Spatio-temporal Genetic Structuring of Leishmania major in Tunisia by Microsatellite Analysis

In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic (Gafsa, Kairouan and Sidi Bouzid governorates) and collected during two periods: 1991–1992 and 2008–2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991–1992 and 2008–2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008–2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations.     

The effect of habitat fragmentation on the genetic structure of a top predator: loss of diversity and high differentiation among remnant populations of Atlantic Forest jaguars (Panthera onca)

Habitat fragmentation may disrupt original patterns of gene flow and lead to drift-induced differentiation among local population units. Top predators such as the jaguar may be particularly susceptible to this effect, given their low population densities, leading to small effective sizes in local fragments. On the other hand, the jaguar's high dispersal capabilities and relatively long generation time might counteract this process, slowing the effect of drift on local populations over the time frame of decades or centuries. In this study, we have addressed this issue by investigating the genetic structure of jaguars in a recently fragmented Atlantic Forest region, aiming to test whether loss of diversity and differentiation among local populations are detectable, and whether they can be attributed to the recent effect of drift. We used 13 microsatellite loci to characterize the genetic diversity present in four remnant populations, and observed marked differentiation among them, with evidence of recent allelic loss in local areas. Although some migrant and admixed individuals were identified, our results indicate that recent large-scale habitat removal and fragmentation among these areas has been sufficiently strong to promote differentiation induced by drift and loss of alleles at each site. Low estimated effective sizes supported the inference that genetic drift could have caused this effect within a short time frame. These results indicate that jaguars' ability to effectively disperse across the human-dominated landscapes that separate the fragments is currently very limited, and that each fragment contains a small, isolated population that is already suffering from the effects of genetic drift.     

Bisphenol A induces otolith malformations during vertebrate embryogenesis

Background  

The plastic monomer and plasticizer bisphenol A (BPA), used for manufacturing polycarbonate plastic and epoxy resins, is produced at over 2.5 million metric tons per year. Concerns have been raised that BPA acts as an endocrine disruptor on both developmental and reproductive processes and a large body of evidence suggests that BPA interferes with estrogen and thyroid hormone signaling. Here, we investigated BPA effects during embryonic development using the zebrafish and Xenopus models.     

Cloning and molecular characterization of a new fungal xylanase gene from Sclerotinia sclerotiorum S2

Sclerotinia sclerotiorum fungus has three endoxylanases induced by wheat bran. In the first part, a partial xylanase sequence gene (90 bp) was isolated by PCR corresponding to catalytic domains (β 5 and β 6 strands of this protein). The high homology of this sequence with xylanase of Botryotinia fuckeliana has permitted in the second part to amplify the XYN1 gene. Sequence analysis of DNA and cDNA revealed an ORF of 746 bp interrupted by a 65 bp intron, thus encoding a predicted protein of 226 amino acids. The mature enzyme (20.06 kDa), is coded by 188 amino acid (pI 9.26). XYN1 belongs to G/11 glycosyl hydrolases family with a conserved catalytic domain containing E(86) and E(178) residues. Bioinformatics analysis revealed that there was no Asn-X-Ser/Thr motif required for N-linked glycosylation in the deduced sequence however, five O-glycosylation sites could intervene in the different folding of xylanses isoforms and in their secretary pathway.     

CLONE STRUCTURE IN FOUR SOLIDAGO ALTISSIMA (ASTERACEAE) POPULATIONS: RHIZOME CONNECTIONS WITHIN GENOTYPES

We studied clone structure and degree of genotypic mixing of Solidago altissima L. (Asteraceae) clones in four old fields near Ithaca, New York. The fields differed in time from agricultural abandonment and were approximately 1, 5, 20, and 35 years old. In the three older fields, three 0.75 m² plots were excavated intact and rhizome connections among ramets were mapped. In the youngest field 30 ramets were dug up singly. The genotype of all ramets was determined using electrophoresis of four polymorphic enzyme systems. Fields differed in the number and dispersion of genotypes within plots, and the degree of connection among ramets in the same clone. The one-year-old field was composed of single ramet genotypes which had probably established from seed the previous year. The five-year-old field contained many small contiguous clones of S. altissima with highly interconnected ramets. In the oldest two fields clones were highly intermixed and ramets of the same genotype were not extensively interconnected. These results demonstrate that clones of S. altissima display considerable phenotypic variability between fields and patterns of clone development may differ. The causes of this variability remain to be identified. We suggest that either selection for different genotypes or changing habitat conditions during succession may lead to changes in clone form.     

Association of VEGF genetic polymorphisms with prostate carcinoma risk and clinical outcome

OBJECTIVES: Vascular endothelial growth factor (VEGF) is a potent stimulus of angiogenesis that has an important role in many human malignancies including prostate carcinoma (PCa). We evaluated the role of the functional VEGF polymorphisms as genetic markers for PCa susceptibility and prognosis. METHODS: The study included 101 patients with PCa and [corrected] 100 age-matched healthy men. The VEGF genotypes -1154G>A were identified by allele-specific polymerase chain reaction (AS-PCR) and the genotypes -634G>C and 936C>T were identified by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). RESULTS: A negative association was found between VEGF -1154AA genotype and PCa risk (OR=0.27; P=0.009). Furthermore, the presence of the VEGF -1154A allele appeared to be associated with a decreased [corrected] risk of higher tumor grade (OR=0.37; P=0.01). A significant increased risk of prostate cancer was associated with the VEGF -634 (GC+CC) combined genotype (OR=1.95; P=0.02). The VEGF -634C allele was associated with the aggressive phenotype of prostate cancer as defined by the high histological grade (OR=3.48; P=0.007). The VEGF -1154A/-634G haplotype was negatively associated with PCa risk (OR=0.48; P=0.005) and high tumor grade compared to low grade (OR=0.37; P=0.02). CONCLUSIONS: Genetic variations in the VEGF may predict not only PCa risk but also tumor aggressiveness.     

Site Mapping and Characterization of O-Glycan Structures on α-Dystroglycan Isolated from Rabbit Skeletal Muscle

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, α-dystroglycan (α-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown α-DG to be modified by both O-GalNAc- and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle α-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on α-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from α-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.     

CENP-E--dependent BubR1 autophosphorylation enhances chromosome alignment and the mitotic checkpoint

How the state of spindle microtubule capture at the kinetochore is translated into mitotic checkpoint signaling remains largely unknown. In this paper, we demonstrate that the kinetochore-associated mitotic kinase BubR1 phosphorylates itself in human cells and that this autophosphorylation is dependent on its binding partner, the kinetochore motor CENP-E. This CENP-E-dependent BubR1 autophosphorylation at unattached kinetochores is important for a full-strength mitotic checkpoint to prevent single chromosome loss. Replacing endogenous BubR1 with a nonphosphorylatable BubR1 mutant, as well as depletion of CENP-E, the BubR1 kinase activator, results in metaphase chromosome misalignment and a decrease of Aurora B-mediated Ndc80 phosphorylation at kinetochores. Furthermore, expressing a phosphomimetic BubR1 mutant substantially reduces the incidence of polar chromosomes in CENP-E-depleted cells. Thus, the state of CENP-E-dependent BubR1 autophosphorylation in response to spindle microtubule capture by CENP-E is important for kinetochore function in achieving accurate chromosome segregation.     

Cloning, characterization and molecular docking of a highly thermostable β-1,4-glucosidase from Thermotoga petrophila

A genomic DNA fragment, encoding a thermotolerant β-glucosidase, of the obligate anaerobe Thermotoga petrophila RKU-1 was cloned after PCR amplification into Escherichia coli strain BL21 CodonPlus. The purified cloned enzyme was a monomeric, 51.5?kDa protein (by SDS-PAGE) encoded by 1.341?kb gene. The estimated K (m) and V (max) values against p-nitrophenyl-β-D-glucopyranoside were 2.8?mM and 42.7?mmol?min(-1)?mg(-1), respectively. The enzyme was also active against other p-nitrophenyl substrates. Possible catalytic sites involved in hydrolyzing different p-nitrophenyl substrates are proposed based on docking studies of enzyme with its substrates. Because of its unique characters, this enzyme is a potential candidate for industrial applications.