Global Functional Atlas of Escherichia coli Encompassing Previously Uncharacterized Proteins

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a “systems-wide” functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.     

IgG Autoantibody to Brain Beta Tubulin III Associated with Cytokine Cluster-II Discriminate Cerebral Malaria in Central India

Background

The main processes in the pathogenesis of cerebral malaria caused by Plasmodium falciparum involved sequestration of parasitized red blood cells and immunopathological responses. Among immune factors, IgG autoantibodies to brain antigens are increased in P. falciparum infected patients and correlate with disease severity in African children. Nevertheless, their role in the pathophysiology of cerebral malaria (CM) is not fully defined. We extended our analysis to an Indian population with genetic backgrounds and endemic and environmental status different from Africa to determine if these autoantibodies could be either a biomarker or a risk factor of developing CM.

Methods/Principal Findings

We investigated the significance of these self-reactive antibodies in clinically well-defined groups of P. falciparum infected patients manifesting mild malaria (MM), severe non-cerebral malaria (SM), or cerebral malaria (CM) and in control subjects from Gondia, a malaria epidemic site in central India using quantitative immunoprinting and multivariate statistical analyses. A two-fold complete-linkage hierarchical clustering allows classifying the different patient groups and to distinguish the CM from the others on the basis of their profile of IgG reactivity to brain proteins defined by PANAMA Blot. We identified beta tubulin III (TBB3) as a novel discriminant brain antigen in the prevalence of CM. In addition, circulating IgG from CM patients highly react with recombinant TBB3. Overall, correspondence analyses based on singular value decomposition show a strong correlation between IgG anti-TBB3 and elevated concentration of cluster-II cytokine (IFNγ, IL1β, TNFα, TGFβ) previously demonstrated to be a predictor of CM in the same population.

Conclusions/Significance

Collectively, these findings validate the relationship between antibody response to brain induced by P. falciparum infection and plasma cytokine patterns with clinical outcome of malaria. They also provide significant insight into the immune mechanisms associated to CM by the identification of TBB3 as a new disease-specific marker and potential therapeutic target.     

Hexafluoroisopropanol-induced helix–sheet transition of stem bromelain: Correlation to function

Stem bromelain is a proteolytic phytoprotein with a variety of therapeutic effects. Understanding its structural properties could provide insight into the mechanisms underlying its clinical utility. Stem bromelain was evaluated for its conformational and folding properties at the pH conditions it encounters when administered orally. It exists as a partially folded intermediate at pH 2.0. The conformational changes to this intermediate state were evaluated using fluorinated alcohols known to induce changes similar to those seen in vivo. Studies using circular dichroism, fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid and mass spectrometry indicate that treatment with 10–30% hexafluoroisopropanol induces the partially folded intermediate to adopt much of the native protein's secondary structure, but only a rudimentary tertiary structure, characteristic of the molten globule state. Addition of slightly higher concentrations of hexafluoroisopropanol caused transformation from an α-helix to a β-sheet and induced formation of a compact nonnative structure. This nonnative form was more inhibitory of cell survival than either the native or the partially folded intermediate forms, as measured by enhanced suppression of proliferative cues (e.g., extracellular-signal-regulated kinase) and initiation of apoptotic events. The nonnative form also showed better antitumorigenic properties, as evaluated using an induced two-stage mouse skin papilloma model. In contrast, the nonnative state showed only a fraction of the proteolytic activity of the native form. This study demonstrates that hexafluoroisopropanol can induce a conformational change in stem bromelain to a form with potentially useful therapeutic properties different from those of the native protein.     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.     

Quantitative analysis of flavonol glycosides in Ginkgo biloba: a comparison of two analytical methods

A comparison of testing methods for constituents of Ginkgo biloba was performed as part of the process for determining which of the many available analytical methods was most appropriate for inclusion in the American Herbal Pharmacopoeia (AHP). Three primary methods were reviewed: those adopted by the European Pharmacopoeia, the National Science Foundation-Institute for Nutraceutical Advancement (NSF-INA) and the United States Pharmacopeia. Methods were compared by evaluating sample preparation and hydrolysis, precision and methods of total flavonol glycoside calculation by two independent laboratories. The AHP adopted the method of NSF-INA for inclusion in the AHP monograph owing to observed superiority of this method in terms of precision, glycoside calculation, ease of sample preparation and chromatographic parameters.     

Propagation and Conservation of Picrorhiza kurrooa Royle ex Benth.: An Endangered Himalayan Medicinal Herb of High Commercial Value

Picrorhiza kurrooa Royle ex Benth., a high value medicinal herb of alpine Himalaya and a source of hepatoprotective picrosides, is listed as ‘endangered’ due to heavy collection from its natural habitat. The present report deals with successful propagation of this species using both conventional and in vitro techniques. Vegetative propagation was achieved by rooting runner cuttings with indole-3-butyric acid (IBA) or α-naphtheleneacetic acid (NAA) treatment before planting. Nearly 87% rooting success was achieved by treatment of cuttings with 50.0 μM IBA. Seeds were given a presoaking treatment with gibberellic acid (GA₃), 6-benzylaminopurine (BAP) or a combination of both to influence germination. More than 11-fold improvement in germination was recorded in seeds treated with 250.0 μM GA₃. In vitro shoot multiplication was achieved through sprouting of axillary buds using nodal segment. Multiple shoots were formed following culture for 3 weeks on Murashige and Skoog (MS; 1962. Physiologia Plantarum 15: 473–497) medium containing 1.0 μM BAP. Cent percent rooting success, without basal callus formation, was observed when individual microshoots were placed in MS medium supplemented with IBA. The plantlets raised using conventional as well as tissue culture methods were hardened and successfully established in the experimental field located at 2450 m elevation. In addition, strategies have been discussed to encourage cultivation and in situ conservation of this highly valued medicinal herb so as to reduce pressure on its natural populations.     

Decolourisation and detoxification of synthetic molasses melanoidins by individual and mixed cultures of Bacillus spp

The decolourisation of synthetic melanoidins (i.e., GGA, GAA, SGA, and SAA) by three Bacillus isolates (Bacillus thuringiensis (MTCC 4714), Bacillus brevis (MTCC 4716) and Bacillus sp. (MTCC 6506)) was studied. Significant reduction in the values of physicochemical parameters was noticed alongwith the decolourisation of all four melanoidins (10% v/v). B. thuringiensis (MTCC 4714) caused maximum decolourisation followed by B. brevis (MTCC 4716) and Bacillus sp. (MTCC 6506). A mixed culture comprised of these three strains was capable of decolourising all four melanoidins. The medium that contained glucose as a sole carbon source showed 15% more decolourisation than that containing both carbon and nitrogen sources. Melanoidin SGA was maximally decolourised (50%) while melanoidin GAA was decolourised least ( approximately 06%) in the presence of glucose as a sole energy source. The addition of 1% glucose as a supplementary carbon source was essential for co-metabolism of melanoidin complex. The decolourisation of synthetic melanoidin by three Bacillus spp. significantly reduced the toxicity to the tubificid worm (Tubifex tubifex, Müller).     

Repellent and insecticidal activities of essential oils from Artemisia princeps and Cinnamomum camphora and their effect on seed germination of wheat and broad bean

Repellent and insecticidal activities of essential oils extracted from leaves of Artemisia princeps Pamp and seeds of Cinnamomum camphora (L.) Presl. against storage pests Sitophillus oryzae L. and Bruchus rugimanus Bohem were investigated. Results showed that the two individual oils displayed good, but their mixture (1:1) exhibited much better repellent activities at concentrations from 250 to 1000 microg g(-1) and insecticidal actions at concentrations 1000 microg g(-1) against the test beetles S. oryzae and B. rugimanus. Oils from A. princeps and C. camphora applied individually were significantly toxic to seed germination of wheat at 500 microg ml(-1). However, no toxic effects were found when the two oils were mixed (1:1 w/w) at the same concentration. These observations indicated that the mixture of the two plant-derived oils had a synergic effect and could be used in the control of storage pests.     

Design and synthesis of potent and selective 1,3,4-trisubstituted-2-oxopiperazine based melanocortin-4 receptor agonists

The design and synthesis of a series of potent 1,3,4-trisubstituted-2-oxopiperazine based MC4 agonists are described. The tripeptidomimetic analogs (12a,b and 23) and the dipeptidomimetic 27 displayed single-nanomolar binding affinity and agonist potency for MC4R and excellent selectivity for MC4R relative to MC1R.