Molecular dynamics study of the KcsA potassium channel

The structural, dynamical, and thermodynamic properties of a model potassium channel are studied using molecular dynamics simulations. We use the recently unveiled protein structure for the KcsA potassium channel from Streptomyces lividans. Total and free energy profiles of potassium and sodium ions reveal a considerable preference for the larger potassium ions. The selectivity of the channel arises from its ability to completely solvate the potassium ions, but not the smaller sodium ions. Self-diffusion of water within the narrow selectivity filter is found to be reduced by an order of magnitude from bulk levels, whereas the wider hydrophobic section of the pore maintains near-bulk self-diffusion. Simulations examining multiple ion configurations suggest a two-ion channel. Ion diffusion is found to be reduced to approximately (1)/(3) of bulk diffusion within the selectivity filter. The reduced ion mobility does not hinder the passage of ions, as permeation appears to be driven by Coulomb repulsion within this multiple ion channel.     

Selection of predicted siRNA as potential antiviral therapeutic agent against influenza virus

Influenza virus A (IVA) infection is responsible for recent death worldwide. Hence, there is a need to develop therapeutic agents against the virus. We describe the prediction of short interfering RNA (siRNA) as potential therapeutic molecules for the HA (Haemagglutinin) and NA (Neuraminidase) genes. We screened 90,522 siRNA candidates for HA and 13,576 for NA and selected 1006 and 1307 candidates for HA and NA, respectively based on the proportion of viral sequences that are targeted by the corresponding siRNA, with complete matches. Further short listing to select siRNA with no off-target hits, fulfilling all the guidelines mentioned in approach, provided us 13 siRNAs for haemagglutinin and 13 siRNAs for neuraminidase. The approach of finding siRNA using multiple sequence alignments of amino acid sequences has led to the identification of five conserved amino acid sequences, three in hemagglutinin i.e. RGLFGAIAGFIE, YNAELLV and AIAGFIE and two in neuraminidase i.e. RTQSEC and EECSYP which on reveres translation provided siRNA sequences as potential therapeutic candidates. The approaches used during this study have enabled us to identify potentially therapeutic siRNAs against divergent IVA strains.     

Analysis of glycosylation motifs and glycosyltransferases in Bacteria and Archaea

The process of glycosylation has been studied extensively in prokaryotes but many questions still remain unanswered. Glycosyltransferase is the enzyme which mediates glycosylation and has its preference for the target glycosylation sites as well as for the type of glycosylation i.e. N-linked and O-linked glycosylation. In this study we carried out the bioinformatics analysis of one of the key enzymes of pgl locus from Campylobacter jejuni, known as PglB, which is distributed widely in bacteria and AglB from archaea. Relatively little sequence similarity was observed in the archaeal AglB(s) as compared to those of the bacterial PglB(s). In addition we tried to the answer the question of as to why not all the sequins Asp-X-Ser/Thr have an equal opportunity to be glycosylated by looking at the influence of the neighboring amino acids but no significant conserved pattern of the flanking sites could be identified. The software tool was developed to predict the potential glycosylation sites in autotransporter protein, the virulence factors of gram negative bacteria, and our results revealed that the frequency of glycosylation sites was higher in adhesins (a subclass of autotransporters) relative to the other classes of autotransporters.     

Molecular typing of Bordetella parapertussis isolates circulating in Pakistan

Although a whole-cell pertussis vaccine was introduced in Pakistan in 1980, little is known about the pertussis prevalence and circulating strains in Pakistan. The aim of this study was to analyze Bordetella parapertussis isolates circulating between 2005 and 2009 in Pakistan and to compare them with those found in other countries during different periods. A total of 59 (7.35%) B.?parapertussis isolates from 802 subjects (median age, 3?years) from Pakistan, with pertussis-like symptoms were investigated. We carried out genotyping and DNA microarray analyses on these isolates and compared them with some international isolates of B.?parapertussis. We found that the allele for pertactin (prn) found in strains studied from Pakistan was identical to the predominant type found in Europe. We showed that B.?parapertussis isolates circulating in Pakistan are part of the same pulsed-field gel electrophoresis group to those circulating in Finland during the period of 1982-2007. Finally, microarray analysis confirmed that the isolates collected in Pakistan, were quite similar to international strains. Overall, these results confirm that B.?parapertussis is extremely monomorphic. The high isolation rate of B.?parapertussis (7.35%) compared to Bordetella pertussis (0.5%) may suggest that the whole-cell vaccine used in Pakistan is effective against B.?pertussis (0.5% infections detected), but much less so against B.?parapertussis.     

Comparative proteomics analysis of the root apoplasts of rice seedlings in response to hydrogen peroxide

Background

Plant apoplast is the prime site for signal perception and defense response, and of great importance in responding to environmental stresses. Hydrogen peroxide (H₂O₂) plays a pivotal role in determining the responsiveness of cells to stress. However, how the apoplast proteome changes under oxidative condition is largely unknown. In this study, we initiated a comparative proteomic analysis to explore H₂O₂-responsive proteins in the apoplast of rice seedling roots.

Methodology/Principal Findings

14-day-old rice seedlings were treated with low concentrations (300 and 600 µM) of H₂O₂ for 6 h and the levels of relative electrolyte leakage, malondialdehyde and H₂O₂ were assayed in roots. The modified vacuum infiltration method was used to extract apoplast proteins of rice seedling roots, and then two-dimensional electrophoresis gel analysis revealed 58 differentially expressed protein spots under low H₂O₂ conditions. Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H₂O₂-responsive proteins. Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs. These proteins identified are involved in a variety of processes, including redox homeostasis, cell wall modification, signal transduction, cell defense and carbohydrate metabolism, indicating a complex regulative network in the apoplast of seedling roots under H₂O₂ stress.

Conclusions/Significance

The present study is the first apoplast proteome investigation of plant seedlings in response to H₂O₂ and may be of paramount importance for the understanding of the plant network to environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H₂O₂ response.     

Mechanisms of sublethal copper toxicity damage to the photosynthetic apparatus of Rhodospirillum rubrum

Magnesium (Mg²⁺) is the ubiquitous metal ion present in chlorophyll and bacteriochlorophyll (BChl), involved in photosystems in photosynthetic organisms. In the present study we investigated targets of toxic copper binding to the photosynthetic apparatus of the anoxygenic purple bacterium Rhodospirillum rubrum. This was done by a combination of in vivo measurements of flash photolysis and fast fluorescence kinetics combined with the analysis of metal binding to pigments and pigment-protein complexes isolated from Cu-stressed cells by HPLC-ICPMS (ICP-sfMS). This work concludes that R. rubrum is highly sensitive to Cu²⁺, with a strong inhibition of the photosynthetic reaction centres (RCs) already at 2 μM Cu²⁺. The inhibition of growth and of RC activity was related to the formation of Cu-containing BChl degradation products that occurred much more in the RC than in LH1. These results suggest that the shift of metal centres in BChl from Mg²⁺ to Cu²⁺ can occur in vivo in the RCs of R. rubrum under environmentally realistic Cu²⁺ concentrations, leading to a strong inhibition of the function of these RCs.     

Efficient regeneration and antioxidative enzyme activities in <Emphasis Type="Italic">Brassica rapa</Emphasis> var. <Emphasis Type="Italic">turnip</Emphasis>

The regeneration potential and antioxidative enzyme activities of economically important Brassica rapa var. turnip were evaluated. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium incorporated with different concentrations of various plant growth regulators (PGRs). The highest leaf explant response (83%) was recorded for 2.0 mg l⁻¹ benzyladenine (BA) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Subsequent subculturing of callus after 3 weeks of culture, on medium with similar compositions of PGRs, induced shoot organogenesis. The highest shoot induction response (83%) was recorded for 5.0 mg l⁻¹ BA after 5 weeks of transfer. However, 7.8 shoots/explant were recorded for 2.0 mg l⁻¹ BA. The transferring of shoots to elongation medium resulted in 5.1-cm-long shoots on 10 mg l⁻¹ of gibberellic acid (GA₃). Rooted plantlets were obtained on MS medium containing different concentrations of indole butyric acid (IBA). The determination of activities of antioxidative enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], glutathione peroxidase [GPX], and peroxidase [POD]) revealed involvement of these enzymes in callus formation and differentiation. All of the activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. This study will help in the advancement of a regeneration protocol for B. rapa var. turnip and the understanding of the functions of antioxidative enzymes in plant differentiation.     

Reassortment Networks and the evolution of pandemic H1N1 swine-origin influenza

Prior research developed Reassortment Networks to reconstruct the evolution of segmented viruses under both reassortment and mutation. We report their application to the swine-origin pandemic H1N1 virus (S-OIV). A database of all influenza A viruses, for which complete genome sequences were available in Genbank by October 2009, was created and dynamic programming was used to compute distances between all corresponding segments. A reassortment network was created to obtain the minimum cost evolutionary paths from all viruses to the exemplar S-OIV A/California/04/2009. This analysis took 35 hours on the Cray Extreme Multithreading (XMT) supercomputer, which has special hardware to permit efficient parallelization. Six specific H1N1/H1N2 bottleneck viruses were identified that almost always lie on minimum cost paths to S-OIV. We conjecture that these viruses are crucial to S-OIV evolution and worthy of careful study from a molecular biology viewpoint. In phylogenetics, ancestors are typically medians that have no functional constraints. In our method, ancestors are not inferred, but rather chosen from previously observed viruses along a path of mutation and reassortment leading to the target virus. This specificity and functional constraint render our results actionable for further experiments in vitro and in vivo.