Analysis of transmembrane domain mutants is consistent with sequential cleavage of Notch by gamma-secretase

gamma-Secretase is a lipid-embedded, intramembrane-cleaving aspartyl protease that cleaves its substrates twice within their transmembrane domains (TMD): once near the cytosolic leaflet (at S3/epsilon) and again in the middle of the TMD (at S4/gamma). To address whether this unusual process occurs in two independent or interdependent steps, we investigated how mutations at the S3/epsilon site in Notch1-based substrates impact proteolysis. We demonstrate that such mutations greatly inhibit not only gamma-secretase-mediated cleavage at S3 but also at S4, independent of their impact on NICD stability. These results, together with our previous observations, suggest that hydrolysis at the center of the Notch transmembrane domain (S4/gamma) is dependent on the S3/epsilon cleavage. Notch (and perhaps all gamma-secretase substrates) may be cleaved by sequential proteolysis starting at S3.     

Maintaining optimal state probabilities in biological systems

A biological problem is usually studied experimentally by reducing it into a number of modules. In contrast, the systems biology approach seeks to address the collective behavior of interacting molecules vis-a-vis the corresponding higher level behavior. Various attributes of a biological system are conditionally dependent on each other, and these conditionalities are usually represented through Bayesian networks for computing easily the joint probability for a state of an attribute. In this article, a genetic algorithm is investigated to a biological system, by representing it through a Bayesian network, for evaluating the optimum state probabilities of different attributes, in order to obtain a desired joint probability for a given state of an attribute. We believe that such a study would be helpful in achieving a desired health condition by maintaining various attributes of a system to their estimated optimum levels.     

Risk Factors for MDR and XDR-TB in a Tertiary Referral Hospital in India

Background

India has a high burden of drug resistant TB, although there are few data on XDR-TB. Although XDR-TB has existed previously in India, the definition has not been widely applied, and surveillance using second line drug susceptibility testing has not been performed. Our objective was to analyze clinical and demographic risk factors associated with isolation of MDR and XDR TB as compared to susceptible controls, at a tertiary center.

Methodology/Findings

Retrospective chart review based on positive cultures isolated in a high volume mycobacteriology laboratory between 2002 and 2007. 47 XDR, 30 MDR and 117 susceptible controls were examined. Drug resistant cases were less likely to be extrapulmonary, and had received more previous treatment regimens. Significant risk factors for XDR-TB included residence outside the local state (OR 7.43, 3.07-18.0) and care costs subsidized (OR 0.23, 0.097-0.54) in bivariate analysis and previous use of a fluoroquinolone and injectable agent (other than streptomycin) (OR 7.00, 95% C.I. 1.14-43.03) and an initial treatment regimen which did not follow national guidelines (OR 5.68, 1.24-25.96) in multivariate analysis. Cavitation and HIV did not influence drug resistance.

Conclusions/Significance

There is significant selection bias in the sample available. Selection pressure from previous treatment and an inadequate initial regimen increases risk of drug resistance. Local patients and those requiring financial subsidies may be at lower risk of XDR-TB.     

The progress and potential of proteomic biomarkers for type 1 diabetes in children

Introduction: Although it is possible to identify the genetic risk for type 1 diabetes (T1D), it is not possible to predict who will develop the disease. New biomarkers are needed that would help understand the mechanisms of disease onset and when to administer targeted therapies and interventions.

Areas covered: An overview is presented of international study efforts towards understanding the cause of T1D, including the collection of several extensive temporal sample series that follow the development of T1D in at risk children. The results of the proteomics analysis of these materials are presented, which have included bodily fluids, such as serum or plasma and urine, as well as tissue samples from the pancreas.

Expert commentary: Promising recent reports have indicated detection of early proteomic changes in the serum of patients prior to diagnosis, potentially providing new measures for risk assessment. Similarly, there has been evidence that post-translational modification (PTM) may result in the recognition of islet cell proteins as autoantigens; modified proteins could thus be used as targets for immunomodulation to overcome the threat of the autoimmune response.     

Cyanobacteria-mediated phenylpropanoids and phytohormones in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) enhance plant growth and stress tolerance

Phenylpropanoids, flavonoids and plant growth regulators in rice (Oryza sativa) variety (UPR 1823) inoculated with different cyanobacterial strains namely Anabaena oryzae, Anabaena doliolum, Phormidium fragile, Calothrix geitonos, Hapalosiphon intricatus, Aulosira fertilissima, Tolypothrix tenuis, Oscillatoria acuta and Plectonema boryanum were quantified using HPLC in pot conditions after 15 and 30 days. Qualitative analysis of the induced compounds using reverse phase HPLC and further confirmation with LC-MS/MS showed consistent accumulation of phenolic acids (gallic, gentisic, caffeic, chlorogenic and ferulic acids), flavonoids (rutin and quercetin) and phytohormones (indole acetic acid and indole butyric acid) in rice leaves. Plant growth promotion (shoot, root length and biomass) was positively correlated with total protein and chlorophyll content of leaves. Enzyme activity of peroxidase and phenylalanine ammonia lyase and total phenolic content was fairly high in rice leaves inoculated with O. acuta and P. boryanum after 30 days. Differential systemic accumulation of phenylpropanoids in plant leaves led us to conclude that cyanobacterial inoculation correlates positively with plant growth promotion and stress tolerance in rice. Furthermore, the study helped in deciphering possible mechanisms underlying plant growth promotion and stress tolerance in rice following cyanobacterial inoculation and indicated the less explored avenue of cyanobacterial colonization in stress tolerance against abiotic stress.     

A propionyloxy derivative of 11-keto-β-boswellic acid induces apoptosis in HL-60 cells mediated through topoisomerase I & II inhibition

Boswellic acids have invariably been reported for their antiproliferative potential in various cell systems. In the present study the growth inhibitory effect of propionyloxy derivative of 11-keto-β-boswellic acid (PKBA; a semisynthetic analogue of 11-keto-β-boswellic acid) on HL-60 promyelocytic leukemia cells is being reported for the first time. In the preliminary studies, in vitro cytotoxicity of PKBA was investigated against eight human cancer cell lines viz., IMR-32, SF-295 (both neuroblastoma), PC-3 (prostate), Colo-205 (colon), MCF-7 (breast), OVCAR-5 (ovary), HL-60, Molt-4 (both leukemia) and their respective IC(50) values were found to be 5.95, 7.11, 15.2, 14.5, 15, 15.9, 8.7 & 9.5μg/ml, respectively. For determining the mechanism of cell death in HL-60 cells, PKBA was subjected to different mechanistic studies. DNA relaxation assay of PKBA revealed inhibition of both topoisomerases I & II. The fragmentation analysis of DNA revealed typical ladders indicating the cytotoxic effect to be mediated by induction of apoptosis. The morphologic studies of PKBA showed the presence of true apoptotic bodies. Apoptosis was confirmed further by flow-cytometric detection of sub-G(1) peaks and enhanced annexin-V-FITC binding of the cells. The activation of apoptotic cascade by PKBA in HL-60 cells was found to be associated with the loss of mitochondrial membrane potential, release of cytochrome c, activation of initiator and executioner caspases and cleavage of poly ADP ribose polymerase (PARP). In vivo studies of PKBA revealed anti-tumoral activity against both ascitic and solid murine tumor models. These studies thus demonstrate PKBA to induce apoptosis in HL-60 cells due to the inhibition of topoisomerases I and II.     

4-(Ethoxycarbonyl) phenyl-1-amino-oxobutanoic acid-chitosan complex as a new matrix for silver nanocomposite film: preparation, characterization and antibacterial activity

The present work describes the preparation of new chitosan complex with 4-(ethoxycarbonyl) phenyl-1-amino-oxobutanoic acid (ETHA), as a matrix for silver nanoparticles to obtain a nanocomposite film by solution casing method. The characterization of the prepared nanocomposite film was made by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetry (TG) and scanning electron microscopy (SEM). The optical property of nanocomposite film was analyzed by UV-Visible and photo-luminescence (PL) spectroscopy. The nanocomposite film was screen for antibacterial activity with Staphylococcus aureus (gram positive), Pseudomonas aurigionasa (gram negative) and Escherichia coli (gram negative) bacteria by adopting the disk diffusion method. The result of antibacterial study revealed that the prepared nanocomposite film may be a promising candidate for wide range of bio-medical applications.     

Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.     

Potent quinoxaline-based inhibitors of PDGF receptor tyrosine kinase activity. Part 2: the synthesis and biological activities of RPR127963 an orally bioavailable inhibitor

RPR127963 demonstrates an excellent pharmacokinetic profile in several species and was found to be efficacious in the prevention of restenosis in a Yucatan mini-pig model upon oral administration of 1-5 mg/kg. The in vitro selectivity profile and SAR of the highly optimized PDGF-R tyrosine kinase inhibitor are highlighted.     

Multifarious plant growth promoting characteristics of chickpea rhizosphere associated Bacilli help to suppress soil-borne pathogens

Wilt and root rot are the major constraints in chickpea production and very difficult to manage through agrochemicals. Hence, for an ecofriendly and biological management, 240 strains of Bacillus and Bacillus derived genera were isolated from chickpea rhizosphere, further narrowed down to 14 strains on the basis of in vitro production of indole acetic acid, siderophore, phosphate solubilization, hydrolytic enzymes and were evaluated for antagonism against chickpea pathogens (Fusarium oxysporum f. sp. ciceri race 1, F. solani and Macrophomina phaseolina). The strains were identified on the basis of physiological characters and 16S RNA gene sequencing. The genotypic comparisons of strains were determined by BOX-polymerase chain reaction profiles and amplified rDNA restriction analysis. These isolates were evaluated in greenhouse assay in which B. subtilis (B-CM191, B-CV235, B-CL-122) proved to be effective in reducing wilt incidence and significant enhancement in growth (root and shoot length) and dry matter of chickpea plants. PCR amplification of bacillomycin (bmyB) and β-glucanase genes suggests that amplified genes from the Bacillus could have a role to further define the diversity, ecology, and biocontrol activities in the suppression of soil-borne pathogens.