A comparative genomic analysis of the alkalitolerant soil bacterium Bacillus lehensis G1

Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil samples. B. lehensis produces cyclodextrin glucanotransferase (CGTase), an enzyme that has enabled the extensive use of cyclodextrin in foodstuffs, chemicals, and pharmaceuticals. The genome sequence of B. lehensis G1 consists of a single circular 3.99 Mb chromosome containing 4017 protein-coding sequences (CDSs), of which 2818 (70.15%) have assigned biological roles, 936 (23.30%) have conserved domains with unknown functions, and 263 (6.55%) have no match with any protein database. Bacillus clausii KSM-K16 was established as the closest relative to B. lehensis G1 based on gene content similarity and 16S rRNA phylogenetic analysis. A total of 2820 proteins from B. lehensis G1 were found to have orthologues in B. clausii, including sodium–proton antiporters, transport proteins, and proteins involved in ATP synthesis. A comparative analysis of these proteins and those in B. clausii and other alkaliphilic Bacillus species was carried out to investigate their contributions towards the alkalitolerance of the microorganism. The similarities and differences in alkalitolerance-related genes among alkalitolerant/alkaliphilic Bacillus species highlight the complex mechanism of pH homeostasis. The B. lehensis G1 genome was also mined for proteins and enzymes with potential viability for industrial and commercial purposes.     

Phylogenetic diversity and genome sizes of Astragalus (Fabaceae) in the Lebanon biogeographical crossroad

The Lebanese mountain range is an important zone of plant species richness and endemism where the genus Astragalus constitutes a principal component of plant biodiversity. Most of endemic Astragalus taxa, living in mountains and arid zones of Mounts Lebanon and anti-Lebanon, are characterized by a cushion, spiny vegetative form, named “tragacanthic”, which is a remarkable example of vegetative convergence evolution. Because of determination difficulties, taxonomic uncertainties, and discrepancy in the number of taxa listed according to authors, new data are hardly needed to improve systematics of Astragalus and to investigate the role of the Lebanese mountain range as refugia of biodiversity. Before this study only two values on the genome size of Astragalus were reported in the literature and no previous molecular studies had been carried out on Astragalus genus in Lebanon. We examined the utility of rDNA ITS molecular markers to distinguish Astragalus species of Lebanese mountain range and the variation range of their genome size. The main results revealed a striking diversity in Lebanese Astragalus species with the emphasis of a huge variation of genome sizes, an important inter-specific chromosome polymorphism and the existence of a high phylogenetic diversity. The strict endemic species of the Lebanese mountains are positioned throughout the phylogeny. These results confirm that the Lebanon and anti-Lebanon mounts constitute a third diversity center for Astragalus and that high altitude areas are important refugia of plant biodiversity despite centuries of exploitation by humans.     

Molecular cloning,expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica

Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.     

Epigallocatechin-3-Gallate (EGCG), a Green Tea Polyphenol,Stimulates Hepatic Autophagy and Lipid Clearance

Epigallocatechin gallate (EGCG) is a major polyphenol in green tea that has been shown to have anti-inflammatory, anti-cancer, anti-steatotic effects on the liver. Autophagy also mediates similar effects; however, it is not currently known whether EGCG can regulate hepatic autophagy. Here, we show that EGCG increases hepatic autophagy by promoting the formation of autophagosomes, increasing lysosomal acidification, and stimulating autophagic flux in hepatic cells and in vivo. EGCG also increases phosphorylation of AMPK, one of the major regulators of autophagy. Importantly, siRNA knockdown of AMPK abrogated autophagy induced by EGCG. Interestingly, we observed lipid droplet within autophagosomes and autolysosomes and increased lipid clearance by EGCG, suggesting it promotes lipid metabolism by increasing autophagy. In mice fed with high-fat/western style diet (HFW; 60% energy as fat, reduced levels of calcium, vitamin D3, choline, folate, and fiber), EGCG treatment reduces hepatosteatosis and concomitantly increases autophagy. In summary, we have used genetic and pharmacological approaches to demonstrate EGCG induction of hepatic autophagy, and this may contribute to its beneficial effects in reducing hepatosteatosis and potentially some other pathological liver conditions.     

Kinetic Modeling and Error Analysis for Decontamination of Different Petroleum Hydrocarbon Components in Biostimulation of Oily Soil Microcosm

A microcosm study was constructed to investigate the effect of complex co-substrate (corn steep liquor, CSL) addition on indigenous bacterial community, rate and extent of petroleum hydrocarbons (PH) degradation in an oily soil with total petroleum hydrocarbons (TPH) content of 63353 mg kg^?1. TPH degradation was found to be characterized by a rapid phase of degradation during the first three weeks where 76% removal of TPH occurred, followed by a slower degradation phase, where further 7% of the initial TPH was removed by the end of incubation period, 35 d. Branched alkanes are more resistant to microbial degradation than n-alkanes. Furthermore, the unresolved complex mixtures (UCM) of hydrocarbons are less degradable than n- and iso-alkanes. Pristane (Pr) was the most recalcitrant aliphatic compound studied in this work. These results in addition to the extensive bacterial growth observed (from 10⁷ to 10¹⁰ CFU g^?1 soil) give strong support that the addition of CSL resulted in increased degradation rates. The indigenous bacteria grew exponentially during the incubation period of 35 d with a growth rate of 0.26 d^?1. Kinetic modeling was performed to estimate the rates of biodegradation of each hydrocarbon type component in the studied system. Five different error functions were used in this study to evaluate the fitness of the model equation to the obtained experimental data. This showed that the degradation of ∑nC₂₀-nC_24, ∑nC₃₅-nC₄₂ and nC₁₈ can be better represented by a second order model, whereas the TPH, total resolvable peaks (TRP), nC₁₇, UCM, ∑nC₁₀-nC₁₄, ∑nC₁₅-nC_19, ∑nC₂₅-nC₂₉, ∑nC₃₀-nC_34, ∑nC_n, and ∑isoC_n and isoprenoids Pr and phytane (Ph) were similarly following the first order model.     

Evaluation of anti-nociceptive,anti-inflammatory and antipyretic potential of Mikania cordata (Burm. f.) Robinson in experimental animal model

Mikania cordata is widely used for the treatment of cuts, wounds, and dengue fever in Bangladesh. In the present study, essential oil (12.5, 25 and 50?mg/kg) and two extracts, viz., chloroform and ethyl acetate extracts (200, 400, 800?mg/kg b.w.) were tested for peripheral and central anti-nociceptive activity by acetic acid-induced writhing and hot plate method, respectively. Carrageenan-induced rat paw edema assay and yeast-induced hyperthermia assay were also carried out to evaluate anti-inflammatory and antipyretic properties of oil and extracts, respectively at aforesaid doses. The essential oil (50?mg/kg), chloroform extract (800?mg/kg) and ethyl acetate extract (800?mg/kg) showed potent peripheral anti-nociceptive activity having 47.33%, 29.33% and 16.65% of writhing inhibition, respectively, comparable with standard diclofenac (52.0%). Essential oil (50?mg/kg), chloroform extract (800?mg/kg) and ethyl acetate extract (800?mg/kg) presented promising central anti-nociceptive activity as well having 95.86%, 79.18% and 42.37% elongation of reaction time, respectively, at 90?min after administration of essential oil, ethyl acetate extract and 60?min after administration of chloroform extract. In anti-inflammatory activity screening, the essential oil (50?mg/kg) produced the highest 72.80% edema inhibition at 4?h after administration of carrageenan which was comparable with that of standard phenylbutazoe (87.87%). On the other hand, chloroform extract (800?mg/kg) and ethyl acetate extract (800?mg/kg) showed up to 34.31% and 15.27% of edema inhibition, respectively, at 4?h after administration of carrageenan. In antipyretic assay, the essential oil and chloroform extract displayed a strong antipyretic effect in yeast-induced rats, whereas the ethyl acetate extract had no antipyretic activity. The present study revealed anti-nociceptive, anti-inflammatory and antipyretic potential of M. cordata which could be the therapeutic option against fever, inflammations as well as painful conditions and confirmed the traditional use of M. cordata.