Energy determines broad pattern of plant distribution in Western Himalaya

Several factors describe the broad pattern of diversity in plant species distribution. We explore these determinants of species richness in Western Himalayas using high‐resolution species data available for the area to energy, water, physiography and anthropogenic disturbance. The floral data involves 1279 species from 1178 spatial locations and 738 sample plots of a national database. We evaluated their correlation with 8‐environmental variables, selected on the basis of correlation coefficients and principal component loadings, using both linear (structural equation model) and nonlinear (generalised additive model) techniques. There were 645 genera and 176 families including 815 herbs, 213 shrubs, 190 trees, and 61 lianas. The nonlinear model explained the maximum deviance of 67.4% and showed the dominant contribution of climate on species richness with a 59% share. Energy variables (potential evapotranspiration and temperature seasonality) explained the deviance better than did water variables (aridity index and precipitation of the driest quarter). Temperature seasonality had the maximum impact on the species richness. The structural equation model confirmed the results of the nonlinear model but less efficiently. The mutual influences of the climatic variables were found to affect the predictions of the model significantly. To our knowledge, the 67.4% deviance found in the species richness pattern is one of the highest values reported in mountain studies. Broadly, climate described by water–energy dynamics provides the best explanation for the species richness pattern. Both modeling approaches supported the same conclusion that energy is the best predictor of species richness. The dry and cold conditions of the region account for the dominant contribution of energy on species richness.     

Stimuli responsive self-assembled hydrogel of a low molecular weight free dipeptide with potential for tunable drug delivery

Bottom-up fabrication by molecular self-assembly is now widely recognized as a potent method for generating interesting and functional nano- and mesoscale structures. Hydrogels from biocompatible molecules are an interesting class of mesoscale assemblies with potential biomedical applications. The self-assembly of a proteolysis resistant aromatic dipeptide containing a conformational constraining residue (DeltaPhe) into a stable hydrogel has been studied in this work. The reported dipeptide has free -N and -C termini. The hydrogel was self-supportive, was fractaline in nature, and possessed high mechanical strength. It was responsive to environmental conditions like pH, temperature, and ionic strength. The gel matrix could encapsulate and release bioactive molecules in a sustained manner. The described hydrogel showed no observable cytotoxicity to the HeLa and L929 cell lines in culture.     

Thiophene containing triarylmethanes as antitubercular agents

A new series of thiophene containing triarylmethane derivatives were synthesized from the Friedel-Crafts alkylation of diarylcarbinols followed by incorporation of amino alkyl chains. These were evaluated against Mycobacterium tuberculosis H37R(v) and showed the activity in the range of 3.12-12.5 microg/mL in vitro.     

Genotoxic stress in plants: shedding light on DNA damage, repair and DNA repair helicases

Plant cells are constantly exposed to environmental agents and endogenous processes that inflict damage to DNA and cause genotoxic stress, which can reduce plant genome stability, growth and productivity. Plants are most affected by solar UV-B radiation, which damage the DNA by inducing the formation of two main UV photoproducts such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). Reactive oxygen species (ROS) are also generated extra- or intra-cellularly, which constitute yet another source of genotoxic stress. As a result of this stress, the cellular DNA-damage responses (DDR) are activated, which transiently arrest the cell cycle and allow cells to repair DNA before proceeding into mitosis. DDR requires the activation of Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) genes, which regulate the cell cycle and transmit the damage signals to downstream effectors of cell-cycle progression. Since genomic protection and stability are fundamental to ensure and sustain plant diversity and productivity, therefore, repair of DNA damages is essential. In plants the bulky DNA lesions, CPDs and 6-4PPs, are repaired by a simple and error-free mechanism: photoreactivation, which is a light-dependent mechanism and requires CPD or 6-4PP specific photolyases. In addition to this direct repair process, the plants also have sophisticated light-independent general repair mechanisms, such as the nucleotide excision repair (NER) and base excision repair (BER). The completed plant genome sequences reveal that most of the genes involved in NER and BER are present in higher plants, which suggests that the network of in-built DNA-damage repair mechanisms is conserved. This article describes the insight underlying the DNA damage and repair pathways in plants. The comet assay to measure the DNA damage and the role of DNA repair helicases such as XPD and XPB are also covered.     

Submergence tolerance in relation to variable floodwater conditions in rice

Flash floods adversely affect rice productivity in vast areas of rainfed lowlands in South and Southeast Asia and tropical Africa. Tolerant landraces that withstand submergence for 1–2 weeks were identified; however, incorporation of tolerance into modern high-yielding varieties through conventional breeding methods has been slow because of the complexity of both the tolerance phenotype and floodwater conditions, and the ensuing discrepancies encountered upon phenotyping in different environments. Designing an effective phenotyping strategy requires a thorough understanding of the specific floodwater characteristics that most likely affect survival during flooding. We investigated the implications of floodwater temperature and light penetration, caused by artificial shading, seasonal variation, or water turbidity, for seedling survival after submergence. Three field experiments were conducted using rice genotypes contrasting in their tolerance of submergence: FR13A and Kusuma (tolerant); Gangasiuli (intermediate); Sabita, CRK-2-6 and Raghukunwar (elongating/avoiding types); and IR42 (sensitive). We tested the hypotheses that warmer floodwater decreases plant survival and that turbid water augment plant mortality by causing effects similar to those caused by shading, by reducing light penetration. Plants survive better when water is cooler, and survival decreased at about 8% per unit increase in water temperature above 26 °C. Lower intensity of light and warmer temperatures seem to reduce biomass and increase mortality under flooding. An increase in the concentrations of O₂ and CO₂ and a decrease in water pH did not improve survival in clear unshaded water. Turbid floodwater was more damaging to rice as plant mortality increased as the percentage of silt increased, and the effects of water turbidity cannot be explained by the reduction in light penetration alone. Even the most tolerant rice cultivar, FR13A, experienced higher mortality when flooded with turbid floodwater. Correlation studies revealed that cultivars with the capacity to maintain higher biomass, higher chlorophyll, and non-structural carbohydrate concentrations after submergence had higher survival. These findings help to understand the variation observed in submergence tolerance when screening is done under different environments. The study could have implications for designing proper screening strategies and assessing the damage submergence causes across different rice-growing regions.     

Genetic Characterization of Hepatitis B Virus in Peripheral Blood Leukocytes: Evidence for Selection and Compartmentalization of Viral Variants with the Immune Escape G145R Mutation

The compartmentalization of viral variants in distinct host tissues is a frequent event in many viral infections. Although hepatitis B virus (HBV) classically is considered hepatotropic, it has strong lymphotropic properties as well. However, unlike other viruses, molecular evolutionary studies to characterize HBV variants in compartments other than hepatocytes or sera have not been performed. The present work attempted to characterize HBV sequences from the peripheral blood leukocytes (PBL) of a large set of subjects, using advanced molecular biology and computational methods. The results of this study revealed the exclusive compartmentalization of HBV subgenotype Ae/A2-specific sequences with a potent immune escape G145R mutation in the PBL of the majority of the subjects. Interestingly, entirely different HBV genotypes/subgenotypes (C, D, or Aa/A1) were found to predominate in the sera of the same study populations. These results suggest that subgenotype Ae/A2 is selectively archived in the PBL, and the high prevalence of G145R indicates high immune pressure and high evolutionary rates of HBV DNA in the PBL. The results are analogous to available literature on the compartmentalization of other viruses. The present work thus provides evidence in favor of the compartment-specific abundance, evolution, and emergence of the potent immune escape mutant. These findings have important implications in the field of HBV molecular epidemiology, transmission, transfusion medicine, organ transplantation, and vaccination strategies.Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family and classically has been described to be hepatotropic, causing a wide range of clinical and subclinical manifestations of liver disease (57). Nevertheless, studies of HBV-infected human subjects and woodchucks infected with Woodchuck hepatitis virus (WHV; an animal model of hepadnaviral infection) have reported different molecular forms of replicative intermediates in the lymphatic cells and have established that hepadnaviruses are strongly lymphotropic in nature (29). Moreover, the results of studies of human subjects as well as with animal models have revealed that the life-long occult persistence of replication- and transmission-competent viruses in lymphatic cells is a strict consequence of hepadnaviral infections (29).More interestingly, in animal models, lymphatic system-restricted occult hepadnaviral infection has been found to be transmissible vertically as an asymptomatic, serologically occult infection exclusively confined to the lymphatic system (29). Earlier we provided evidence that occult HBV persisting in the lymphatic cells are transmissible, specifically to the PBL through horizontal intrafamilial modes (9). These observations clearly indicate important immunological, pathogenic, and epidemiological implications of lymphatic system-restricted hepadnaviral infections. Although the involvement of specific viral variants has been suggested to explain this lymphatic system-restricted hepadnaviral infection and transmission (29), the classical belief that hepatocytes are the primary target and only reservoir of HBV has precluded the genetic characterization of hepadnaviruses from extrahepatic sites.Fascinatingly, despite being classically considered a hepatotropic virus, hepatitis C virus (HCV), belonging to the family Flaviviridae, also shows occult persistence and lymphotropism very similar to that of hepadnaviruses (37). Similarly to WHV, HBV, and HCV, other viruses, including HIV (human immunodeficiency virus), small ruminant lentivirus, and Epstein-Barr virus, also have been shown to infect and persist in different anatomical compartments of the body in addition to their classical target cells (38, 40, 43, 45, 50). Furthermore, recent molecular evolutionary analyses based on envelope sequences of these viruses (e.g., HIV, HCV, small ruminant lentivirus, Epstein-Barr virus, etc.) have established clearly that these viruses undergo selection and independent evolution in diverse tissues, leading to the tissue-specific compartmentalization of viral populations (38, 40, 43, 45, 50). In contrast to other viruses, to the best of our knowledge, methodical molecular evolutionary studies to characterize HBV sequences isolated from extrahepatic sites of HBV-infected subjects have not been reported in the literature.We hypothesized that similar to other viruses, HBV also undergo independent evolution in different compartments of the body under the influence of differential immune pressure. To examine our hypothesis, we used the most easily available lymphatic cells, the peripheral blood leukocytes (PBL), determined the HBV envelope sequences from HBV DNA isolated from these cells, and performed advanced genetic, phylogenetic, and mutational analysis. The results of this work demonstrate a highly compartment-specific preponderance of HBV genetic variants in serum and PBL of the same study population, providing evidence in favor of the compartmentalization of HBV genetic variants. The results and important implications of these findings are discussed in this work.     

A Y526Q Mutation in the Newcastle Disease Virus HN Protein Reduces Its Functional Activities and Attenuates Virus Replication and Pathogenicity

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein that plays a crucial role in virus infectivity. In this study, using the mesogenic strain Beaudette C (BC), we mutated three conserved amino acids thought to be part of the binding/catalytic active site in the HN protein. We also mutated five additional residues near the proposed active site that are nonconserved between BC and the avirulent strain LaSota. The eight recovered NDV HN mutants were assessed for effects on biological activities. While most of the mutations had surprisingly little effect, mutation at conserved residue Y526 reduced the neuraminidase, receptor binding, and fusion activities and attenuated viral virulence in eggs and young birds.Newcastle disease virus (NDV) is an avian pathogen of the genus Avulavirus in the family Paramyxoviridae (10). The envelope of NDV contains two surface glycoproteins, the fusion (F) protein and the HN (hemagglutinin-neuraminidase [NA]) protein. The F protein mediates viral penetration and requires cleavage-activation by host protease. Cleavability of the F protein is a major determinant of virulence. However, other viral proteins, including HN, also contribute to virulence (5). HN is a multifunctional glycoprotein. It recognizes sialic acid-containing receptors on cell surfaces; promotes the fusion activity of F protein, thereby allowing the virus to penetrate the cell surface; and acts as an NA that removes sialic acid from progeny virus particles to prevent viral self-aggregation (9).HN is a type II homotetrameric glycoprotein with a monomer length of 577 amino acids for most NDV strains (14). The ectodomain of the HN protein consists of a 95-amino-acid stalk region supporting a 428-amino-acid terminal globular head. Although mutations in the transmembrane and stalk regions of the HN protein can affect the structure and activities of the protein (11, 15), the antigenic, receptor recognition, and NA active sites are all localized in the globular head (12, 16). The X-ray crystal structure of the globular head of the NDV HN protein has identified residues that appear to contribute to receptor recognition, NA, and fusion activities (4). Previous studies have proposed that conserved residues R174, I175, D198, K236, R416, R498, Y526, and E547 are important in receptor recognition and NA activities and that residues R174 and E547 influence the fusion promotion activity of the HN protein (3, 4, 6). Although transfection studies using plasmids expressing HN mutants of NDV have highlighted the importance of these residues in different biological functions of the HN protein, their contribution to NDV biology and pathogenesis in the context of the complete virus was not known.In this study, we examined the roles of three of the above-named conserved residues, R416, R498, and Y526 (all located near the sialic acid binding site), in the biological activities and pathogenesis of the HN protein of NDV in the context of infectious virus. In addition, comparison of the HN protein sequence between the avirulent strain LaSota and the moderately virulent strain Beaudette C (BC) identified 12 amino acid differences in the globular head region of the HN protein (H203, T214, I219, S228, L269, A271, E293, G310, S494, E495, T502, and N568, named according to the BC amino acid assignment). We also examined five of these nonconserved residues, T214, I219, S494, E495, and N568, located in close proximity to residues identified earlier by crystal structure studies, to determine whether these might affect HN function and contribute to the difference in pathogenicity between the LaSota and BC strains (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Three-dimensional structure of the NDV HN protein showing the positions of amino acid residues that were substituted in the present study. The residues are shown in space-filling mode and represented in different colors. The MacPymol (DeLano Scientific) software was used to generate the model of the globular domain of the NDV HN monomer. The structure was derived from the crystal structure of the NDV HN protein reported by Crennell et al. (4).We used site-directed mutagenesis (2) to introduce individual amino acid substitutions into a cDNA of the HN gene of strain BC. For the conserved residues, we changed arginine at positions 416 and 498 and tyrosine at position 526 to polar glutamine. For the nonconserved residues, the assignments T214, I219, S494, E495, and N568 of strain BC were altered to the corresponding assignments of strain LaSota: S214, V219, G494, V495, and D568, respectively. Each mutagenized HN gene was then inserted into a full-length cDNA clone of the BC antigenome. These clones were transfected into HEp2 cells, and mutant viruses were recovered as previously described (8). These viruses were designated according to the substitutions introduced: T214S, I219V, R416Q, S494G, E495V, R498Q, Y526Q, and N568D. The HN genes from recovered viruses were sequenced. This confirmed the presence of each introduced mutation and the lack of adventitious mutations in the HN gene. To determine the stability of each HN mutation, the recovered viruses were passaged five times in 9-day-old embryonated chicken eggs and five times in chicken embryo fibroblast DF-1 cells. Sequence analysis of the HN gene of the mutant viruses at each passage showed that the introduced mutations were unaltered (data not shown). To rule out the possibility that change in the HN protein sequence could be compensated for by a mutation in the F protein, the F gene from each recovered virus was sequenced. No compensatory mutations in the F gene were observed (data not shown). The HN protein content of each mutant virus, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining, was very similar to that of the parental BC virus (pBC) (Table ​(Table1).1). The multicycle growth kinetics of the recombinant HN mutant viruses in DF-1 cells (Fig. ​(Fig.2)2) showed that the replication kinetics of all of the HN mutant viruses were similar to those of pBC, with the exception of the Y526Q mutant, which showed delayed growth and had a lower virus yield (1.5 to 2.0 log₁₀ PFU/ml) than the parental and other mutant viruses. In addition, the Y526Q mutant produced syncytia at 72 h, whereas the parental and other mutant viruses initiated syncytia at 24 h postinfection. These studies showed the importance of amino acid residue Y526 at the active site of the HN protein of NDV.Open in a separate windowFIG. 2.Multicycle growth kinetics of HN mutants of NDV in chicken embryo fibroblast (DF-1) cells. Cells were infected with the indicated parental or mutant virus at an multiplicity of infection of 0.01. Supernatant samples were collected at 8-h intervals until 64 h postinfection, and virus titers were determined at different time points by plaque assay. Values are averages from three independent experiments.

TABLE 1.

Biological activities of HN mutants of NDV

Neutralizing a Surface Charge on the FMN Subdomain Increases the Activity of Neuronal Nitric-oxide Synthase by Enhancing the Oxygen Reactivity of the Enzyme Heme-Nitric Oxide Complex

Nitric-oxide synthases (NOSs) are calmodulin-dependent flavoheme enzymes that oxidize l-Arg to nitric oxide (NO) and l-citrulline. Their catalytic behaviors are complex and are determined by their rates of heme reduction (k_r), ferric heme-NO dissociation (k_d), and ferrous heme-NO oxidation (k_ox). We found that point mutation (E762N) of a conserved residue on the enzyme''s FMN subdomain caused the NO synthesis activity to double compared with wild type nNOS. However, in the absence of l-Arg, NADPH oxidation rates suggested that electron flux through the heme was slower in E762N nNOS, and this correlated with the mutant having a 60% slower k_r. During NO synthesis, little heme-NO complex accumulated in the mutant, compared with ∼50–70% of the wild-type nNOS accumulating as this complex. This suggested that the E762N nNOS is hyperactive because it minimizes buildup of an inactive ferrous heme-NO complex during NO synthesis. Indeed, we found that k_ox was 2 times faster in the E762N mutant than in wild-type nNOS. The mutational effect on k_ox was independent of calmodulin. Computer simulation and experimental measures both indicated that the slower k_r and faster k_ox of E762N nNOS combine to lower its apparent K_m,O2 for NO synthesis by at least 5-fold, which in turn increases its V/K_m value and enables it to be hyperactive in steady-state NO synthesis. Our work underscores how sensitive nNOS activity is to changes in the k_ox and reveals a novel means for the FMN module or protein-protein interactions to alter nNOS activity.Nitric oxide (NO)² is a biological mediator that is produced in animals by three NO synthase isozymes (NOS, EC 1.14.13.39): inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) (1, 2). The NOS are modular enzymes composed of an N-terminal oxygenase domain and a C-terminal flavoprotein domain, with a calmodulin (CaM)-binding site connecting the two domains (3). During NO synthesis, the flavoprotein domain transfers NADPH-derived electrons through its FAD and FMN cofactors to a heme located in the oxygenase domain. The FMN-to-heme electron transfer enables heme-dependent oxygen activation and a stepwise conversion of l-Arg to NO and citrulline (4, 5). Heme reduction also requires that CaM be bound to NOS and is rate-limiting for NO biosynthesis (6–9).NOS enzymes operate under the constraint of having their newly made NO bind to the ferric heme before it can exit the enzyme (10). How this intrinsic heme-NO binding event impacts NOS catalytic cycling is shown in Fig. 1 and has previously been discussed in detail (10–13). The l-Arg to NO biosynthetic reaction (Fe^III to Fe^IIINO in Fig. 1) is limited by the rate of ferric heme reduction (k_r), because all biosynthetic steps downstream are faster than k_r. However, once the ferric heme-NO complex forms at the end of each catalytic cycle, it can either dissociate to release NO into the medium (at a rate k_d as shown in Fig. 1) or become reduced by the flavoprotein domain (at a rate k′_r in Fig. 1; equal to k_r) to form the enzyme ferrous heme-NO species (Fe^IINO), which releases NO very slowly (11, 12). Consequently, two cycles compete during steady-state NO synthesis (Fig. 1); NO dissociation from the ferric heme (k_d) is part of a “productive cycle” that releases NO and is essential for NOS bioactivity, whereas reduction of the ferric heme-NO complex (k_r′) channels the enzyme into a “futile cycle” that actually represents a NO dioxygenase activity. The rate of futile cycling is also determined by the rate of O₂ reaction with the ferrous heme-NO complex (at a rate k_ox in Fig. 1), which regenerates the ferric enzyme. Surprisingly, NOS enzymes have evolved to have a broad range of k_r (varies 40×), k_ox (varies 15×), and k_d (varies 30×) values (Table S1) (12). This causes each NOS to distribute quite differently during steady-state NO synthesis and gives each NOS a unique catalytic profile (12).Open in a separate windowFIGURE 1.Global kinetic model for NOS catalysis. Ferric enzyme reduction (k_r) is rate-limiting for the biosynthetic reactions (central linear portion). k_cat1 and k_cat2 are the conversion rates of the enzyme Fe^IIO₂ species to products in the l-Arg and N^ω-hydroxy-l-arginine (NOHA) reactions, respectively. The ferric heme-NO product complex (Fe^IIINO) can either release NO (k_d) or become reduced (k′_r) to a ferrous heme-NO complex (Fe^IINO), which reacts with O₂ (k_ox) to regenerate ferric enzyme. Enzyme partitioning and NO release are determined by the relative rates of k_r, k_ox, and k_d. This figure is adapted from Ref. 12.The enzyme physical and electronic factors that may set and regulate each of the three kinetic parameters (k_r, k_ox, and k_d) in NOS enzymes remain to be fully described. At present, the composition of the NOS flavoprotein domain and CaM appear to be primarily responsible for determining the k_r (14–17), whereas the composition of the NOS oxygenase domain is presumed to determine the k_d and k_ox (18, 19). Indeed, our recent point mutagenesis study identified a patch of electronegative residues on the FMN subdomain that are required to maintain a normal k_r and NO synthesis activity in nNOS, suggesting that subdomain electrostatic interactions are important in the process (20). We found particularly large effects when the negative charge at Glu⁷⁶² was neutralized via mutation to Asn. Remarkably, the NO synthesis activity of E762N nNOS was double that of wild-type nNOS, despite the mutant displaying a slow k_r that was half of wild type. In the current report, we show that the E762N mutation has an additional, unsuspected effect on the k_ox kinetic parameter of nNOS. How this effect alters distribution of the nNOS enzyme during steady-state catalysis, impacts the apparent K_m,O2, and leads to hyperactive NO synthesis is described. Our finding that the nNOS flavoprotein domain can tune a key kinetic parameter that defines the rate of a heme-based reaction in the nNOS oxygenase domain is unusual and suggests a means by which protein-protein interactions could regulate the catalytic behavior of nNOS.     

A novel ACO algorithm for optimization via reinforcement and initial bias

In this paper, we introduce the MAF-ACO algorithm, which emulates the foraging behavior of ants found in nature. In addition to the usual pheromone model present in ACO algorithms, we introduce an incremental learning component. We view the components of the MAF-ACO algorithm as stochastic approximation algorithms and use the ordinary differential equation (o.d.e.) method to analyze their convergence. We examine how the local stigmergic interaction of the individual ants results in an emergent dynamic programming framework. The MAF-ACO algorithm is also applied to the multi-stage shortest path problem and the traveling salesman problem. Research of Prof. V.S. Borkar was supported in part by grant no. III.5(157)/99-ET and a J.C. Bose Fellowship from the Department of Science and Technology, Government of India.     

Flanking Microsatellite Markers for Breeding Varieties Against Asian Rice Gall Midge

The Asian rice gall midge, Orseolia oryzae Wood-Mason (Cecidomyiidae: Diptera) is a serious pest of wet season rice in South and Southeast Asia. Due to internal feeding habit and presence of biotypes of the pest, the most feasible way to control is breeding varieties resistant against multiple biotypes through marker-assisted breeding (MAB). But very few versatile co-dominant markers linked to the gall midge resistance genes are available. We used a set of F₉ recombinant inbred lines (RILs) of the cross TN1/PTB10 and identified microsatellite markers for the gall midge resistance gene in cv. PTB10 on short arm of rice chromosome 8. Markers RM22550 and RM547 flank the gene at a distance of 0.9 and 1.9 cM, respectively. Amplification of the markers in gall midge resistant and susceptible cultivars showed that these markers can be successfully used in MAB for development of gall midge resistant varieties.