Identifying Leptospira interrogans putative virulence factors with a yeast protein expression screen.

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., with global implications primarily in tropical countries. However, the mechanisms of leptospiral pathogenesis are still not fully known and not all virulence factors (VFs) have been identified. Budding yeast, Saccharomyces cerevisiae is a popular eukaryotic model which has been used to identify bacterial VFs that target the conserved eukaryotic cellular processes. In this study, we screened for putative VFs of L. interrogans, one of the dominant species causing leptospirosis, by expressing candidate VFs in budding yeast and examining their impact on yeast growth in a high-throughput format. From an initial selection of 288 L. interrogans ORFs, we screened 226 candidate VFs in a yeast growth inhibition assay and identified nine putative VFs in four categories (adhesion, enzymatic, host structure interaction, and immunogenicity). Notably, LIC10280 was highly toxic even when expressed at low copies. We also observed specific subcellular localization for several putative VFs. This study shows that there are still potential L. interrogans VFs that await discovery. KEY POINTS: • High-throughput cloning and expression of leptospiral proteins in yeast. • Heterologous expression of nine leptospiral proteins inhibited yeast growth. • An uncharacterized protein LIC10280 maybe a putative VF for further validation.

Characterization of polyhydroxyalkanoate production capacity, composition and weight synthesized by Burkholderia cepacia JC-1 from various carbon sources.

Polyhydroxyalkanoates (PHA) are microbial polymers that have received widespread attention in recent decades as potential alternatives to some petrochemical-based plastics. However, widespread use of PHA is often impeded by its cost of production. Therefore, the search for and systematic investigation of versatile microbial PHA producers capable of using various carbon sources, even in the form of animal fats, for PHA biosynthesis is desirable. This study highlights the PHA production capacity, monomer composition and molecular weight synthesized by Burkholderia cepacia JC-1, a locally isolated strain from soil, from various carbon sources. In the category of simple sugars and plant oils, the use of glucose and palm oil at C:N ratio of 40 resulted in the highest accumulation of 52 wt% and 36 wt% poly(3-hydroxybutyrate) [P(3HB)] homopolymer and dry cell weight of 2.56 g/L and 3.17 g/L, respectively. Interestingly, B. cepacia JC-1 was able to directly utilize animal-derived lipid in the form of crude and extracted chicken fat, resulting in appreciable dry cell weight and PHA contents of up to 3.19 g/L and 47 wt% respectively, surpassing even that of palm oil in the group of triglycerides as substrates. The presence of antibiotics (streptomycin) in cultivation medium did not significantly affect cell growth and polymer production. The supply of sodium pentanoate as a co-substrate resulted in the incorporation of 3-hydroxyvalerate (3HV) monomer at fractions up to 37 mol%. The molecular weight of polymers produced from glucose, palm oil and chicken fat were in the range of 991-2118 kDa, higher than some reported studies involving native strains. The results from this study form an important basis for possible improvements in using B. cepacia JC-1 and crude chicken fats in solid form for PHA production in the future.

Total Phenolics, Total Flavonoids, Antioxidant Capacities, and Volatile Compounds Gas Chromatography-Mass Spectrometry Profiling of Moringa oleifera Ripe Seed Polar Fractions.

BACKGROUND: Academic reports have confirmed Moringa oleifera leaves to possess significant antioxidant capacities; however, such studies are unavailable for its ripe seeds even though they are more desirous for consumption due to their sweet taste. OBJECTIVE: In this study, we investigated antioxidant capacities of four polar extracts (crude water, ethanol, butanol, and aqueous residue) from the plant's ripe seeds. MATERIALS AND METHODS: Phytochemicals were extracted from the ripe seeds of M. oleifera using ethanol and water solvents at initial stage. Butanol and aqueous residue were then subsequently fractioned out from the ethanol extract. Phenolic and flavonoid contents of the polar extracts were determined. Then, their antioxidant capacities were quantified by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. Finally, gas chromatography-mass spectrometry (GC-MS) analyses of the extracts were performed. RESULTS: DPPH and ABTS tests showed that the polar extracts possess significant antioxidant capacities that ranged from 29 to 35.408 µM Trolox equivalence antioxidant capacity (TEAC)/mg sample and 7 to 29 µM TEAC/mg sample, respectively. The antioxidant capacities of the extracts corresponded to their phenolic and flavonoid contents that varied from 13.61 to 20.42 mg gallic acid equivalence/g sample and 0.58 to 9.81 mg quercetin equivalence/g sample, respectively. Finally, GC-MS analyses revealed antimicrobial phenolic compounds, 4-hydroxybenzaldehyde in crude water extract and 4-hydroxybenzene acetonitrile in the ethanol and butanol extracts, and aqueous residue. CONCLUSION: Our results established that M. oleifera ripe seeds have significant antioxidant activity which may be due to its phenolic and nonphenolic compounds content. SUMMARY: In this study, polar phytochemicals from ripe seeds of Moringa oleifera were extracted by water and ethanol solvents, and butanol extract and aqueous residue were subsequently fractioned out of the ethanol extract. The four polar extracts were shown to have significant antioxidant capacities which correspond to their phenolic contents. Further, antimicrobial compounds 4-hydroxybenzaldehyde and 4-hydroxybenzene acetonitrile were identified in the extracts by gas chromatography-mass spectrometry analyses. Abbreviations used: ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH: 2,2-diphenyl-1-picrylhydrazyl; TEAC: Trolox equivalence antioxidant capacity; QE: Quercetin equivalence; GAE: Gallic acid equivalence; GC-MS: Gas chromatography-mass spectrometry.

ANTIPROLIFERATIVE EFFECT ON BREAST CANCER (MCF7) OF MORINGA OLEIFERA SEED EXTRACTS.

BACKGROUND: Moringa oleifera belongs to plant family, Moringaceae and popularly called "wonderful tree", for it is used traditionally to cure many diseases including cancer in Africa and Asia, however, there is limited knowledge on cytotoxic activity of Moringa oleifera seeds on MCF7 breast cancer cell. The present study evaluated antiproliferative effect on MCF7 of the seed. MATERIALS AND METHODS: Seeds of Moringa oleifera were grinded to powder and its phytochemicals were extracted using water and 80% ethanol solvents, part of the ethanolic extract were sequentially partitioned to fractions with four solvents (hexane, dichloromethane, chloroform, and n-butanol). Antiproliferative effects on MCF7 of the samples were determined. Finally, potent samples that significantly inhibited MCF7 growth were tested on MCF 10A. RESULTS: Crude water extract, hexane and dichloromethane fractions of the seeds inhibited the proliferation of MCF7 with the following IC50 values 280 µg/ml, 130 µg/ml and 26 µg/ml respectively, however, of the 3 samples, only hexane fraction had minimal cytotoxic effect on MCF 10A (IC50 > 400µg/ml). CONCLUSION: Moringa oleifera seed has antiproliferative effect on MCF7.

EcoliOverExpressionDB: a database of recombinant protein overexpression in E. coli.

UNLABELLED: Recombinant protein production is a significant biotechnological process as it allows researchers to produce a specific protein in desired quantities. Escherichia coli (E. coli) is the most popular heterologous expression host for the production of recombinant proteins due to its advantages such as low cost, high-productivity, well-characterized genetics, simple growth requirements and rapid growth. There are a number of factors that influence the expression level of a recombinant protein in E. coli which are the gene to be expressed, the expression vector, the expression host, and the culture condition. The major motivation to develop our database, EcoliOverExpressionDB, is to provide a means for researchers to quickly locate key factors in the overexpression of certain proteins. Such information would be a useful guide for the overexpression of similar proteins in E. coli. To the best of the present researchers' knowledge, in general and specifically in E. coli, EcoliOverExpressionDB is the first database of recombinant protein expression experiments which gathers the influential parameters on protein overexpression and the results in one place. AVAILABILITY: EcoliOverExpressionDB is freely available and accessible using all major browsers at http://birg4.fbb.utm.my:8080/EcoliOverExpressionDB/.

Sequence analysis and structure prediction of type II Pseudomonas sp. USM 4-55 PHA synthase and an insight into its catalytic mechanism.

BACKGROUND: Polyhydroxyalkanoates (PHA), are biodegradable polyesters derived from many microorganisms such as the pseudomonads. These polyesters are in great demand especially in the packaging industries, the medical line as well as the paint industries. The enzyme responsible in catalyzing the formation of PHA is PHA synthase. Due to the limited structural information, its functional properties including catalysis are lacking. Therefore, this study seeks to investigate the structural properties as well as its catalytic mechanism by predicting the three-dimensional (3D) model of the Type II Pseudomonas sp. USM 4-55 PHA synthase 1 (PhaC1P.sp USM 4-55). RESULTS: Sequence analysis demonstrated that PhaC1P.sp USM 4-55 lacked similarity with all known structures in databases. PSI-BLAST and HMM Superfamily analyses demonstrated that this enzyme belongs to the alpha/beta hydrolase fold family. Threading approach revealed that the most suitable template to use was the human gastric lipase (PDB ID: 1HLG). The superimposition of the predicted PhaC1P.sp USM 4-55 model with 1HLG covering 86.2% of the backbone atoms showed an RMSD of 1.15 A. The catalytic residues comprising of Cys296, Asp451 and His479 were found to be conserved and located adjacent to each other. In addition to this, an extension to the catalytic mechanism was also proposed whereby two tetrahedral intermediates were believed to form during the PHA biosynthesis. These transition state intermediates were further postulated to be stabilized by the formation of oxyanion holes. Based on the sequence analysis and the deduced model, Ser297 was postulated to contribute to the formation of the oxyanion hole. CONCLUSION: The 3D model of the core region of PhaC1P.sp USM 4-55 from residue 267 to residue 484 was developed using computational techniques and the locations of the catalytic residues were identified. Results from this study for the first time highlighted Ser297 potentially playing an important role in the enzyme's catalytic mechanism.

Phylogeny and characterization of three nifH-homologous genes from Paenibacillus azotofixans.

In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.

Molecular characterisation of six alternatively spliced variants and a novel promoter in human peroxisome proliferator-activated receptor alpha.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcriptional factor that governs many biological processes, including lipid metabolism, inflammation, and atherosclerosis. We demonstrate here the existence of six variants and multiple transcriptional start sites of the 5(') untranslated region (UTR) of hPPARalpha gene, originating from the use of alternative splicing mechanisms and four different promoters. Three new novel exons at the 5(')-untranslated region of human PPARalpha gene were also identified and designated as Exon A, Exon B, and Exon 2b. In addition, 1.2kb promoter fragment which drives the transcription of 2 variants with Exon B (hPPARalpha4 and 6) was successfully cloned and characterised. Sequencing results revealed promoter B did not contain a conservative TATA box within the first 100 nucleotides from transcriptional start site but has several GC-rich regions and putative Sp1 sites. Using luciferase reporter constructs transfected into HepG2 and Hep3B cell lines, promoter B was shown to be functionally active. Basal transcriptional activity was significantly high in the promoter fragment -341/+34, but lower in the region -341/-1147 as compared to the fragment -341/+34, indicating the presence of an element conferring transcriptional activation between positions -341 and +34 or alternatively, the presence of transcriptional repression between positions -341 and -1147 in the promoter B of hPPARalpha.