DENV-1 and 14 DENV-3 strains were sequenced in the study. All the DENV-1 strains grouped with the American African genotype. All DENV-3 strains were found to belong to Genotype III. Nucleotide substitution rates of dengue 1 and 3 viruses were determined in the study. Time to the most recent common ancestor (TMRCA) of dengue 1 viruses was determined to be 132 years. TMRCA of DENV-3 viruses was estimated to be 149 years. Bayesian skyline plots were constructed for Indian DENV-1 and 3 strains which showed a decrease in population size since 2005 in case of DENV- 1 strains while no change was observed in recent years in case of DENV-3 strains. The study also revealed a change in the dominating serotype in Delhi, India in recent years. The study will be helpful in formulating control strategies for the outbreaks. In addition, it will also assist in tracking the movement and evolution of this emerging virus.     

High-temperature biotrickling filtration of hydrogen sulphide

Biofiltration of malodorous reduced sulphur compounds such as hydrogen sulphide has been confined to emissions that are at temperatures below 40°C despite the fact that there are many industrial emissions (e.g. in the pulp and paper industry) at temperatures well above 40°C. This paper describes our study on the successful treatment of hydrogen sulphide gas at temperatures of 40, 50, 60 and 70°C using a microbial community obtained from a hot spring. Three biotrickling filter (BTF) systems were set up in parallel for a continuous run of 9 months to operate at three different temperatures, one of which was always at 40°C as a mesophilic control and the other two were for exploring high-temperature operation up to 70°C. The continuous experiment and a series of batch experiments in glass bottles (250 ml) showed that addition of glucose and monosodium glutamate enhanced thermophilic biofiltration of hydrogen sulphide gas and a removal rate of 40 g m⁻³ h⁻¹ was achieved at 70°C. We suggest that the glucose is acting as a carbon source for the existing microbial community in the BTFs, whereas glutamate is acting as a compatible solute. The use of such organic compounds to enhance biodegradation of hydrogen sulphide, particularly at high temperatures, has not been demonstrated to our knowledge and, hence, has opened up a range of possibilities for applying biofiltration to hot gas effluent.     

Immunosuppressive and anti-angiogenic sphingosine 1-phosphate receptor-1 agonists induce ubiquitinylation and proteasomal degradation of the receptor

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and beta-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.     

Beneficial effects of banana (<Emphasis Type="Italic">Musa</Emphasis> sp. var. elakki bale) flower and pseudostem on hyperglycemia and advanced glycation end-products (AGEs) in streptozotocin-induced diabetic rats

Diabetes is a chronic health problem and major cause of death in most of the countries. Diet management plays an important role in controlling diabetes and its complications along with insulin and drugs. We have examined the effect of banana (Musa sp. var. elakki bale) flower and pseudostem on hyperglycemia and advanced glycation end-products (AGEs) in streptozotocin-induced diabetic rats. Our results indicated that banana flower and pseudostem have low glycemic index and have a high content of dietary fiber and antioxidants. Diabetic symptoms like hyperglycemia, polyuria, polyphagia, polydipsia, urine sugar, and body weight were ameliorated in banana flower- and pseudostem-treated rats. Increased glomerular filtration rate in the diabetic group (5.1 ± 0.22 ml/min) was decreased in banana flower-fed (2.5 ± 0.37 ml/min) and pseudostem-fed (3.0 ± 0.45 ml/min) groups and were significant at P < 0.001 and P < 0.01, respectively. Fructosamine and AGEs formed during diabetes were inhibited in treated groups when compared with the diabetic group. The diabetic group showed 11.5 ± 0.64 μg of AGEs/mg protein in kidney, whereas, in banana flower- and pseudostem-fed groups, it was reduced to 9.21 ± 0.32 and 9.29 ± 0.24 μg/mg protein, respectively, and were significant at P < 0.01. These findings suggest that banana flower and pseudostem have anti-diabetic and anti-AGEs properties and are beneficial as food supplements for diabetics.     

Development and validation of functional marker targeting an InDel in the major rice blast disease resistance gene Pi54 (Pik h )

Rice blast is one of the most devastating diseases affecting the rice crop throughout the world. In molecular breeding for host plant resistance, functional markers are very useful for enhancing the precision and accuracy in marker-assisted selection (MAS) of target gene(s) with minimum effort, time and cost. Pi54 (which was earlier known as Pik ^h ) is one of the major blast resistance genes and has been observed to show resistance against many isolates of the blast pathogen in India. The gene has been cloned through map-based strategy and encodes a nucleotide-binding site?Cleucine-rich repeat (NBS?CLRR) domain-containing protein. In the present study, we carried out allele mining for this gene and identified a 144-bp insertion/deletion (InDel) polymorphism in the exonic region of the gene. A PCR-based co-dominant molecular marker targeting this InDel, named Pi54 MAS, was developed. Pi54 MAS was observed to perfectly co-segregate with blast resistance in a mapping population with no recombinants. Validation of this marker in 105 genotypes which are either susceptible or resistant to rice blast disease showed that the marker is polymorphic in most of the resistant?Csusceptible genotype combinations and is more accurate than the earlier reported markers for Pi54. Hence this functional, co-dominant marker is suggested for routine deployment in MAS of Pi54 in breeding programs.     

QTL analysis for grain quality traits in 2 BC2F2 populations derived from crosses between Oryza sativa cv Swarna and 2 accessions of O. nivara

The appearance and cooking quality of rice determine its acceptability and price to a large extent. Quantitative trait loci (QTLs) for 12 grain quality traits were mapped in 2 mapping populations derived from Oryza sativa cv Swarna × O. nivara. The BC(2)F(2) population of the cross Swarna × O. nivara IRGC81848 (population 1) was evaluated during 2005 and that from Swarna × O. nivara IRGC81832 (population 2) was evaluated during 2006. Linkage maps were constructed using 100 simple sequence repeat (SSR) markers in population 1 and 75 SSR markers in population 2. In all, 21 QTLs were identified in population 1 (43% from O. nivara) and 37 in population 2 (38% QTLs from O. nivara). The location of O. nivara-derived QTLs mp1.2 for milling percent, kw6.1 for kernel width, and klac12.1 for kernel length after cooking coincided in the 2 populations and appear to be useful for Marker Assisted Selection (MAS). Four QTLs for milling percent, 1 QTL each for amylose content, water uptake, elongation ratio, 2 QTLs for kernel width, and 3 QTLs for gel consistency, each explained more than 20% phenotypic variance. Three QTL clusters for grain quality traits were close to the genes/QTLs for shattering and seed dormancy. QTLs for 4 quality traits were associated with 5 of the 7 major yield QTLs reported in the same 2 mapping populations. Useful introgression lines have been developed for several agronomic traits. It emerges that 40% O. nivara alleles were trait enhancing in both populations, and QTLs for grain quality overlapped with yield meta-QTLs and QTLs for dormancy and seed shattering.