Evaluating the Use of Multilocus Variable Number Tandem Repeat Analysis of Shiga Toxin-Producing Escherichia coli O157 as a Routine Public Health Tool in England

Multilocus variable number tandem repeat analysis (MLVA) provides microbiological support for investigations of clusters of cases of infection with Shiga toxin-producing E. coli (STEC) O157. All confirmed STEC O157 isolated in England and submitted to the Gastrointestinal Bacteria Reference Unit (GBRU) during a six month period were typed using MLVA, with the aim of assessing the impact of this approach on epidemiological investigations. Of 539 cases investigated, 341 (76%) had unique (>2 single locus variants) MLVA profiles, 12% of profiles occurred more than once due to known household transmission and 12% of profiles occurred as part of 41 clusters, 21 of which were previously identified through routine public health investigation of cases. The remaining 20 clusters were not previously detected and STEC enhanced surveillance data for associated cases were retrospectively reviewed for epidemiological links including shared exposures, geography and/or time. Additional evidence of a link between cases was found in twelve clusters. Compared to phage typing, the number of sporadic cases was reduced from 69% to 41% and the diversity index for MLVA was 0.996 versus 0.782 for phage typing. Using MLVA generates more data on the spatial and temporal dispersion of cases, better defining the epidemiology of STEC infection than phage typing. The increased detection of clusters through MLVA typing highlights the challenges to health protection practices, providing a forerunner to the advent of whole genome sequencing as a diagnostic tool.     

Bio indices for 2,4-D sensitivity between two plant species: <Emphasis Type="Italic">Azolla pinnata</Emphasis> R.Br. and <Emphasis Type="Italic">Vernonia cinerea</Emphasis> L. with their cellular responses

In the present experiment a pteridophytic species Azolla and an angiospermic species Vernonia were evaluated on the basis of cellular reactivity for herbicidal action through ongoing concentrations. Initially, both the species recorded a significant activity of IAA-oxidase as mark of IAA metabolism with herbicidal sensitivity. Still, Vernonia species were more affected on 2,4-D mediated auxin catabolism. The loss of auxin concentrations on the tissues by 2,4-D reaction was also reflected on growth parameters including relative growth rate and chlorophyll biosynthesis. In a dose dependent manner Vernonia plants were more affected with loss of chlorophyll content and decline in relative growth rate. On the other hand, both those parameters were adjusted significantly with 2,4-D accumulation in Azolla. The stability of cellular metabolism was documented by significant down regulation of protein and lipid peroxidation with concomitant moderation to superoxide and hydrogen peroxide accumulation. The later two were more vulnerable to damage in the Vernonia plant with profuse accumulation of protein and lipid peroxidation products. Similarly, tissue specific reaction to superoxide and hydrogen peroxide accumulation were distinctly demarcated in two species significantly. As a whole, the cellular responses and metabolite distribution to 2,4-D sensitization are the features to describe bio-indices for aquatic fern species Azolla with comparison to angiospermic species Vernonia.     

Uric Acid Excretion in Isoparorchis hypselobagri (Billet, 1898)

The nature and amount of excretory products of Isoparorchis hypselobagri, a digenetic trematode inhabiting the swim bladder of Wallago attu have been studied. The parasites were kept in PBS media without glucose (control) and with glucose (treated) successfully up to 300 h and different excretory products were estimated at an interval of 12 h. The quantitative estimation of excreted uric acid in I. hypselobagri was revealed less in amount in the flukes which were kept in vitro in the control incubation media than those considered from the treated incubation media. The highest amount of uric acid excreted in control media was 6.72 mg % at 204 h, and in treated condition 16.658 mg % after 216 h of incubation. The lowest amount of uric acid excreted, 0.186 mg % in control media after 252 h of incubation and 2.896 mg % in treated media after 192 h of incubation. The rate of excretion of uric acid in treated condition after every 12 h of incubation is much higher than the control. Significant amount of uric acid have also been recorded in the swim bladder washings of host. It is apparent that the amount of excreted uric acid depends on the number and weight of the parasites harbor. Results suggest that I. hypselobagri is ammonotelic, ureotelic and also uricotelic trematode.     

A proximal tryptophan in NO synthase controls activity by a novel mechanism

The heme of neuronal nitric oxide synthase (nNOS) participates in O2 activation but also binds self-generated NO, resulting in reversible feedback inhibition. We utilized mutagenesis to investigate if a conserved tryptophan residue (Trp409), which engages in pi-stacking with the heme and hydrogen bonds to its axial cysteine ligand, helps control catalysis and regulation by NO. Mutants W409F and W409Y were hyperactive regarding NO synthesis without affecting cytochrome c reduction, reductase-independent N-hydroxyarginine oxidation, or Arg and tetrahydrobiopterin binding. In the absence of Arg electron flux through the heme was slower in the W409 mutants than in wild-type. However, less NO complex accumulated during NO synthesis by the mutants. To understand the mechanism, we compared the kinetics of heme-NO complex formation, rate of heme reduction, kcat prior to and after NO complex formation, NO binding affinity, NO complex stability, and its reaction with O2. During the initial phase of NO synthesis, heme-NO complex formation was three and five times slower in W409F and W409Y, which corresponded to a slower heme reduction. NO complex formation inhibited wild-type turnover 7-fold but reduced mutant turnover less than 2-fold, giving mutants higher steady-state activities. NO binding kinetics were similar among mutants and wild type, although mutants also formed a 417 nm ferrous-NO complex. Oxidation of ferrous-NO complex was seven times faster in mutants than in wild type. We conclude that mutant hyperactivity primarily derives from slower heme reduction and faster oxidation of the heme-NO complex by O2. In this way Trp409 mutations minimize NO feedback inhibition by limiting buildup of the ferrous-NO complex during the steady state. Conservation of W409 among NOS suggests that this proximal Trp may regulate NO feedback inhibition and is important for enzyme physiologic function.     

Ascorbate Peroxidase from Leishmania major Controls the Virulence of Infective Stage of Promastigotes by Regulating Oxidative Stress

Background

Peroxidase represents a heterogeneous group of distinct enzyme family that plays extremely diverse biological functions. Ascorbate peroxidase from Leishmania major (LmAPX) has been shown to be central to the redox defense system of Leishmania. To investigate further its exact physiological role in Leishmania, we attempted to create LmAPX -knockout mutants by gene replacement in L. major strains.

Methodology/Principal Findings

The null mutant cell culture contains a higher percentage of metacyclic and apoptotic cells compared to both wild type and LmAPX overexpressing cells. Flowcytometric analysis reveals the presence of a higher concentration of intracellular H₂O₂, indicative of increased oxidative stress in parasites lacking LmAPX. IC₅₀ value for exogenously added H₂O₂ shows that deletion of LmAPX in L. major renders the cell more susceptible to H₂O₂. Real time PCR studies demonstrate an elevated mRNA level of non-selenium glutathione peroxidase in LmAPX null mutant cell line, suggesting that these enzymes were induced to compensate the LmAPX enzyme. The null mutant cells exhibit hypervirulence after infection with macrophages as well as inoculation into BALB/c mice; in contrast, overexpressing cells show avirulence.

Conclusions/Significance

Collectively, these data provide strong evidence that LmAPX is an important factor for controlling parasite differentiation and survival within macrophages.     

Effect of drought stress implemented at pre- or post-anthesis stage on some physiological parameters as screening criteria in chickpea cultivars

Drought is one of the most important factors limiting chickpea production in arid and semi-arid regions. There is little information regarding genotypic variation for drought tolerance in chickpea cultivars. Screening for drought tolerance is very important. It is essential to identify the physiological mechanisms of drought tolerance to complete conventional breeding program. Glasshouse experiment was carried out to study the genotypic variation among 11 chickpea (Cicer arietinum L.) cultivars. Plants were grown either under optimum conditions or drought stress was implemented at pre-or post-anthesis stages. The drought susceptibility index (DSI) was used as the measure of drought tolerance. Relationships between DSI and excised-leaf water loss (RWL), relative water content (RWC), membrane permeability, ascorbic acid, proline, and chlorophyll contents, lipid peroxidation, and hydrogen peroxide concentrations were determined in order to find out whether these physiological parameters could be used as the genotypic selection criteria for drought tolerance. The results of this study indicated that there was a wide variation in tolerance to drought stress among the chickpea cultivars, which could be exploited in breeding new chickpea cultivars with high drought tolerance. The results also demonstrated that drought-tolerant cultivars had a higher RWC, ascorbic acid and proline concentrations, but lower RWL and membrane permeability in comparison to drought-sensitive cultivars. The significant and a well defined relationships between DSI and RWC, RWL, ascorbic acid, proline, and membrane permeability were found. It was concluded that these parameters could be instrumental in predicting the drought tolerance of chickpea cultivars. This text was submitted by the authors in English.     

Alterations in polypeptides pattern in malaria vector Anopheles stephensi, fed upon immunized blood causing fecundity reduction

Changes in polypeptides pattern of haemolymph, midgut, ovary and salivary glands of female mosquito A. stephensi were studied when fed upon anti-mosquito haemolymph antibodies. The expression of almost all polypeptides was reduced in haemolymph and ovary of the immune fed mosquitoes as compared to control. However, there was no significant difference in case of midgut and salivary glands. Seven polypeptides 100, 90, 84, 80, 62, 19 and 12.5 kDa were absent in haemolymph and five 92, 90, 80, 60 and 55 kDa were absent in ovaries. Changes in the polypeptide pattern have been correlated with the fecundity reduction due to immunized blood feeding.     

A morphometric study of the Thingbu-pa and population comparison with neighbouring Monpa tribes of Arunachal Pradesh, India

In the present study an attempt has been made to examine the morphometric characteristics of the adult males of the Thinbu-pa and the nature and extent of morphometric variation among four neighbouring Monpa groups of Arunachal Pradesh (India), namely the Panchen-pa, Dirang Monpa, Tawang Monpa and Kalaktang Monpa. For the sake of investigation altogether 14 anthropometric measurements (11 pertaining to head and face and 3 on the body as a whole) have been taken and 5 indices have been calculated. To obtain the distance values the methods of Penrose (1947, 1954) and El-Najjar (1978) have been followed. It is observed that the Thingbu-pa maintain far distance with the four neighbouring Monpa groups as well as the Panchen-pa also maintain far distance with the other four groups, while the Dirang Monpa, Tawang Monpa and Kalaktang Monpa maintain close distance among each other.     

Variation in anthropometric characters and their relationship with climatic factors: a study on the peoples of Arunachal Pradesh, India

In the present study an attempt has been made to examine the anthropometric variation and the relationship of anthropometric variables with climatic factors among the peoples of Arunachal Pradesh, India. The distance analysis does not seem to provide any interesting corroboration between anthropometric data and the intra- and intergroup relationship. A similar trend has also been noticed while analysing the correlation coefficient between two indices (i.e. the cephalic and total facial index). The present study suggests that the climatic factors are playing an important role in regulating the variation of anthropometric characters among the different population groups of Arunachal Pradesh.     

Role of proximal methionine residues in Leishmania major peroxidase

The active site architecture of Leishmania major peroxidase (LmP) is very similar with both cytochrome c peroxidase and ascorbate peroxidase. We utilized point mutagenesis to investigate if the conserved proximal methionine residues (Met248 and Met249) in LmP help in controlling catalysis. Steady-state kinetics of methionine mutants shows that ferrocytochrome c oxidation is <2% of wild type levels without affecting the second order rate constant of first phase of Compound I formation, while the activity toward a small molecule substrate, guaiacol or iodide, increases. Our diode array stopped-flow spectral studies show that the porphyrin π-cation radical of Compound I in mutant LmP is more stable than wild type enzyme. These results suggest that the electronegative sulfur atoms of the proximal pocket are critical factors for controlling the location of a stable Compound I radical in heme peroxidases and are important in the oxidation of ferrocytochrome c.