A novel fuzzy programming approach for piece selection problem in P2P content distribution network.

Piece selection policy in dynamic P2P networks play crucial role and avoid the last piece problem. BitTorrent uses rarest-first piece selection mechanism to deal with this problem, but its efficacy is limited because each peer only has a local view of piece rareness. The problem of piece section is multiple objectives. A novel fuzzy programming approach is introduced in this article to solve the multiple objectives piece selection problem in P2P network, in which some of the factors are fuzzy in nature. Piece selection problem has been prepared as a fuzzy mixed integer goal programming piece selection problem that includes three primary goals such as minimizing the download cost, time, maximizing speed and useful information transmission subject to realistic constraints regarding peer's demand, capacity and dynamicity. The proposed approach has the ability to handle practical situations in a fuzzy environment and offers a better decision tool to each peer to select optimal pieces to download from other peers in dynamic P2P network. Extensive simulations are carried out to demonstrate the effectiveness of the proposed model. It is proved that proposed system outperforms existing with respect to download cost, time and meaningful exchange of useful information.

Sympatric occurrence of sibling Phytophthora species associated with foot rot disease of black pepper in India.

Foot rot disease caused by Phytophthora capsici is a serious threat to black pepper cultivation in India and globally. High diversity exists among the Phytophthora isolates of black pepper and hence detailed investigations of their morphology and phylogenetic taxonomy were carried out in the present study. In order to resolve the diversity, 182 isolates of Phytophthora, collected from different black pepper-growing tracts of South India during 1998-2013 and maintained in the National Repository of Phytophthora at ICAR-Indian Institute of Spices Research, Kozhikode, were subjected to morphological, molecular and phylogenetic characterization. Morphologically all the isolates were long pedicellate with umbellate/simple sympodial sporangiophores and papillate sporangia with l/b ranging from 1.63 to 2.55 µm. Maximum temperature for the growth was ~ 34 °C. Chlamydospores were observed in "tropicalis" group, whereas they were absent in "capsici" group. Initial molecular studies using internal transcribed spacer (ITS) marker gene showed two clear cut lineages-"capsici-like" and "tropicalis-like" groups among them. Representative isolates from each group were subjected to host differential test, multilocus sequence typing (MLST) and phylogeny studies. MLST analysis of seven nuclear genes (60S ribosomal protein L10, beta-tubulin, elongation factor 1 alpha, enolase, heat shock protein 90, 28S ribosomal DNA and TigA gene fusion protein) clearly delineated black pepper Phytophthora isolates into two distinct species-P. capsici and P. tropicalis. On comparing with type strains from ATCC, it was found that the type strains of P. capsici and P. tropicalis differed from black pepper isolates in their infectivity on black pepper. The high degree of genetic polymorphism observed in black pepper Phytophthora isolates is an indication of the selection pressure they are subjected to in the complex habitat which ultimately may lead to speciation. So based on the extensive analysis, it is unambiguously proved that the foot rot disease of black pepper in India is predominantly caused by two species of Phytophthora, viz. P. capsici and P. tropicalis. Presence of multiple species of Phytophthora in the black pepper agro-ecosystem warrants a revisit to the control strategy being adopted for managing this serious disease. The silent molecular evolution taking place in such an ecological niche needs to be critically studied for the sustainable management of foot rot disease.

Identification of stable reference gene for transcript normalization in black pepper-Phytophthora capsici pathosystem.

A systematic validation of reference genes is a pre-requisite for the proper normalization of gene transcripts. In the present study, the annotated sequences from black pepper (Piper nigrum L.) leaf transcriptome were used as reference genes namely actin (PnACT), glyceraldehyde phosphate dehydrogenase (PnGAPDH), ß-tubulin (PnTUB), ubiquitin conjugating enzyme (PnUBCE), 18srRNA and elongation factor-1-α (PnElF) to identify the stable reference gene. We focused the selection of stable reference gene on important biotic stress (Phytophthora) with different algorithms (geNorm, NormFinder and BestKeeper) along with Reffinder which resulted in identification of PnGAPDH and PnUBCE as stable genes. Norm qPCR (R package) was also used to estimate the stability of the selected genes. We elucidated the expression patterns of a target gene PnBGLU which codes for 1,3 beta glucanase with most stable as well as least stable reference genes by which the importance of selecting the stable gene for gene expression studies in this system was emphasized. The mean expression levels of PnBGLU was significantly overestimated and misinterpreted when least stable reference gene was used as normalizer. The selected reference genes on further analysis of the expression dynamics of PnBGLU among resistant and susceptible genotypes showed PnGAPDH as the suitable reference gene for P. nigrum-P. capsici pathosystem.

Proteomics assisted profiling of antimicrobial peptide signatures from black pepper (Piper nigrum L.).

Plant antimicrobial peptides are the interesting source of studies in defense response as they are essential components of innate immunity which exert rapid defense response. In spite of abundant reports on the isolation of antimicrobial peptides (AMPs) from many sources, the profile of AMPs expressed/identified from single crop species under certain stress/physiological condition is still unknown. This work describes the AMP signature profile of black pepper and their expression upon Phytophthora infection using label-free quantitative proteomics strategy. The differential expression of 24 AMPs suggests that a combinatorial strategy is working in the defense network. The 24 AMP signatures belonged to the cationic, anionic, cysteine-rich and cysteine-free group. As the first report on the possible involvement of AMP signature in Phytophthora infection, our results offer a platform for further study on regulation, evolutionary importance and exploitation of theses AMPs as next generation molecules against pathogens.

Interplay of genes in plant-pathogen interactions: In planta expression and docking studies of a beta 1,3 glucanase gene from Piper colubrinum and a glucanase inhibitor gene from Phytophthora capsici.

Oomycete pathogen, Phytophthora capsici is devastating for black pepper (Piper nigrum L.) and causes foot rot disease at all stages of plant growth. Phytophthora secretes a glucanase inhibitor protein (GIP), which is capable of inhibiting defence proteins like endoglucanases. In this particular study Quantitative PCR analysis, molecular docking studies and analysis of sequences of Glucanase inhibitor protein and beta-1,3 glucanse genes were done mainly depending on the data derived from Phytophthora capsici whole genome sequencing and Piper colubrinum RNA-sequencing (RNA-Seq). Amino acid sequence length of GIP gene from P. capsici was about 353 amino acids and that of glucanase pcEGase gene from P. colubrinum was about 312 amino acids. GIP gene from P. capsici showed high level of expression at early hours of the inoculation time period and pcEGase gene showed high level of expression at 16 hpi. High level of expression of pcEGase gene at 16 hpi is an indication that the GIP gene is successfully inhibited by the glucanase protein from the plant. Moreover insilico studies gave some hint on the importance of certain sites on the surfaces of both interacting proteins that might be having a role in binding of the two proteins and subsequent reactions thereof. Insilico analysis also conclusively proved that inhibition of glucanase inhibitor protein is mainly caused by recognition of an arginine as well as an isoleucine residue during the interaction of the two proteins.

Influence of temperature on symptom expression, detection of host factors in virus infected Piper nigrum L.

Expression of symptoms in black pepper plants (Piper nigrum) infected with Piper yellow mottle virus (PYMoV) vary depending on the season, being high during summer months. Here, we explored the influence of temperature on symptom expression in PYMoV infected P. nigrum. Our controlled environment study revealed increase in virus titer, total proteins, IAA and reducing sugars when exposed to temperature stress. There was change in the 2-D separated protein before and after exposure. The 2-D proteomics LC-MS identified host and viral proteins suggesting virus-host interaction during symptom expression. The analysis as well as detection of host biochemical compounds may help in understanding the detailed mechanisms underlying the viral replication and damage to the crop, and thereby plan management strategies.

Mycobacterial r32-kDa antigen-specific T-cell responses correlate with successful treatment and a heightened anti-microbial response in human leprosy patients.

BACKGROUND: Immunological characterization of mycobacterial peptides may help not only in the preparation of a vaccine for leprosy but also in developing in vitro T-cell assays that could perhaps be used as an in vitro correlate for treatment outcome. The main goal of this study was to evaluate the use of Mycobacterium bovis recombinant 32-kDa protein (r32-kDa) antigen-stimulated T-cell assay as a surrogate marker for treatment outcome and monitor vitamin D receptor (VDR)-mediated anti-microbial responses during multidrug therapy (MDT) in leprosy. METHODS: Newly diagnosed tuberculoid and lepromatous leprosy patients were enrolled and followed up during their course of MDT at 6 and 12 months. IFN-γ, IL-10, IL-17 and IL-23 levels in culture supernatants and expression of VDR, TLR2, LL37 and DEFB in r32-kDa-stimulated PBMCs were measured. Controls comprised household contacts (HHCs) and healthy endemic subjects (HCs). RESULTS: Significant differences were observed in the levels of IFN-γ, IL-17, IL-23, VDR and anti-microbial peptides LL37 and DEFB after treatment and when compared with that of HHCs and HCs, respectively. CONCLUSIONS: These findings suggest that responses to r32-kDa antigen reflect an improved immunological and anti-microbial response in leprosy patients during therapy, thereby indicating its potential use as an immune correlate in the treatment of leprosy patients.

An Efficient Framework for Large Scale Multimedia Content Distribution in P2P Network: I2NC.

Network coding (NC) makes content distribution more effective and easier in P2P content distribution network and reduces the burden of the original seeder. It generalizes traditional network routing by allowing the intermediate nodes to generate new coded packet by combining the received packets. The randomization introduced by network coding makes all packets equally important and resolves the problem of locating the rarest block. Further, it reduces traffic in the network. In this paper, we analyze the performance of traditional network coding in P2P content distribution network by using a mathematical model and it is proved that traffic reduction has not been fully achieved in P2P network using traditional network coding. It happens due to the redundant transmission of noninnovative information block among the peers in the network. Hence, we propose a new framework, called I2NC (intelligent-peer selection and incremental-network coding), to eliminate the unnecessary flooding of noninnovative coded packets and thereby to improve the performance of network coding in P2P content distribution further. A comparative study and analysis of the proposed system is made through various related implementations and the results show that 10-15% of traffic reduced and improved the average and maximum download time by reducing original seeder's workload.

Association of Taq I, Fok I and Apa I polymorphisms in Vitamin D Receptor (VDR) gene with leprosy.

BACKGROUND: Vitamin D Receptor (VDR) is a transacting transcription factor which mediates immunomodulatory function and plays a key role in innate and adaptive immune responses through its ligand and polymorphisms in VDR gene may affect its regulatory function. OBJECTIVE: To investigate the association of three VDR gene polymorphisms (TaqI rs731236, FokI rs2228570 and ApaI rs7975232) with leprosy. METHODS: The study group includes 404 participants of which 222 were leprosy patients (paucibacillary=87, multibacillary=135) and 182 healthy controls. Genotyping was done using PCR-RFLP technique. Statistical analysis was performed using SNP Stats and PLINK software. RESULTS: The VDR FokI (rs2228570) ff genotype, ApaI (rs7975232) AA, Aa genotype and haplotype T-f-a, T-F-A were positively associated with leprosy when compared to healthy controls. CONCLUSION: The two variants at Fok and Apa positions in VDR gene are significantly associated with leprosy. Genotypes at FokI (ff), ApaI (aa) and haplotype (T-F-a, T-f-a) may contribute to the risk of developing leprosy by altering VDR phenotype/levels subsequently modulation of immune response.

Mating type genes and genetic markers to decipher intraspecific variability among Fusarium udum isolates from pigeonpea.

To ascertain the variability in Fusarium udum (Fu) isolates associated with pigeonpea wilt is a difficult task, if based solely on morphological and cultural characters. In this respect, the robustness of five different genetic marker viz., random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC), BOX elements, mating type locus, and microsatellite markers were employed to decipher intra-specific variability in Fu isolates. All techniques yielded intra-specific polymorphism, but different levels of discrimination were obtained. RAPD-PCR was more discriminatory, enabling the detection of thirteen variants among twenty Fu isolates. By microsatellite, ERIC- and BOX-PCR fingerprinting, the isolates were categorized in seven, five, and two clusters, respectively. Cluster analysis of the combined data also showed that the Fu isolates were grouped into ten clusters, sharing 50-100% similarity. The occurrence of both mating types in Fu isolates is reported for the first time in this study. All examined isolates harbored one of the two mating-type idiomorphs, but never both, which suggests a heterothallic mating system of sexual reproduction among them. Information obtained from comparing results of different molecular marker systems should be useful to organize the genetic variability and ideally, will improve disease management practices by identifying sources of inoculum and isolate characteristics.

Genetic association of G896A polymorphism of TLR4 gene in leprosy through family-based and case-control study designs.

BACKGROUND: Polymorphisms in TLR4 may change the function of the protein and alter the efficiency of immune response of host to infection. The high relevance of host gene polymorphisms with outcome of Mycobacterium leprae infection led us to study the genetic association of TLR4 G896A polymorphism in order to identify its risk among contacts of affected leprosy patients. METHODS: For case-control study design a total of 628 individuals were recruited; 17 multicase leprosy families which included 32 case-parent trios were considered for family-based study. Genotyping was done using PCR-RFLP method. RESULTS: In case-control study AA genotype was positively associated while GA genotype was negatively associated with leprosy. In family based transmission disequilibrium test (TDT) analysis allele G was found to be over transmitted to the affected individuals. CONCLUSION: Case-control study suggests that homozygous AA genotype may confer susceptibility and heterozygous GA genotype may confer resistance to leprosy, while allele A was observed to increase risk and that of allele G may confer resistance to leprosy. No strong transmission disequilibrium was detected in family-based TDT analysis, possibly due to lower number of trios. In contrast to case-control data allele G was over transmitted to the affected ones in TDT analysis. To conclude, the frequencies of genotypes in household contacts were almost the same as in leprosy patients, suggesting that contacts with AA genotype may be at higher risk of leprosy and may therefore require prophylactic inputs.

Influence of Intron II microsatellite polymorphism in human toll-like receptor 2 gene in leprosy.

Leprosy is a chronic granulomatous infection caused by the obligate intracellular organism Mycobacterium leprae. TLR2 plays a key role when activated by M. leprae lipoproteins initiating protective responses which induce bacterial killing and therefore control of disease spread. Microsatellite polymorphisms in intron2 of TLR2 gene have been reported to be associated with development of clinical features of several infectious diseases. The study aims to evaluate the influence of GT microsatellite on the expression of TLR2 which could make humans prone to M. leprae infections. A total of 279 individuals were enrolled in the study, 88 were leprosy patients, 95 were house hold contacts (HHC) and 96 were healthy controls (HC). Genotyping was done using PCR-Sequencing method. TLR2 mRNA expression was analyzed by RT-PCR. IL-10 and IFN-γ levels were measured using ELISA in MLSA stimulated cell culture supernatants. Statistical analysis was performed using Chi-Square (χ(2)) test and t-tests. Allele/genotype of TLR2 microsatellite which includes longer GT repeats was associated with low TLR2 mRNA expression and high IL-10 production while that including shorter GT repeats was associated with high TLR2 mRNA expression and low IL-10 production. High IL10 producing allele of TLR2 microsatellite might predispose house hold contacts to leprosy.

Distribution of CGG/GCC repeats at the FMR1 and FMR2 genes in an Indian population with mental retardation of unknown etiology.

AIMS: Fragile X syndrome is one of the X-linked disorders associated with moderate to severe mental retardation. Fragile X A syndrome (FRAXA) and fragile X E syndrome (FRAXE) are caused by trinucleotide repeat expansion of CGG and GCC repeats at the 5' untranslated region of the FMR1 and FMR2 genes, respectively. The present study was undertaken to identify the repeat polymorphism and to estimate the risk of transmission in Andhra Pradesh and surrounding states of South India. RESULTS: The FRAXA and FRAXE allelic polymorphisms were studied by radioactive polymerase chain reaction that revealed 25 FRAXA among 344 X-chromosomes and 20 FRAXE allelic variants among 212 X-chromosomes in our population. The most frequent FRAXA allele size was of 29 CGG repeats (27.5%) followed by allele sizes of 28 (20.8%) and 31 (7.2%), and that of FRAXE was 15 GCC repeats (24.0%) followed by allele containing 18 repeats (18.4%) and 16 repeats (11.3%). CONCLUSIONS: CGG/GCC repeat polymorphism at the FMR1 and FMR2 loci observed in this study demonstrated a racial and ethnic variation among the populations.

Assessment of a clinical checklist in the diagnosis of fragile X syndrome in India.

Fragile X syndrome (FRAXA) is one of the most common forms of mental retardation. It is caused by the expansion of cytosine-guanine-guanine (CGG) repeats in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene, located at Xq27.3. The number of CGG repeats in the FMR1 gene occurs in four distinct ranges: 2-50 (normal), 50-60 (gray zone), 60-200 (premutation), and > 200 (full mutation). When the number of CGG repeats exceeds 200, the gene becomes hypermethylated and transcriptionally silenced, which results in the loss of FMR protein and causes FRAXA. The key clinical features of FRAXA are mental retardation, macro-orchidism, long face, prominent jaw, connective tissue abnormalities, and behavioral problems. A modified 15-item checklist was used to assess the clinical features in 337 individuals (316 males and 21 females) who have mental retardation of unknown etiology. These patients were in institutions. Molecular diagnosis was performed using polymerase chain reaction and Southern blot analysis and revealed that 14 males were positive for FRAXA. Studies of the families of the affected males revealed an additional 11 affected males and 20 carrier females. Retrospective analysis of clinical features was performed in a total of 327 males and 41 females. Six clinical features were statistically significant in FRAXA individuals when compared to non-FRAXA individuals. These features were hyperactivity (p<0.05), poor eye contact (p<0.001), hyper extensibility of joints (p<0.001), large ears (p<0.001), macro-orchidism (p<0.001), and a family history of mental retardation (p<0.001). When a total score of 5 out of 15 was used as the threshold clinical score, 73.18% of the patients with total scores < 5 could be eliminated as FRAXA-negative patients, thereby improving the reliability of FRAXA testing using the clinical checklist.

Protein phosphorylation pattern in the immune cells of leprosy affected individuals.

BACKGROUND: Leprosy is an infectious disease in which the susceptibility to the pathogen Mycobacterium leprae and the clinical manifestations are attributed to host immune cell response. Receptor mediated events and signalling in the immune cells are mediated by protein phosphorylation. The main signalling pathways and protein kinases known to be involved in the regulation of immune cells are cAMP dependent kinases, calcium/calmodulin dependent kinases, protein kinase C and mitogen activated protein kinases. The cumulative consequence of alterations in signalling pathways can be evaluated by intrinsic cellular protein phosphorylation by gamma-P32 ATP. The present study was designed to assess the protein phosphorylation in the immune cells of leprosy patients as compared with normal individuals. METHODOLOGY: Lymphocyte protein phosphorylation was conducted in 15 leprosy patients and 9 normal individuals. Protein phosphorylation of lymphocytes was carried out in the presence/absence of protein kinase modulators. The phosphorylation patterns were documented and analysed consequent to SDS-PAGE, staining, destaining, drying and autoradiography. RESULTS: The major phosphorylated proteins in lymphocytes were of molecular weights 20-22, 24-29, 30-35, 43, 46-50 and 66-68 kDa. In general, the major phosphorylated proteins were similar in the controls and in the patients. The phosphorylatability of these proteins varied with different modulators. Variations in the phosphorylation pattern were observed in 25% of the leprosy patients where there was a decrease of the 66 kDa protein and a decrease of 20-22 kDa protein phosphorylation. CONCLUSION: The observed alterations in the protein phosphorylation pattern could be due to alteration in kinases and/or their substrates or due to the effect of M. leprae on immune cells.

Decreased calcineurin activity in circulation of Duchenne muscular dystrophy.

OBJECTIVES: In an analysis of enzymes in easily accessible tissues like blood cells, serum can provide a valuable information and a simple tool for disease and carrier detection. In the study presented we have analyzed calcineurin activity in Duchenne muscular dystrophy (DMD) and carrier sera and lymphocytes for its diagnostic value and its status in DMD pathology. DESIGN AND METHODS: We have monitored calcineurin activity in sera and lymphocytes of DMD, in carriers and in controls using colorimetric method by following the p-nitrophenol released in the presence and absence of Trifluoperazine (TFP), an inhibitor of calcineurin. RESULTS: Results showed a significant decrease in serum and lymphocyte calcineurin activity in DMD (p<0.001) without alteration in carriers compared to normal. CONCLUSION: Further studies are required to understand possible alterations mediated by calcineurin with reference to DMD lymphocytes as any alteration in phosphorylation/dephosphorylation pathway can disturb the normal functioning of these cells. The decreased calcineurin activity observed in DMD serum compared with controls could be further examined for its diagnostic utility.

Altered expression, intracellular distribution and activity of lymphocyte calpain II in Duchenne muscular dystrophy.

BACKGROUND: Calpain II is an calcium-dependent cysteine protease involved in essential regulatory or processing functions of the cell, mediated by physiological concentrations of Ca(2+). However, in an environment of abnormal intracellular calcium as in Duchenne muscular dystrophy (DMD), calpain is suggested to cause membrane alterations. METHODS: Twelve individuals with dystrophin gene deletion and an equal number of age and sex matched controls were chosen for the study. The expression pattern of calpain II (both at RNA and protein levels), its cellular location upon activation and its activity in lymphocytes were specifically assessed to know if our earlier report of increased calpain activity in DMD lymphocytes is a result of de novo synthesis or is due to basic defect in calcium handling. RESULTS: We found a significant increase in the expression, alteration in calpain II distribution and increased activity of this enzyme. CONCLUSION: Membrane abnormalities and altered signaling pathways observed in DMD lymphocytes may be due to increased association of calpain II onto membrane and cytosol.

Activity of novel protein kinase C and distribution of protein kinase C theta in subcellular fractions of normal and Duchenne muscular dystrophic muscle.

Phospholipid-dependent, Ca(2+)-independent isoenzymes termed novel protein kinase C or nPKC, include PKC delta, epsilon, eta, theta and mu. Status and role of nPKC and PKC theta in Duchenne muscular dystrophic (DMD) condition is unknown. In the present study, we have shown that most of the nPKC isoforms are translocated to the membrane fraction of DMD tissue specimen. It is well established that translocation plays a key role in signal transduction by individual PKC isoforms. In our experiment, the increased association of nPKC isoform PKC theta to membrane was further confirmed by Western blot. Increased expression of PKC theta mRNA was identified by dot blot analysis. The above results suggest that, the alterations in nPKC location and increased expression of PKC theta observed is a result of modification of PKC-mediated signal transduction and cell function.

Deletion analysis & calpain status for carrier detection in a family with Duchenne muscular dystrophy.

Eight females with a family history of Duchenne muscular dystrophy (DMD) were analysed for their carrier status by m-calpain test, which monitors the m-calpain (milli-calpain), a proteolytic enzyme in the platelets, using an ELISA technique. Four of the eight females were identified as carriers by virtue of their elevated enzyme levels as compared to control. DNA samples of these members were analysed to ascertain the carrier status, by PCR followed by dosage analysis by densitometry. DNA analysis confirmed the findings by calpain test, which underlines the reliability of this phenotypic test for carrier detection in DMD. Calpain test has been informative in a large group of patients and carriers tested so far. Since the calpain test is cost and labour effective, it is suited for routine and widespread screening purposes.