Dioxygenase from Aspergillus fumigatus MC8: molecular modelling and in silico studies on enzyme–substrate interactions

Flavoenzymes have been extensively studied for their structural and mechanistic properties because they find potential application as industrial biocatalysts. They are attractive for biocatalysis because of the selectivity, controllability and efficiency of their reactions. Some of these enzymes catalyse the oxidative modification of protein substrates. Among them oxygenases (monoxoygenases and dioxygenases) are of special interest because they are highly entantio as well as regio-selective and can be used for oxyfunctionalisation. Dioxygenase enzymes catalyse oxygenation reactions in which both di-oxygen atoms are incorporated into the product. A dioxygenase enzyme purified from Aspergillus fumigatus MC8 was subjected to protein digestion followed by peptide sequencing. The sequence analysis of the peptide fragments resulted in identifying its match with that of an extracellular dioxygenase sequence from the same species of fungus existing in the protein database. The sequence was submitted to protein homology/analogy recognition engine online server for homology modelling and the 3D structure was predicted. Subsequently, the in silico studies of the enzyme–substrate (protein–ligand) interaction were carried out by using the method of molecular docking simulations wherein the modelled dioxygenase enzyme (protein) was docked with the substrates (ligands), catechin and epicatechin.     

Deep sequencing identified potential miRNAs involved in defence response,stress and plant growth characteristics of wild genotypes of cardamom

• Cardamom has long been used as a food flavouring agent and in ayurvedic medicines for mouth ulcers, digestive problems and even depression. Extensive occurrence of pests and diseases adversely affect its cultivation and result in substantial reductions in total production and productivity. Numerous studies revealed the significant role of miRNAs in plant biotic stress responses.
• In the current study, miRNA profiling of cultivar and wild cardamom genotypes was performed using an Ion Proton sequencer.
• We identified 161 potential miRNAs representing 42 families, including monocot/tissue‐specific and 14 novel miRNAs in both genotypes. Significant differences in miRNA family abundance between the libraries were observed in read frequencies. A total of 19 miRNAs (from known miRNAs) displayed a twofold difference in expression between wild and cultivar genotypes. We found 1168 unique potential targets for 40 known miRNA families in wild and 1025 potential targets for 42 known miRNA families in cultivar genotypes. The differential expression analysis revealed that most miRNAs identified were highly expressed in cultivars and, furthermore, lower expression of miR169 and higher expression of miR529 in wild cardamom proved evidence that wild genotypes have stronger drought stress tolerance and floral development than cultivars.
• Potential targets predicted for the newly identified miRNAs from the miRNA libraries of wild and cultivar cardamom genotypes involved in metabolic and developmental processes and in response to various stimuli. qRT‐PCR confirmed miRNAs were differentially expressed between wild and cultivar genotypes. Furthermore, four target genes were validated experimentally to confirm miRNA–mRNA target pairing using RNA ligase‐mediated 5′ Rapid Amplification of cDNA Ends (5′RLM‐RACE) PCR.

     

Extracellular production of L-glutaminase by alkalophilic Beauveria bassiana BTMF S10 isolated from marine sediment

Beauveria sp. BTMF S10 isolated from marine sediment produced extracellular L-glutaminase. Maximal L-glutaminase yield (46.9U/ml) was obtained in a medium supplemented with 1% (w/v) yeast extract and sorbitol, 9% (w/v) sodium chloride and 0.2% (w/v) methionine, initial pH 9.0 and at 27 ^°C after 108h. This enzyme was inducible and growth-associated.     

Production of phytase by Mucor racemosus in solid-state fermentation

Phytase production was studied by three Mucor and eight Rhizopus strains by solid-state fermentation (SSF) on three commonly used natural feed ingredients (canola meal, coconut oil cake, wheat bran). Mucor racemosus NRRL 1994 (ATCC 46129) gave the highest yield (14.5 IU/g dry matter phytase activity) on coconut oil cake. Optimizing the supplementation of coconut oil cake with glucose, casein and (NH(4))(2)SO(4), phytase production in solid-state fermentation was increased to 26 IU/g dry matter (DM). Optimization was carried out by Plackett-Burman and central composite experimental designs. Using the optimized medium phytase, alpha-amylase and lipase production of Mucor racemosus NRRL 1994 was compared in solid-state fermentation and in shake flask (SF) fermentation. SSF yielded higher phytase activity than did SF based on mass of initial substrate. Because this particular isolate is a food-grade fungus that has been used for sufu fermentation in China, the whole SSF material (crude enzyme, in situ enzyme) may be used directly in animal feed rations with enhanced cost efficiency.     

Elevated amniotic fluid F₂-isoprostane: a potential predictive marker for preeclampsia

In the complex mechanism of preeclampsia, oxidative stress is an important pathogenic factor, and F₂-isoprostane is a marker of oxidative stress and lipid peroxidation. The objective of this study was to identify if the amniotic fluid (AF) levels of F₂-isoprostanes were elevated in women who later developed preeclampsia. In this study, we analyzed AF F₂-isoprostane concentrations with enzyme immunoassay (EIA), and the EIA results could be validated by quantitative mass spectrometry. The mean AF concentration of F₂-isoprostanes was significantly higher in pregnancies with subsequent development of preeclampsia (123.1 ± 57.6 pg/ml, n = 85) than in controls (73.8 ± 36.6 pg/ml, n = 85). The AF elevation of F₂-isoprostanes was even higher in the preeclampsia with intrauterine growth restriction group (138.3 ± 65.2 pg/ml, n = 39). The area under the curve of the receiver operating characteristics analysis for AF F₂-isoprostanes assay was 0.81, supporting its potential as a biomarker for preeclampsia. These results indicate that oxidative stress existed before the onset of clinical preeclampsia, further suggesting that the elevation of AF F₂-isoprostanes may be used as a guide for antioxidant supplementation to reduce the risk and/or severity of preeclampsia.     

Lamin A/C-dependent localization of Nesprin-2, a giant scaffolder at the nuclear envelope

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.     

Tamarind seed powder and palm kernel cake: two novel agro residues for the production of tannase under solid state fermentation by Aspergillus niger ATCC 16620

Palm kernel cake (PKC), the residue obtained after extraction of palm oil from oil palm seeds and tamarind seed powder (TSP) obtained after removing the fruit pulp from tamarind fruit pod were tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger ATCC 16620. The fungal strain was grown on the substrates without any pretreatment. In PKC medium, a maximum enzyme yield of 13.03 IU/g dry substrate (gds) was obtained when SSF was carried out at 30 degrees C, 53.5% initial substrate moisture, 33 x 10(9) spores/5 g substrate inoculum size and 5% tannic acid as additional carbon source after 96 h of fermentation. In TSP medium, maximum tannase yield of 6.44 IU/gds was obtained at 30 degrees C, 65.75% initial substrate moisture, 11 x 10(9) spores/5 g substrate inoculum, 1% glycerol as additional carbon source and 1% potassium nitrate as additional nitrogen source after 120 h of fermentation. Results from the study are promising for the economic utilization and value addition of these important agro residues, which are abundantly available in many tropical and subtropical countries.     

Men's approval of family planning in Bangladesh

This article attempts to evaluate men's approval of family planning in Bangladesh using the couple data set from the recent Bangladesh Demographic and Health Survey (BDHS), 1999-2000. Family planning approval is addressed both from individual and couple perspectives. Analysis of BDHS data shows that about 85% of the wives report that their husbands approve of family planning, which is lower than the wives' own approval rate (95%). Using the couple data set, husbands' characteristics were matched to the wives' responses on family planning approval. Regression analyses show that age, education, access to TV, inter-spousal communication, current use of family planning and the number of living children significantly determine family planning approval among both men as well as couples. Family planning approval was found to be much lower in Sylhet than in the other administrative divisions. Multilevel modelling analysis suggests almost negligible variation at the community level. The findings indicate the need for careful evaluation of the DHS questions in order to measure appropriately men's family planning attitudes.     

Rapidly evolving sex-specific sequences in <Emphasis Type="Italic">Calamus travancoricus</Emphasis> Bedd. ex. Becc. and <Emphasis Type="Italic">Calamus nagbettai</Emphasis> R.R.Fernald &amp; Dey

Gene sequences mediating sexual reproduction are more divergent within and between closely related species. Microsatellite or simple sequence repeat (SSR) markers are valuable molecular tools for analysis of genetic variability, phylogeny, and also for identifying sex at seedling stage in dioecious plant species. Calamus travancoricus Bedd. ex. Becc. and Calamus nagbettai R.R.Fernald & Dey are economically important rattan species. The dioecious nature of the Calamus spp. limits its breeding and cultivation. The sex of rattans can only be identified after attaining reproductive maturity which ranges from 5 to 15 years. A study was carried out in this background and 9 putative sex-specific PCR products were identified as sex markers for C. travancoricus and C. nagbettai and sequenced by Sanger method. The sequence homology search revealed occurrence of identical sequences in many plant species across different families indicating the conserved nature of the sequences. However, these sequences were not present in opposite sex in the studied species, indicating divergent evolution favoring sex determination. Annotation of these sequences revealed that most of these are mediating sexual reproduction by and large. An adequate sex ratio is to be maintained for these dioecious palms in natural habitat for producing offsprings having equal gene complements for continual evolution and sustainable utilization. Developing scientific management strategies and improved utilization of canes could help to generate employment locally and thus contribute to the socioeconomics sustainably.     

Is traditional contraceptive use in Moldova associated with poverty and isolation?

This study investigates the correlates of traditional contraceptive use in Moldova, a poor country in Europe with one of the highest proportions of traditional contraceptive method users. The high reliance on traditional methods, particularly in the context of sub-replacement level fertility rate, has not been systematically evaluated in demographic research. Using cross-sectional data on a sub-sample of 6039 sexually experienced women from the 2005 Moldovan Demographic and Health Survey, this study hypothesizes that (a) economic and spatial disadvantages increase the likelihood of traditional method use, and (b) high exposure to family planning/reproductive health (FP/RH) programmes increases the propensity to modern method use. Multilevel multinomial models are used to examine the correlates of traditional method use controlling for exposure to sexual activity, socioeconomic and demographic characteristics and data structure. The results show that economic disadvantage increases the probability of traditional method use, but the overall effect is small. Although higher family planning media exposure decreases the reliance on traditional methods among younger women, it has only a marginal effect in increasing modern method use among older women. Family planning programmes designed to encourage women to switch from traditional to modern methods have some success--although the effect is considerably reduced in regions outside of the capital Chisinau. The study concludes that FP/RH efforts directed towards the poorest may have limited impact, but interventions targeted at older women could reduce the burden of unwanted pregnancies and abortions. Addressing differentials in accessing modern methods could improve uptake in rural areas.