Fatty acid patterns of film-forming yeasts and new evidence for the heterogeneity of Pichia membranaefaciens

P. NORONHA-DA-COSTA, C. RODRIGUES, I. SPENCER-MARTINS AND V. LOUREIRO. 1996. Total fatty acids (C14 : 0 to C18 : 3) of 50 strains assigned by classical identification methods to Pichia membranaefaciens (28), P. anomala (15), Dekkera anomala (2), D. bruxellensis (2) and Candida vini (3) were determined and data analysed by multivariate statistical procedures. Principal components cluster analysis defined six groups of strains. Thirteen strains of P. anomala formed a well-defined cluster, whereas P. membranaefaciens was split into three groups. The taxonomic status of the P. membranaefaciens strains was evaluated by determination of the G+C content and DNA-DNA hybridization. The results provided evidence in support of P. membranaefaciens encompassing distinct species. Only eight strains were certified as P. membranaefaciens and six were included in the same cluster, indicating that numerical analysis of fatty acid profiles points to genetic differences which remain undetectable by conventional phenotypic tests.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.      

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.     

Ultra-Deep Sequencing of HIV-1 near Full-Length and Partial Proviral Genomes Reveals High Genetic Diversity among Brazilian Blood Donors

Background

Here, we aimed to gain a comprehensive picture of the HIV-1 diversity in the northeast and southeast part of Brazil. To this end, a high-throughput sequencing-by-synthesis protocol and instrument were used to characterize the near full length (NFLG) and partial HIV-1 proviral genome in 259 HIV-1 infected blood donors at four major blood centers in Brazil: Pro-Sangue foundation (São Paulo state (SP), n 51), Hemominas foundation (Minas Gerais state (MG), n 41), Hemope foundation (Recife state (PE), n 96) and Hemorio blood bank (Rio de Janeiro (RJ), n 70).

Materials and Methods

A total of 259 blood samples were obtained from 195 donors with long-standing infections and 64 donors with a lack of stage information. DNA was extracted from the peripheral blood mononuclear cells (PBMCs) to amplify the HIV-1 NFLGs from five overlapping fragments. The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol.

Results

Of the 259 samples studied, 208 (80%) NFLGs and 49 (18.8%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Of these 257 samples, 183 (71.2%) were pure subtypes consisting of clade B (n = 167, 65%), C (n = 10, 3.9%), F1 (n = 4, 1.5%), and D (n = 2, 0.7%). Recombinant viruses were detected in 74 (28.8%) samples and consist of unique BF1 (n = 41, 15.9%), BC (n = 7, 2.7%), BCF1 (n = 4, 1.5%), CF1 and CDK (n = 1, 0.4%, each), CRF70_BF1 (n = 4, 1.5%), CRF71_BF1 (n = 12, 4.7%), and CRF72_BF1 (n = 4, 1.5%). Evidence of dual infection was detected in four patients coinfected with the same subtype (n = 3) and distinct subtype (n = 1).

Conclusion

Based on this work, subtype B appears to be the prevalent subtype followed by a high proportion of intersubtype recombinants that appeared to be arising continually in this country. Our study represents the largest analysis of the viral NFLG ever undertaken worldwide and provides insights into the understanding the genesis of the HIV-1 epidemic in this particular area of South America and informs vaccine design and clinical trials.     

An unusual suspect in cocaine addiction

Most Mage family members code for antigens on melanoma tumor cells. Maged1 is the black sheep, promiscuously found in normal adult cells, including neurons of the basal ganglia and prefrontal cortex. In this issue of EMBO reports, De Backer et al 1 propose an unexpected role for Maged1. Acute effects of cocaine, such as enhanced locomotion and reinforcement, are gone in mice in which the gene is deleted. In a painstaking combinatorial approach comparing several conditional gene knockout (KO) mouse lines, the authors parse the relevant neural circuits.     

Follicle populations and gene expression profiles of Nelore and Angus heifers with low and high ovarian follicle counts

The number of antral follicles counted (AFC) by ultrasound is associated with fertility in cattle. Cows with higher follicle count (HFC) have higher performance in reproductive‐assisted technologies than cows with lower follicle count (LFC). In this study, we aimed to define the preantral follicle count by histology and to identify differentially expressed genes (DEGs) using a microarray in Nelore and Angus heifers with HFC and LFC. The ovaries of each animal were scanned with an ultrasound device 12 to 24 hr after estrus. The groups were formed based on the average number of total follicles (≥3 mm) counted in each breed consistently ± the standard deviation. For the histological analysis, preantral follicles were counted and classified under a stereo microscope, and follicle density was determined. Microarray analysis was performed on pools of three follicles dissected from the ovaries of 15 Nelore (6 HFC and 9 LFC) and 17 Angus heifers (9 HFC and 8 LFC). Angus heifers have increased total and primordial follicle density. Nelore heifers have increased antral follicle count. Different patterns of gene expression regulate follicle recruitment and development in Angus and Nelore heifers and may be associated with the different follicle densities observed in Angus versus Nelore heifers. Furthermore, HFC heifers presented increased expression of genes associated with cellular development and metabolism.     

Complete genome sequence of a sucrose-nonfermenting epidemic strain of Vibrio cholerae O1 from Brazil

We report the genome sequence of Vibrio cholerae strain IEC224, which fails to ferment sucrose. It was isolated from a cholera outbreak in the Amazon. The defective sucrose phenotype was determined to be due to a frameshift mutation, and a molecular marker of the Latin American main epidemic lineage was identified.     

Green coconut fiber: a novel carrier for the immobilization of commercial laccase by covalent attachment for textile dyes decolourization

Commercial laccase formulation was immobilized on modified green coconut fiber silanized with 3-glycidoxypropyltrimethoxysilane, aiming to achieve a cheap and effective biocatalyst. Two different strategies were followed: one point (pH 7.0) and multipoint (pH 10.0) covalent attachment. The influence of immobilization time on enzymatic activity and the final reduction with sodium borohydride were evaluated. The highest activities were achieved after 2?h of contact time in all situations. Commercial laccase immobilized at pH 7.0 was found to have higher activity and higher affinity to the substrate. However, the immobilization by multipoint covalent attachment improved the biocatalyst thermal stability at 50?°C, when compared to soluble enzyme and to the immobilized enzyme at pH 7.0. The Schiff's bases reduction by sodium borohydride, in spite of causing a decrease in enzyme activity, showed to contribute to the increase of operational stability through bonds stabilization. Finally, these immobilized enzymes showed high efficiency in the continuous decolourization of reactive textile dyes. In the first cycle, the decolourization is mainly due to dyes adsorption on the support. However, when working in successive cycles, the adsorption capacity of the support decreases (saturation) and the enzymatic action increases, indicating the applicability of this biocatalyst for textile wastewater treatment.     

Pollen-mediated gene flow in wheat (Triticum aestivum L.) in a semiarid field environment in Spain

Transgenic wheat (Triticum aestivum L.) varieties are being developed and field-tested in various countries. Concerns regarding gene flow from genetically modified (GM) crops to non-GM crops have stimulated research to estimate outcrossing in wheat prior to the release and commercialization of any transgenic cultivars. The aim is to ensure that coexistence of all types of wheat with GM wheat is feasible in accordance with current regulations. The present study describes the result of a field experiment under the semi-arid climate conditions of Madrid, Spain, at two locations (??La Canaleja?? and ??El Encin?? experimental stations) in Madrid over a 3-year period, from 2005 to 2007. The experimental design consisted of a 50?×?50?m wheat pollen source sown with wheat cultivars resistant to the herbicide chlortoluron (??Deganit?? and ??Castan?? respectively) and three susceptible receptor cultivars (??Abental??, ??Altria?? and ??Recital??) sown in replicated 1?×?1?m plots at different distances (0, 1, 3, 5, 10, 20, 40, 80 and 100?m) and four directions. Outcrossing rates were measured as a percentage of herbicide-resistant hybrids using an herbicide-screening assay. Outcrossing was greatest near the pollen source, averaging 0.029?% at 0?m distance at ??La Canaleja?? and 0.337?% at ??El Encin??, both below the 0.9?% European Union regulated threshold, although a maximum outcrossing rate of 3.5?% was detected in one recipient plot. These percentages declined rapidly as the distance increased, but hybrids were detected at different rates at distances of up to 100?m, the maximum distance of the experiment. Environmental conditions, as drought in 2004?C2005 and 2005?C2006, may have influenced the extent of outcrossing. These assays carried out in wheat under semi-arid conditions in Europe provide a more complete assessment of pollen-mediated gene flow in this crop.