Two nor-triterpene lactones from Caltha palustris

The chemical constituents present in the chloroform-soluble fraction of the ethanolic extract of Caltha palustris have been investigated. Two of these, caltholide and epicaltholide have been characterized as 24-nor-3β-hydroxylupan-13β,28-lactone and its 3α-isomer, respectively.     

Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean

The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.     

Evaluation of models for the evolution of protein sequences and functions under structural constraint

In the field of evolutionary structural genomics, methods are needed to evaluate why genomes evolved to contain the fold distributions that are observed. In order to study the effects of population dynamics in the evolved genomes we need fast and accurate evolutionary models which can analyze the effects of selection, drift and fixation of a protein sequence in a population that are grounded by physical parameters governing the folding and binding properties of the sequence. In this study, various knowledge-based, force field, and statistical methods for protein folding have been evaluated with four different folds: SH2 domains, SH3 domains, Globin-like, and Flavodoxin-like, to evaluate the speed and accuracy of the energy functions. Similarly, knowledge-based and force field methods have been used to predict ligand binding specificity in SH2 domain. To demonstrate the applicability of these methods, the dynamics of evolution of new binding capabilities by an SH2 domain is demonstrated.     

Isolation and characterization of a new osmotolerant, non-virulent Klebsiella pneumoniae strain SAP for biosynthesis of succinic acid

In the present study, isolation of anaerobic bacteria from 24 different eco-niches was carried out. A total number of 300 bacterial isolates, including 230 obligate and 70 facultative anaerobes were obtained using anaerobic techniques. All the isolates were initially screened for succinic acid production by Fluorescein test and TLC method. During screening, 10 isolates found to produce succinic acid were further examined by HPLC and then finally confirmed for succinic acid by LC-MS analysis. Amongst 10 isolates, isolate SAP, a facultative anaerobe isolated from buffalo rumen fluid, showed maximum yield of 2.1 g/l of succinic acid from 10 g of glucose in 24 hr under anaerobic condition. This isolate was identified as Klebsiella pneumoniae strain SAP by 16S rDNA sequence and signature sequence analysis. Mouse lethality test for the strain SAP showed LD50 value of 3.3 x 10(8) CFU/ml, which shows non-virulent nature of the strain. This strain may become a candidate strain for succinic acid production because of its osmotolerant nature and higher succinate:acetate ratio.     

Molecular characterisation of Mycobacterium tuberculosis isolates in the First National Survey of Anti-tuberculosis Drug Resistance from Venezuela

Background

Molecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD).

Results

Clinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised. Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis.

Conclusion

This study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies.     

Food availability affects circadian clock-controlled activity and Zugunruhe in the night migratory male blackheaded bunting (Emberiza melanocephala)

This study investigated the functional linkage between food availability and activity behavior in the Palaearctic Indian night migratory blackheaded bunting (Emberiza melanocephala) subjected to artificial light-dark (LD) cycles. Two experiments were performed on photosensitive birds. In the first one, birds were exposed to short days (LD 10/14; Experiment 1A), long days (LD 13/11; Experiment 1B), or increasing daylengths (8 to 13?h light/d; Experiment 1C) and presented with food either for the whole or a restricted duration of the light period. In Experiments 1A and 1B, illumination of the light and dark periods or of the dark period, alone, was changed to assess the influence of the light environment on direct and circadian responses to food cycles. In the second experiment, birds were exposed to LD 12/12 or LD 8/16 with food availability overlapping with the light (light and food presence in phase) or dark period (light and food presence in antiphase). Also, birds were subjected to constant dim light (LL(dim)) to examine the phase of the activity rhythms under synchronizing influence of the food cycles. Similarly, the presentation of food ad libitum (free food; FF) during an experiment examined the effects of the food-restriction regimes on activity rhythms. A continuous measurement of the activity-rest pattern was done to examine both the circadian and direct effects of the food and LD cycles. Measurement of activity at night enabled assessment of the migratory phenotype, premigratory restlessness, or Zugunruhe. The results show that (i) light masked the food effects if they were present together; (ii) birds had a higher anticipatory activity and food intake during restricted feeding conditions; and (iii) food at night alone reduced both the duration and amount of Zugunruhe as compared to food during the day alone. This suggests that food affects both the daily activity and seasonal Zugunruhe, and food cycles act as a synchronizer of circadian rhythms in the absence of dominant natural environmental synchronizers, such as the light-dark cycle.     

Enhanced phytase production from Achromobacter sp. PB‐01 using wheat bran as substrate: Prospective application for animal feed

This article deals with the optimization of the various parameters for production of phytase using Achromobacter sp. PB‐01 in submerged fermentation (SmF). A semisynthetic medium containing ingredients of phytase screening media (PSM) supplemented with 2% (w/v) sucrose, 1% (w/v) peptone, and 10% (w/v) wheat bran was found to be the best production medium among the various combinations tried. Among various surfactants added to SmF, Triton X‐100 (0.1%) exhibited a 16% increase in phytase activity. An overall 11.2 fold enhancement in enzyme activity (0.79 U/mL→8.84 U/mL) was attained when SmF was carried out using 0.5% (v/v) inoculum of a 15 h old culture of Achromobacter sp. PB‐01 at an initial pH of 5.5, temperature 30°C and allowed to grow for 48 h. Presence of accessory hydrolytic enzymes in the crude extract further added value as feed additive by mediating efficient degradation of non‐starch polysaccharides (NSP). In addition, we also investigated the efficacy of phytase on different agro‐industrial residues using in vitro experiments that simulated the conditions of the digestive tract. Results indicate that phytase from our source hydrolyze phytate efficiently with the concomitant liberation of inorganic phosphate, protein, reducing sugar, and calcium. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012