Identification and Characterization of Three Novel Lipases Belonging to Families II and V from Anaerovibrio lipolyticus 5ST

Following the isolation, cultivation and characterization of the rumen bacterium Anaerovibrio lipolyticus in the 1960s, it has been recognized as one of the major species involved in lipid hydrolysis in ruminant animals. However, there has been limited characterization of the lipases from the bacterium, despite the importance of understanding lipolysis and its impact on subsequent biohydrogenation of polyunsaturated fatty acids by rumen microbes. This study describes the draft genome of Anaerovibrio lipolytica 5ST, and the characterization of three lipolytic genes and their translated protein. The uncompleted draft genome was 2.83 Mbp and comprised of 2,673 coding sequences with a G+C content of 43.3%. Three putative lipase genes, alipA, alipB and alipC, encoding 492-, 438- and 248- amino acid peptides respectively, were identified using RAST. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they were thermally unstable and had higher activities at neutral to alkaline pH. Substrate specificity assays indicated that the enzymes had higher hydrolytic activity against caprylate (C8), laurate (C12) and myristate (C14)

Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78 % following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-014-6355-6) contains supplementary material, which is available to authorized users

Piezoelectric coefficients of mNA organic nonlinear optical material using synchrotron X-ray multiple diffraction

Distortions produced in the unit cell of a nonlinear organic crystal under the influence of an applied electric field E are investigated by using synchrotron x-ray multiple diffraction (MD). A typical MD pattern shows numerous (hkl) secondary peaks and the position of each one is basically a function of the unit cell lattice parameters. Thus small changes in any parameter due to a strain produced by E give rise to a corresponding variation in the (hkl) peak position. The method was applied to the meta-nitroaniline (mNA) crystal and we were able to determine three piezoelectric coefficients. [S0031-9007(98)07912-5].81245426542

Synchrotron-radiation x-ray multiple diffraction applied to the study of electric-field-induced strain in an organic nonlinear optical material

In this work, distortions produced in the unit cell of a MBANP [(-)-2-(alpha-methylbenzylamino)-5-nitropyridine] nonlinear organic crystal under the influence of an applied electric field, (E) over bar, are investigated by using synchrotron-radiation x-ray multiple diffraction (XRMD). The method is based in the inherent sensitivity of this technique to determine small changes in the crystal lattice, which provide peak position changes in the XRMD pattern (Renninger scan). A typical Renninger scan shows numerous secondary peaks, each one carrying information on one particular direction within the crystal. The (hkl) peak position in the pattern, for a fixed wavelength, is basically a function of the unit cell lattice parameters. Thus small changes in any parameter due to a strain produced by (E) over right arrow give rise to a corresponding variation in the (hkl) peak position and the observed strain is related to the piezoelectric coefficients. The advantage of this method is the possibility of determining more than one piezoelectric coefficient from a single Renninger scan measurement [L. H. Avanci, L. P. Cardoso, S. E. Girdwood, D. Pugh, J. N. Sherwood, and K. J. Roberts, Phys. Rev. Lett. 81, 5426 (1998)]. The method has been applied to the MBANP (monoclinic, point group 2) crystal and we were able to determine four piezoelectric coefficients: \d(21)\ = 0.2(1) X 10(-11) CN-1, \d(22)\ = 24.8(3) X 10(-11) CN-1, \d(23)\ = 1.3(1) x 10(-11) CN-1, and \d(25)\ = 5.9(1) X 10(-11) CN-1. The measurements were carried out using the SRS stations 16.3, Daresbury Laboratory, Warrington, UK.61106507651

Characterisation of the Faecal Bacterial Community in Adult and Elderly Horses Fed a High Fibre, High Oil or High Starch Diet Using 454 Pyrosequencing

Faecal samples were collected from seventeen animals, each fed three different diets (high fibre, high fibre with a starch rich supplement and high fibre with an oil rich supplement). DNA was extracted and the V1–V2 regions of 16SrDNA were 454-pyrosequenced to investigate the faecal microbiome of the horse. The effect of age was also considered by comparing mature (8 horses aged 5–12) versus elderly horses (9 horses aged 19–28). A reduction in diversity was found in the elderly horse group. Significant differences between diets were found at an OTU level (52 OTUs at corrected Q<0.1). The majority of differences found were related to the Firmucutes phylum (37) with some changes in Bacteroidetes (6), Proteobacteria (3), Actinobacteria (2) and Spirochaetes (1). For the forage only diet,with no added starch or oil, we found 30/2934 OTUs (accounting for 15.9% of sequences) present in all horses. However the core (i.e. present in all horses) associated with the oil rich supplemented diet was somewhat smaller (25/3029 OTUs, 10.3% ) and the core associated with the starch rich supplemented diet was even smaller (15/2884 OTUs, 5.4% ). The core associated with samples across all three diets was extremely small (6/5689 OTUs accounting for only 2.3% of sequences) and dominated by the order Clostridiales, with the most abundant family being Lachnospiraceae. In conclusion, forage based diets plus starch or oil rich complementary feeds were associated with differences in the faecal bacterial community compared with the forage alone. Further, as observed in people, ageing is associated with a reduction in bacterial diversity. However there was no change in the bacterial community structure in these healthy animals associated with age

SUMO-1 possesses DNA binding activity

<p>Abstract</p> <p>Background</p> <p>Conjugation of small ubiquitin-related modifiers (SUMOs) is a frequent post-translational modification of proteins. SUMOs can also temporally associate with protein-targets via SUMO binding motifs (SBMs). Protein sumoylation has been identified as an important regulatory mechanism especially in the regulation of transcription and the maintenance of genome stability. The precise molecular mechanisms by which SUMO conjugation and association act are, however, not understood.</p> <p>Findings</p> <p>Using NMR spectroscopy and protein-DNA cross-linking experiments, we demonstrate here that SUMO-1 can specifically interact with dsDNA in a sequence-independent fashion. We also show that SUMO-1 binding to DNA can compete with other protein-DNA interactions at the example of the regulatory domain of Thymine-DNA Glycosylase and, based on these competition studies, estimate the DNA binding constant of SUMO1 in the range 1 mM.</p> <p>Conclusion</p> <p>This finding provides an important insight into how SUMO-1 might exert its activity. SUMO-1 might play a general role in destabilizing DNA bound protein complexes thereby operating in a bottle-opener way of fashion, explaining its pivotal role in regulating the activity of many central transcription and DNA repair complexes.</p

Investigating the potential of novel nonwoven fabrics for efficient pollination control in plant breeding

Plant breeding is achieved through the controlled self- or cross-pollination of individuals and typically involves isolation of floral parts from selected parental plants. Paper, cellulose or synthetic materials are used to avoid self pollination or cross contamination. Low seed set limits the rate of breeding progress and increases costs. We hypothesized that a novel ‘nonwoven’ fabric optimal for both pollination and seed set in multiple plant species could be developed. After determining the baseline pollen characteristics and usage requirements we established iterative three phase development and biological testing. This determined (1) that white fabric gave superior seed return and informed the (2) development of three non-woven materials using different fibre and layering techniques. We tested their performance in selfing and hybridisation experiments recording differences in performance by material type within species. Finally we (3) developed further advanced fabrics with increased air permeability and tested biological performance. An interaction between material type and species was observed and environmental decoupling investigated, showing that the non-woven fabrics had superior water vapour transmission and temperature regulation compared to controls. Overall, non-woven fabrics outperformed existing materials for both pollination and seed set and we found that different materials can optimize species-specific, rather than species-generic performance

Neoliberalism and University Education in Sub-Saharan Africa

This article reviews the history of university development in Sub-Saharan Africa (SSA) and discusses the impact of neoliberal policies. This will be followed by an examination of the problems facing universities in the region. The following questions will be explored: (a) Are the existing universities in SSA serving the development needs of the region? (b) Are these universities up to the task of moving SSA out of the predicaments it faces such as famine, HIV/AIDS, poverty, diseases, debt, and human rights abuses? Finally, the article argues that for universities to play a role in the development of the region, a new paradigm that makes university education a public good should be established

Transcriptional Regulation of Human Dual Specificity Protein Phosphatase 1 (DUSP1) Gene by Glucocorticoids

Background: Glucocorticoids are potent anti-inflammatory agents commonly used to treat inflammatory diseases. They convey signals through the intracellular glucocorticoid receptor (GR), which upon binding to ligands, associates with genomic glucocorticoid response elements (GREs) to regulate transcription of associated genes. One mechanism by which glucocorticoids inhibit inflammation is through induction of the dual specificity phosphatase-1 (DUSP1, a.k.a. mitogen-activated protein kinase phosphatase-1, MKP-1) gene. Methodology/Principal Findings: We found that glucocorticoids rapidly increased transcription of DUSP1 within 10 minutes in A549 human lung adenocarcinoma cells. Using chromatin immunoprecipitation (ChIP) scanning, we located a GR binding region between 21421 and 21118 upstream of the DUSP1 transcription start site. This region is active in a reporter system, and mutagenesis analyses identified a functional GRE located between 21337 and 21323. We found that glucocorticoids increased DNase I hypersensitivity, reduced nucleosome density, and increased histone H3 and H4 acetylation within genomic regions surrounding the GRE. ChIP experiments showed that p300 was recruited to the DUSP1 GRE, and RNA interference experiments demonstrated that reduction of p300 decreased glucocorticoid-stimulated DUSP1 gene expression and histone H3 hyperacetylation. Furthermore, overexpression of p300 potentiated glucocorticoid-stimulated activity of a reporter gene containing the DUSP1 GRE, and this coactivation effect was compromised when the histone acetyltransferase domain was mutated. ChIP-reChIP experiments using GR followed by p300 antibodies showed significant enrichment of the DUSP1 GRE upon glucocorticoid treatment, suggesting that GR and p300 are in the same protein complex recruited to the DUSP1 GRE. Conclusions/Significance: Our studies identified a functional GRE for the DUSP1 gene. Moreover, the transcriptional activation of DUSP1 by glucocorticoids requires p300 and a rapid modification of the chromatin structure surrounding the GRE. Overall, understanding the mechanism of glucocorticoid-induced DUSP1 gene transcription could provide insights into therapeutic approaches against inflammatory diseases. © 2010 Shipp et al

Bioinformatic Analysis and Post-Translational Modification Crosstalk Prediction of Lysine Acetylation

Recent proteomics studies suggest high abundance and a much wider role for lysine acetylation (K-Ac) in cellular functions. Nevertheless, cross influence between K-Ac and other post-translational modifications (PTMs) has not been carefully examined. Here, we used a variety of bioinformatics tools to analyze several available K-Ac datasets. Using gene ontology databases, we demonstrate that K-Ac sites are found in all cellular compartments. KEGG analysis indicates that the K-Ac sites are found on proteins responsible for a diverse and wide array of vital cellular functions. Domain structure prediction shows that K-Ac sites are found throughout a wide variety of protein domains, including those in heat shock proteins and those involved in cell cycle functions and DNA repair. Secondary structure prediction proves that K-Ac sites are preferentially found in ordered structures such as alpha helices and beta sheets. Finally, by mutating K-Ac sites in silico and predicting the effect on nearby phosphorylation sites, we demonstrate that the majority of lysine acetylation sites have the potential to impact protein phosphorylation, methylation, and ubiquitination status. Our work validates earlier smaller-scale studies on the acetylome and demonstrates the importance of PTM crosstalk for regulation of cellular function