Exopolysaccharide production by the cyanobacterium Anabaena sp. ATCC 33047 in batch and continuous culture

The halotolerant, filamentous, heterocystous cyanobacterium Anabaena sp. ATCC 33047 released, during the stationary growth phase in batch culture and, at low dilution rate, in continuous culture, large amounts of an exopolysaccharide (EPS) to the culture medium. Different environmental, nutritional and physical parameters affected production and accumulation of the EPS. The presence of either a combined nitrogen source or NaCl at high concentration led to decreased EPS production, without affecting cell growth. In contrast, generation of the EPS was markedly enhanced in response to an increase in either air flow rate, temperature or irradiance. In continuous culture, accumulation of EPS in the medium increased in response to a decrease in the dilution rate, with maximal EPS productivity being reached at a dilution rate of 0.03 h⁻¹.     

Mycobacterium tuberculosis Genotypes Determined by Spoligotyping to Be Circulating in Colombia between 1999 and 2012 and Their Possible Associations with Transmission and Susceptibility to First-Line Drugs

Introduction

Tuberculosis (TB) remains a primary public health problem worldwide. The number of multidrug-resistant tuberculosis (MDR TB) cases has increased in recent years in Colombia. Knowledge of M. tuberculosis genotypes defined by spoligotyping can help determine the circulation of genotypes that must be controlled to prevent the spread of TB.

Objective

To describe the genotypes of M. tuberculosis using spoligotyping in resistant and drug-sensitive isolates and their possible associations with susceptibility to first-line drugs.

Methods

An analytical observational study was conducted that included 741 isolates of M. tuberculosis from patients. The isolates originated from 31 departments and were obtained by systematic surveillance between 1999 and 2012.

Results

In total 61.94% of the isolates were resistant to 1 or more drugs, and 147 isolates were MDR. In total, 170 genotypes were found in the population structure of Colombian M. tuberculosis isolates. The isolates were mainly represented by four families: LAM (39.9%), Haarlem (19%), Orphan (17%) and T (9%). The SIT42 (LAM 9) was the most common genotype and contained 24.7% of the isolates, followed by the genotypes SIT62 (Haarlem1), SIT53 (T1), and SIT50 (H3). A high clustering of isolates was evident with 79.8% of the isolates classified into 32 groups. The Beijing family was associated with resistant isolates, whereas the Haarlem and T families were associated with sensitive isolates. The Haarlem family was also associated with grouped isolates (p = 0.031).

Conclusions

A high proportion (approximately 80%) of isolates was found in clusters; these clusters were not associated with resistance to first-line drugs. The Beijing family was associated with drug resistance, whereas the T and Haarlem families were associated with susceptibility in the Colombian isolates studied.     

Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus and Cortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatants of termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensional gel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.     

Protecting capacity against malaria of chemically defined tetramer forms based on the Plasmodium falciparum apical sushi protein as potential vaccine components

Developing novel generations of subunit-based antimalarial vaccines in the form of chemically-defined macromolecule systems for multiple antigen presentation represents a classical problem in the field of vaccine development. Many efforts involving synthesis strategies leading to macromolecule constructs have been based on dendrimer-like systems, the condensation of large building blocks and conventional asymmetric double dimer constructs, all based on lysine cores.     

Study of possible interactions of tubulin, microtubular network, and STOP protein with mitochondria in muscle cells

We studied possible connections of tubulin, microtubular system, and microtubular network stabilizing STOP protein with mitochondria in rat and mouse cardiac and skeletal muscles by confocal microscopy and oxygraphy. Intracellular localization and content of tubulin was found to be muscle type-specific, with high amounts in oxidative muscles, and much lower in glycolytic skeletal muscle. STOP protein localization and content in muscle cells was also muscle type-specific. In isolated heart mitochondria, addition of 1 μM tubulin heterodimer increased apparent K _m for ADP significantly. Dissociation of microtubular system into free tubulin by colchicine treatment only slightly decreased initially high apparent K _m for ADP in permeabilized cells, and diffusely distributed free tubulin stayed inside the cells, obviously connected to the intracellular structures. To identify the genes that are specific for oxidative muscle, we developed and applied a method of kindred DNA. The results of sequencing and bioinformatic analysis of isolated cDNA pool common for heart and m. soleus showed that in adult mice the β-tubulin gene is expressed predominantly in oxidative muscle cells. It is concluded that whereas dimeric tubulin may play a significant role in regulation of mitochondrial outer membrane permeability in the cells in vivo, its organization into microtubular network has a minor significance on that process.     

Distribución de la circunferencia de la cintura y de la relación circunferencia de la cintura con respecto a la talla según la categoría del índice de masa corporal en los pacientes atendidos en consultas de endocrinología y nutrición

IntroductionWaist circumference (WC) and the waist-to-height ratio (WHtR) are anthropometric measures widely used in clinical practice to evaluate visceral fat and the consequent cardiovascular risk. However, risk thresholds should be standardized according to body mass index (BMI).ObjectiveTo determine the distribution of WC and WHtR according to the BMI cut-points currently used to describe overweight and obesity.Materials and methodsWC, WHtR and BMI were measured in 3521 adult patients (>18 years) attended in Endocrinology and Nutrition units.ResultsA total of 20.8% (734 patients) were diabetic. Obesity was found in 82.1% of diabetic patients and in 75% of non-diabetic patients. The WC thresholds proposed by the National Institute of Health (102 cm in men, 88 cm in women), Bray (100 cm in men, 90 cm in women) and the International Diabetes Federation (94 cm in men, 80 cm in women) were exceeded by 92.9%, 94.8% and 98.4% of obese men, 96.8%, 95.5% and 99.7% of obese women, 79.1%, 83.1% and 90% of diabetic men and 95.5%, 81.5% and 97.4% of diabetic women, respectively. Thresholds adapted to the degree of obesity (90, 100, 110 and 125 cm in men and 80, 90, 105 and 115 cm in women for normal BMI, overweight, obesity I and obesity greater than I) were exceeded by 58.4% of obese men, 54.2% of obese women, 57.5% of diabetic men and 60.7% of diabetic women. WC was higher in men, and BMI and the WHtR were higher in women. The WC of diabetic women equalled that of men, and WC, WHtR and BMI were higher in diabetic than in non-diabetic women (p<0.001). WC (p<0.005), WHtR (p<0.001) and BMI (p<0.5) were also higher in diabetic than in non-diabetic men.ConclusionWC and WHtR thresholds by BMI discriminated diabetic and obese patients better than single thresholds, and can be represented graphically by the distribution of percentile ranks of WC and WHtR by BMI.ik     

Optimizing a Proteomics Platform for Urine Biomarker Discovery

Biomarker discovery approaches in urine have been hindered by concerns for reproducibility and inadequate standardization of proteomics protocols. In this study, we describe an optimized quantitative proteomics strategy for urine biomarker discovery, which is applicable to fresh or long frozen samples. We used urine from healthy controls to standardize iTRAQ (isobaric tags for relative and absolute quantitation) for variation induced by protease inhibitors, starting protein and iTRAQ label quantities, protein extraction methods, and depletion of albumin and immunoglobulin G (IgG). We observed the following: (a) Absence of protease inhibitors did not affect the number or identity of the high confidence proteins. (b) Use of less than 20 μg of protein per sample led to a significant drop in the number of identified proteins. (c) Use of as little as a quarter unit of an iTRAQ label did not affect the number or identity of the identified proteins. (d) Protein extraction by methanol precipitation led to the highest protein yields and the most reproducible spectra. (e) Depletion of albumin and IgG did not increase the number of identified proteins or deepen the proteome coverage. Applying this optimized protocol to four pairs of long frozen urine samples from diabetic Pima Indians with or without nephropathy, we observed patterns suggesting segregation of cases and controls by iTRAQ spectra. We also identified several previously reported candidate biomarkers that showed trends toward differential expression, albeit not reaching statistical significance in this small sample set.With ongoing advances in mass spectrometry (MS) and proteomics technology, proteomics analysis is progressively occupying a central position in biomarker discovery platforms. Biofluids such as urine and blood are the preferred media for proteomics analysis because of their ease of collection and extensive history of use in clinical laboratory practice. Urine, in particular, is an information-rich fluid that can be collected non-invasively and in large quantities. Many urine proteins are produced or shed in the kidney and urogenital tract (1), making urine a promising proximal source of biomarkers for diseases affecting these structures.However, proteomics-based biomarker discovery in urine faces multiple challenges. Urine proteomics is complicated by low urine protein concentration, variations in pH, and high concentrations of salts and urea or other urine components that interfere with sample processing. The urine proteome can also change with individual variables such as hydration, diurnal change, diet, and physical activity as well as variation in sample collection, processing, and storage. In addition, urine proteomics shares the usual challenges of biomarker discovery in other biofluids such as throughput, cost, and the need for a reproducible and quantitative work flow.Isotopic or isobaric labeling methods to reduce variation, increase throughput, and enable quantitative analysis have been developed to address some of these challenges. One such method, isobaric tags for relative and absolute quantitation (iTRAQ)¹ (2), combines relative and absolute peptide quantification with multiplexing ability to enable an increased throughput as well as simultaneous comparison of up to eight samples within one experimental run. Variations induced by urine sample processing have been systematically evaluated for proteomics analyses using two-dimensional gel electrophoresis (3–6), differential gel electrophoresis (7), and liquid chromatography-coupled mass spectrometry (LC-MS) (5, 8, 9). However, no systematic analyses of urine sample collection and processing have been reported for iTRAQ.Before utilizing iTRAQ-based quantitative proteomics for urine biomarker discovery, we evaluated the impact of variation in several processing steps (addition of protease inhibitors, the starting protein quantities, quantity of the iTRAQ label, protein extraction methods, and depletion of abundant proteins) on iTRAQ protein identification and quantitation. Applying this optimized biomarker discovery protocol to small quantities of long frozen urine samples from the Pima longitudinal study of diabetic nephropathy, we observed patterns suggestive of segregation of cases and controls by iTRAQ spectra. We also observed trends toward differential expression in several proteins that had been identified as putative biomarkers in previous studies. However, given the small sample size, none of these proteins retained statistical significance after multiple testing correction.