Population coverage analysis of T-Cell epitopes of Neisseria meningitidis serogroup B from Iron acquisition proteins for vaccine design

Although the concept of Reverse Vaccinology was first pioneered for sepsis and meningococcal meningitidis causing bacterium, Neisseria meningitides, no broadly effective vaccine against serogroup B meningococcal disease is yet available. In the present investigation, HLA distribution analysis was undertaken to select three most promiscuous T-cell epitopes out of ten computationally validated epitopes of Iron acquisition proteins from Neisseria MC58 by using the population coverage tool of Immune Epitope Database (IEDB). These epitopes have been determined on the basis of their binding ability with maximum number of HLA alleles along with highest population coverage rate values for all the geographical areas studied. The comparative population coverage analysis of moderately immunogenic and high immunogenic peptides suggests that the former may activate T-cell response in a fairly large proportion of people in most geographical areas, thus indicating their potential for development of epitope-based vaccine.     

<Emphasis Type="Italic">O</Emphasis>-GlcNAcylation of the <Emphasis Type="Italic">Plum pox virus</Emphasis> capsid protein catalyzed by SECRET AGENT: characterization of <Emphasis Type="Italic">O</Emphasis>-GlcNAc sites by electron transfer dissociation mass spectrometry

The capsid protein of Plum pox virus (PPV-CP) is modified with O-linked β-N-acetylglucosamine (O-GlcNAc). In Arabidopsis thaliana this modification is made by an O-GlcNAc transferase named SECRET AGENT (SEC). Modification of PPV-CP by SEC is hypothesized to have a direct role in the infection process, because virus titer and rate of spread are reduced in SEC mutants. Previous studies used deletion mapping and site-directed mutagenesis to identify four O-GlcNAc sites on the capsid protein that are modified by Escherichia coli-expressed SEC. The infection process was not affected when two of these sites were mutated suggesting that O-GlcNAcylation of these sites does not have a significant role in the infection process or that a subset of the modifications is sufficient. Since it is possible that the mutational mapping approach missed or incorrectly identified O-GlcNAc sites, the modifications produced by E. coli-expressed SEC were characterized using mass spectrometry. O-GlcNAcylated peptides were enzymatically tagged with galactose, the products were enriched on immobilized Ricinus communis agglutinin I and sequenced by electron transfer dissociation (ETD) mass spectrometry. Five O-GlcNAc sites on PPV-CP were identified. Two of these sites were not identified in by the previous mutational mapping. In addition, one site previously predicted by mutation mapping was not detected, but modification of this site was not supported when the mutation mapping was repeated. This study suggests that mapping modification sites by ETD mass spectrometry is more comprehensive and accurate than mutational mapping.     

Engineering bioactive surfaces with Fischer carbene complex: protein A on self-assembled monolayer for antibody sensing

A Fischer carbene complex was grafted onto self-assembled monolayers (SAMs) on gold or glass by a copper-free "click" reaction. Pendant lysine residues of protein A obtained from Staphylococcus aureus rapidly reacted with the electrophilic metal complex on SAM effecting a covalent attachment of protein A with the surface. The protein A coated surface further led to bioaffinity immobilization of rabbit IgG in an oriented manner, a feature that also permits its purification from rabbit serum. Rabbit IgG could be removed from protein A coated surface by pH adjustment. The regenerated protein A surface was reused three times without loss of activity.     

Modeling host-pathogen interactions: H. sapiens as a host and C. difficile as a pathogen

Many complex mechanisms in immunological studies cannot be measured by experiments, but can be analyzed by mathematical simulations. Using theoretical modeling techniques, general principles of host–pathogen system interactions can be explored and clinical treatment schedules can be optimized to lower the microbial toxin burden and side effects in the host system. In this study, we use a computational modeling technique that aims to explain the host–pathogen interactions and suggests how the host system tries to survive from the pathogen attack. The method generates data on reaction fluxes in a pathway at steady state. A set of constraints is incorporated and an objective function for the minimization of toxin expression, with respect to some parameters such as concentration of signaling molecules, is formulated. We have integrated the toxin expression regulatory pathway in Clostridium difficile, apoptosis and mitogen‐activated protein kinase pathways in an infected host (Homo sapiens). We have found that due to the minimization of the toxin expression, the signal flow values for most of the survival genes are at the higher side, whereas it is the reverse for most of the proapoptotic genes. We have observed increased signal flow values of the molecules for extracellular regulated kinase as compared with the molecules present in c‐Jun NH2‐terminal kinase/p38 pathways. In light of these observations, we can hypothesize that lower toxin level in a pathogen implies higher chance of host survival. Copyright © 2012 John Wiley & Sons, Ltd.     

Isolation and characterization of Staphylococcus sp. strain NBRIEAG-8 from arsenic contaminated site of West Bengal

Arsenic contaminated rhizospheric soils of West Bengal, India were sampled for arsenic resistant bacteria that could transform different arsenic forms. Staphylococcus sp. NBRIEAG-8 was identified by16S rDNA ribotyping, which was capable of growing at 30,000?mg?l(-1) arsenate [As(V)] and 1,500?mg?l(-1) arsenite [As(III)]. This bacterial strain was also characterized for arsenical resistance (ars) genes which may be associated with the high-level resistance in the ecosystems of As-contaminated areas. A comparative proteome analysis was conducted with this strain treated with 1,000?mg?l(-1) As(V) to identify changes in their protein expression profiles. A 2D gel analysis showed a significant difference in the proteome of arsenic treated and untreated bacterial culture. The change in pH of cultivating growth medium, bacterial growth pattern (kinetics), and uptake of arsenic were also evaluated. After 72?h of incubation, the strain was capable of removing arsenic from the culture medium amended with arsenate and arsenite [12% from As(V) and 9% from As(III)]. The rate of biovolatilization of As(V) was 23% while As(III) was 26%, which was determined indirectly by estimating the sum of arsenic content in bacterial biomass and medium. This study demonstrates that the isolated strain, Staphylococcus sp., is capable for uptake and volatilization of arsenic by expressing ars genes and 8 new upregulated proteins which may have played an important role in reducing arsenic toxicity in bacterial cells and can be used in arsenic bioremediation.     

Identification of putative SNPs in progressive retinal atrophy affected Canis lupus familiaris using exome sequencing

Progressive retinal atrophy (PRA) is one of the major causes of retinal photoreceptor cell degeneration in canines. The inheritance pattern of PRA is autosomal recessive and genetically heterogeneous. Here, using targeted sequencing technology, we have performed exome sequencing of 10 PRA-affected (Spitz = 7, Cocker Spaniel = 1, Lhasa Aphso = 1 and Spitz-Labrador cross breed = 1) and 6 normal (Spitz = 5, Cocker Spaniel = 1) dogs. The high-throughput sequencing using 454-Roche Titanium sequencer generated about 2.16 Giga bases of raw data. Initially, we have successfully identified 25,619 single nucleotide polymorphisms (SNPs) that passed the stringent SNP calling parameters. Further, we performed association study on the cohort, and the highly significant (0.001) associations were short-listed and investigated in-depth. Out of the 171 significant SNPs, 113 were previously unreported. Interestingly, six among them were non-synonymous coding (NSC) SNPs, which includes CPPED1 A>G (p.M307V), PITRM1 T>G (p.S715A), APP G>A (p.T266M), RNF213 A>G (p.V1482A), C>A (p.V1456L), and SLC46A3 G>A (p.R168Q). On the other hand, 35 out of 113 unreported SNPs were falling in regulatory regions such as 3′-UTR, 5′-UTR, etc. In-depth bioinformatics analysis revealed that majority of NSC SNPs have damaging effect and alter protein stability. This study highlighted the genetic markers associated with PRA, which will help to develop genetic assay-based screening in effective breeding.

     

Frequency of fokI and taqI polymorphism of vitamin D receptor gene in Indian population and its association with 25-hydroxyvitamin D levels

BACKGROUND:

The VDR protein is at the centre of the vitamin D endocrine system, a complex physiological system with substantial feedback regulatory mechanisms involved in maintaining serum calcium and 1, 25 dihydroxy vitamin D3. Variations in VDR gene are shown to have implications in several diseases and have also been implicated as an important genetic factor affecting bone mass.

AIM:

To determine the frequency of Fok I and Taq I variants in healthy Indian individuals and its association with 25-OH-Vitamin D levels.

SETTINGS AND DESIGN:

Blood samples were collected from 143 unrelated normal individuals (Male-84 and Female-59) and their genotypes determined.

MATERIALS AND METHODS:

After amplification by polymerase chain reaction, each polymorphism was genotyped by restriction fragment length polymorphism. For 100 normal healthy individuals 25-hydroxyvitamin D estimation was done using DiaSorin kit method.

STATISTICAL ANALYSIS:

Graph pad software was used to calculate the P values from the Chi-square.

RESULTS:

Out of 143 samples analyzed for FokI and TaqI polymorphisms the following genotypic frequency was obtained FF 59%, Ff 36%, ff 5% and TT 49%, Tt 43%, tt 8% respectively.

CONCLUSIONS:

Results indicate that the distribution of the polymorphic loci Fok I and Taq I vary considerably not only in different populations, but also within India. Furthermore, when the genotypes were analyzed with respect to 25-OH-Vitamin D levels, a significant association was seen for the Taq 1 SNP but not with the Fok I.     

Evaluation of Mycobacterium smegmatis as a possible surrogate screen for selecting molecules active against multi-drug resistant Mycobacterium tuberculosis

New and better drugs are needed for tuberculosis (TB), particularly for the multi-drug resistant (MDR) disease. However, the highly infectious nature of MDR Mycobacterium tuberculosis restricts its use for large scale screening of probable drug candidates. We have evaluated the potential of a screen based on a 'fast grower' mycobacterium to shortlist compounds which could be active against MDR M. tuberculosis. Sensitivity profiles of M. smegmatis, M. phlei and M. fortuitum as well as MDR clinical isolates of M. tuberculosis were determined against anti-TB drugs isoniazid and rifampicin. Among the three fast growers, M. smegmatis was found to display a profile similar to MDR M. tuberculosis. Subsequently we evaluated the performance of M. smegmatis as a 'surrogate' screen for 120 compounds which were synthesized for anti-TB activity. Fifty of these molecules were active against M. tuberculosis H(37)Rv at a minimum inhibitory concentration (MIC) cutoff of 相似文献