A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue

A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-Amp™-based DNA isolation technique with rolling circle amplification (RCA) of viral DNA, is presented. Using this method an attempt was made to isolate and clone full geminivirus genomes/genome components from 102 plant samples, including dried leaves stored at room temperature for between 6 months and 10 years, with an average hands-on-time to RCA-ready DNA of 15 min per 20 samples. While storage of dried leaves for up to 6 months did not appreciably decrease cloning success rates relative to those achieved with fresh samples, efficiency of the method decreased with increasing storage time. However, it was still possible to clone virus genomes from 47% of 10-year-old samples. To illustrate the utility of this simple method for high-throughput geminivirus diversity studies, six Maize streak virus genomes, an Abutilon mosaic virus DNA-B component and the DNA-A component of a previously unidentified New Word begomovirus species were fully sequenced. Genomic clones of the 69 other viruses were verified as such by end sequencing. This method should be extremely useful for the study of any circular DNA plant viruses with genome component lengths smaller than the maximum size amplifiable by RCA. © 2008 Elsevier B.V. All rights reserved.

Antibacterial & antiplasmid activities of Helicteres isora L.

Background & objectives: The multiple drug resistance (MDR) is a serious health problem and major challenge to the global drug discovery programmes. Most of the genetic determinants that confer resistance to antibiotics are located on R-plasmids in bacteria. The present investigation was undertaken to investigate the ability of organic extract of the fruits of Helicteres isora to cure R-plasmids from certain clinical isolates. mMethods: Active fractions demonstrating antibacterial and antiplasmid activities were isolated from the acetone extracts of shade dried fruits of H. isora by bioassay guided fractionation. Minimal inhibitory concentration (MIC) of antibiotics and organic extracts was determined by agar dilution method. Plasmid curing activity of organic fractions was determined by evaluating the ability of bacterial colonies (pre treated with organic fraction for 18 h) to grow in the presence of antibiotics. The physical loss of plasmid DNA in the cured derivatives was further confirmed by agarose gel electrophoresis. Results: The active fraction did not inhibit the growth of either the clinical isolates or the strains harbouring reference plasmids even at a concentration of 400 mu g/ml. However, the same fraction could cure plasmids from Enterococcus faecalis, Escherichia coli, Bacillus cereus and E. coli (RP4) at curing efficiencies of 14, 26, 22 and 2 per cent respectively. The active fraction mediated plasmid curing resulted in the subsequent loss of antibiotic resistance encoded in the plasmids as revealed by antibiotic resistance profile of cured strains. The physical loss of plasmid was also confirmed by agarose gel electrophoresis. Interpretation & conclusions: The active fraction of acetone extract of H. isora fruits cured R-plasmids from Gram-positive and Gram-negative clinical isolates as well as reference strains. Such plasmid loss reversed the multiple antibiotic resistance in cured derivatives making them sensitive to low concentrations of antibiotics. Acetone fractions of H. isora may be a source to develop antiplasmid agents of natural origin to contain the development and spread of plasmid borne multiple antibiotic resistance.

Activity against Mycobacterium smegmatis and M. tuberculosis by extract of South African medicinal plants

Seven ethnobotanically selected medicinal plants were screened for their antimycobacterial activity. The mininium inhibitory concentration (MIC) of four plants namely Artemisia afra, Dodonea angustifolia, Drosera capensis and Galenia africana ranged from 0.781 to 6.25 mg/mL against Mycobacterium smegmatis. G. africana showed the best activity exhibiting an MIC of 0.78 mg/mL and a minimum bactericidal concentration (MBC) of 1.56 mg/mL. The MICs of ethanol extracts of A angustifolia and G. africana against M. tuberculosis were found to be 5.0 and 1.2 mg/mL respectively. The mammalian cytotoxicity IC50 value of the most active antimycobacterial extract, from G. africana, was found to be 101.3 mu g/mL against monkey kidney Vero cells. Since the ethanol G. africana displayed the best antimycobacterial activity, it was subjected to fractionation which led to the isolation of a flavone, 5,7,2'-trihydroxyflavone. The MIC of this compound was found to be 0.031 mg/mL against M. smegmatis and 0.10 mg/mL against M. tuberculosis. This study gives some scientific basis to the 14 traditional use of these plants for TB-related symptoms. Copyright (C) 2008 John Wiley & Sons, Ltd.

Investigating the Biological Effects of Rosemary (Rosmarinus officinalis L.) Extract on Skeletal Muscle Glucose Uptake

Skeletal muscle (SKM) is the most important tissue in maintaining glucose homeostasis and impairments in this tissue leads to insulin resistance (IR). Activation of 5’ AMP-activated kinase (AMPK) is viewed as a targeted approach to counteract IR. Rosemary extract (RE) has been reported to decrease blood glucose levels but its effects on SKM are not known. We hypothesized that RE acts directly on SKM to increase glucose uptake (GU). We found an increase in GU (184±5.07% of control, p<0.001) in L6 myotubes by RE to levels similar to insulin and metformin. Carnosic acid (CA) and rosmarinic acid (RA), major polyphenols found in RE, increased GU. RE, CA, and RA significantly increased AMPK phosphorylation and their effects on GU was reduced by an AMPK inhibitor. Our study is the first to show a direct effect of RE, CA and RA on SKM GU by a mechanism that involves AMPK activation.

A unified learning algorithm to extract principal and minor components

Recently, many unified learning algorithms have been developed to solve the task of principal component analysis (PCA) and minor component analysis (MCA). These unified algorithms can be used to extract principal component and if altered simply by the sign, it can also serve as a minor component extractor. This is of practical significance in the implementations of algorithms. Convergence of the existing unified algorithms is guaranteed only under the condition that the learning rates of algorithms approach zero, which is impractical in many practical applications. In this paper, we propose a unified PCA & MCA algorithm with a constant learning rate, and derive the sufficient conditions to guarantee convergence via analyzing the discrete-time dynamics of the proposed algorithm. The achieved theoretical results lay a solid foundation for the applications of our proposed algorithm.

Testing the Efficacy of Panax ginseng Extract as a Novel Anti-Quorum Sensing Agent

Quorum sensing (QS) is the phenomenon by which microorganisms regulate gene expression in response to cell-population density. These microorganisms synthesize and secrete small molecules known as autoinducers that increase in concentration as a function of cell density. Once the cell detects the minimal threshold concentration of an autoinducer, the gene expression is altered accordingly. Although the cellular circuitry responding to QS is relatively conserved, the cellular processes regulated by QS, for example are conjugation, motility, sporulation, biofilm formation and production of virulence factors importamt for infection. Since many pathogens have developed resistance to available antibiotics, novel therapies are required to further treat these pathogens. Inhibitors of QS are potential therapeutic candidates since they would inhibit virulence without selecting for antibiotic resistant strains. Previous studies have shown that the Chinese herb Panax ginseng contains compounds that inhibit QS activity. To further characterize this activity, a highly sensitive quantitative liquid assay was developed using a Chromobacterium violaceum AHL mutant, CV026 as a biomonitor strain. After confirmation that P. ginseng aqueous extracts had anti-QS activity, the extract was fractionated on a reverse phase C18 Sep Pak column. Most anti-QS activity was present in the flow through, and compounds eluted with ten percent acetonitrile in water antibacterial activity. We thus conclude that P. ginseng has anti-QS activity and the active compound is water soluble. Compounds from P. ginseng, could be used as a lead structure to design compound with higher anti-QS activity for therapeutic treatments.

How to extract and expand randomness : a summary and explanation of existing results

We examine the use of randomness extraction and expansion in key agreement (KA) pro- tocols to generate uniformly random keys in the standard model. Although existing works provide the basic theorems necessary, they lack details or examples of appropriate cryptographic primitives and/or parameter sizes. This has lead to the large amount of min-entropy needed in the (non-uniform) shared secret being overlooked in proposals and efficiency comparisons of KA protocols. We therefore summa- rize existing work in the area and examine the security levels achieved with the use of various extractors and expanders for particular parameter sizes. The tables presented herein show that the shared secret needs a min-entropy of at least 292 bits (and even more with more realistic assumptions) to achieve an overall security level of 80 bits using the extractors and expanders we consider. The tables may be used to �nd the min-entropy required for various security levels and assumptions. We also �nd that when using the short exponent theorems of Gennaro et al., the short exponents may need to be much longer than they suggested.

Kava Anxiety Depression Spectrum Study (KADSS): a mixed methods RCT using an aqueous extract of Piper methysticum

Objectives: To report on the design, significance and potential impacts of the first documented human clinical trial assessing the anxiolytic and thymoleptic efficacy of an aqueous monoextract of Piper methysticum (kava). The significance of the qualitative element of our clinical trial is also explored. The Kava Anxiety Depression Spectrum Study (KADSS) is a 3-week placebocontrolled, double-blind, cross-over trial involving 60 adult participants (18—65) with elevated stable anxiety and varying levels of depressive symptoms. Aims: The aims of KADSS are: (1) to determine whether an aqueous standardised extract of kava is effective for the treatment of anxiety; (2) to assess the effects of kava on differing levels of depression; and (3) to explore participants’ experience of taking kava via qualitative research. The study also provides preliminary assessment of the safety of an aqueous extract of kava in humans. Conclusion: If results reveal that the aqueous kava preparation exerts significant anxiolytic effects and appears safe, potentially beneficial impacts may occur. Data supporting a safe and effective kava extract may encourage a re-introduction of kava to Europe, UK and Canada. This may provide a major socioeconomic benefit to Pacific Island nations, and to sufferers of anxiety disorders.

The Kava Anxiety Depression Spectrum Study (KADSS): A randomized, placebo-controlled, cross-over trial using an aqueous extract of Piper methysticum.

Rationale: Piper methysticum (Kava) has been withdrawn in European, British, and Canadian markets due to concerns over hepatotoxic reactions. The WHO recently recommended research into “aqueous” extracts of Kava. Objective: The objective of this study was to conduct the first documented human clinical trial assessing the anxiolytic and antidepressant efficacy of an aqueous extract of Kava. Design and participants: The Kava Anxiety Depression Spectrum Study was a 3-week placebo-controlled, double-blind crossover trial that recruited 60 adult participants with 1 month or more of elevated generalized anxiety. Five Kava tablets per day were prescribed containing 250 mg of kavalactones/day. Results: The aqueous extract of Kava reduced participants' Hamilton Anxiety Scale score in the first controlled phase by −9.9 (CI = 7.1, 12.7) vs. −0.8 (CI = −2.7, 4.3) for placebo and in the second controlled phase by −10.3 (CI = 5.8, 14.7) vs. +3.3 (CI = −6.8, 0.2). The pooled effect of Kava vs. placebo across phases was highly significant (p < 0.0001), with a substantial effect size (d = 2.24, η² [sub]p[sub] = 0.428). Pooled analyses also revealed highly significant relative reductions in Beck Anxiety Inventory and Montgomery–Asberg Depression Rating Scale scores. The aqueous extract was found to be safe, with no serious adverse effects and no clinical hepatotoxicity. Conclusions: The aqueous Kava preparation produced significant anxiolytic and antidepressant activity and raised no safety concerns at the dose and duration studied. Kava appears equally effective in cases where anxiety is accompanied by depression. This should encourage further study and consideration of globally reintroducing aqueous rootstock extracts of Kava for the management of anxiety.