Telomere length analysis in residents of a community exposed to arsenic

Differentiated cells telomere length is an indicator of senescence or lifespan; however, in peripheral blood leukocytes the relative shortening of the telomere has been considered as a biological marker of aging, and lengthening telomere as an associated risk to cancer. Individual’s age, type of tissue, lifestyle, and environmental factors make telomere length variable. The presence of environmental carcinogens such as arsenic (As) influence as causal agents of these alterations, the main modes of action for As described are oxidative stress, reduction in DNA repair capacity, overexpression of genes, alteration of telomerase activity, and damage to telomeres. The telomeres of leukocytes resulting a finite capacity of replication due to the low or no activity of the telomerase enzyme, therefore, elongation telomere in this kind of cells is a potential biological marker associated with the development of chronic diseases and carcinogenesis.     

Bacterial Community Structure in the Hyperarid Core of the Atacama Desert, Chile

Soils from the hyperarid Atacama Desert of northern Chile were sampled along an east-west elevational transect (23.75 to 24.70°S) through the driest sector to compare the relative structure of bacterial communities. Analysis of denaturing gradient gel electrophoresis (DGGE) profiles from each of the samples revealed that microbial communities from the extreme hyperarid core of the desert clustered separately from all of the remaining communities. Bands sequenced from DGGE profiles of two samples taken at a 22-month interval from this core region revealed the presence of similar populations dominated by bacteria from the Gemmatimonadetes and Planctomycetes phyla.     

Fitness effects of beneficial mutations: the mutational landscape model in experimental evolution

The mutational landscape model is a theoretical model describing sequence evolution in natural populations. However, recent experimental work has begun to test its predictions in laboratory populations of microbes. Several of these studies have focused on testing the prediction that the effects of beneficial mutations should be roughly exponentially distributed. The prediction appears to be borne out by most of these studies, at least qualitatively. Another study showed that a modified version of the model was able to predict, with reasonable accuracy, which of a ranked set of beneficial alleles will be fixed next. Although it remains to be seen whether the mutational landscape model adequately describes adaptation in organisms other than microbes, together these studies suggest that adaptive evolution has surprisingly general properties that can be successfully captured by theoretical models.     

Molecular Characterization of Sewage-Borne Pathogens and Detection of Sewage Markers in an Urban Stream in Caracas,Venezuela

Molecular characterization of two sewage-borne pathogens identified hepatitis A virus (HAV) subgenotype IA and Giardia duodenalis assemblages A and B as predominant genotypes circulating in an urban area of Venezuela. This study reveals epidemiological features of human pathogens of worldwide distribution and the efficacy of molecular methods for accurate assessment of sewage pollution.Multiple microbial pathogens may frequently be found in surface waters that receive uncontrolled municipal sewage discharges. The range and diversity of sewage-borne pathogens in surface waters are geographically specific and strongly dependent on the burden of infectious diseases in the population, the seasonal patterns of infectious diseases in the community, and the availability of sewage treatment processing (5, 7). The metropolitan city of Caracas, the capital and largest city of Venezuela, is located in northern South America near the Caribbean coast. Water pollution is a big issue in Caracas, like in many other cities in South America, where most of the human sewage (∼97%) from overpopulated urbanized areas is discharged without any treatment into nearby rivers and coastal environments. Despite these facts, the seriousness of sewage-related health issues is not at the forefront of public concern in this country.Giardia is the protozoan parasite most frequently detected in human fecal samples submitted to diagnostic laboratories from major cities in Venezuela. The frequency of giardiasis reported in the population varies between 21% and 45% but may increase up to 75% among school age children (8). Notwithstanding, the epidemiology of giardiasis in Venezuela remains unknown, and no previous studies have documented the distribution of species and genotype assemblages associated with human infections.Hepatitis A virus (HAV), the etiological agent of hepatitis A in humans, has distinguishable epidemiological patterns of distribution and endemicity closely related to socioeconomic development (9, 12). Water- and food-borne outbreaks of HAV have been well documented worldwide (6, 19). Seroepidemiological studies conducted in selected populations in Venezuela have demonstrated high endemicity of hepatitis A infection among low socioeconomic population strata, with seroprevalences between 48% and 98% (13). Nevertheless, studies on HAV genotype circulation in major urban areas of the country are scarce, as is research on predominant exposure routes and potential transmission patterns through the environment.The analysis of nucleic acid sequences of sewage-borne pathogens may provide relevant information on predominant species and genotypes of human-pathogenic viruses and parasites circulating in specific geographical areas (11, 12, 15). The molecular approach may be of relevance for countries lacking reliable disease surveillance programs and proper understanding of the potential transmission of specific human pathogens through the environment. In this research, Giardia cysts and HAV recovered from an urban stream were characterized by multiple molecular methods along with nucleotide sequence analysis to identify predominant genotypes circulating in a major urban area of Venezuela''s capital. The strength and efficacy of multiple molecular methods for accurate assessment of human sewage pollution and risks of exposure to sewage-borne pathogens were also investigated.Dry season sampling (October through March) was conducted in a heavily polluted urban stream (>10⁶ fecal coliforms/100 ml) that flows in a southeast direction through the metropolitan city of Caracas (16). Water sample volumes of 100 ml were collected in three sterile centrifuge tubes two to four times per month. Giardia cysts were concentrated by centrifugation (100 ml at 1,500 × g for 15 min) followed by DNA extraction by the freeze-thaw method in the presence of Chelex-100 (3) for sucrose-purified cysts. Human-pathogenic assemblage occurrence was determined by nested PCR amplification and sequence analysis of the triosephosphate isomerase (tpi) gene (18). Multiple sequence alignments were performed with ClustalW (21), and phylogenetic analyses were conducted using MEGA4 software (20). The genetic diversity of Giardia isolates was inferred by the neighbor-joining method (17) using a bootstrap test of 1,000 replicates. Giardia cysts counts were obtained by fluorescence microscopy using BTF EasyStain monoclonal antibody stain (BTF Precise Microbiology, Inc.) and 4′6-diamidino-2-phenylindole (DAPI). The recovery efficiency of cysts was determined in five experiments using ColorSeed C&G spike suspensions as internal quality controls (14).HAV particles were concentrated from 35 ml by ultracentrifugation and elution with 0.25 N glycine buffer following procedures previously described (16). Viral RNA was extracted from sample concentrates with Trizol (Invitrogen, Inc., Carlsbad, CA) following the manufacturer''s instructions. General detection of HAV was based on amplification of the 5′ nontranslated region (5′ NTR), while analysis of genetic diversity involved sequencing and phylogenetic analysis of the VP1 amino terminus and the full VP1 gene (2, 10). Sequence alignment was conducted with the DNAman software 5.2.2 (Lynnon BioSoft, Quebec, Canada) followed by phylogenetic analysis.Molecular detection of sewage pollution was accomplished by PCR amplification of Bacteroidales human-specific 16S rRNA genes, Bacteroides thetaiotamicron 16S rRNA genes, and the nifH gene of Methanobrevibacter smithii using primers and PCR conditions originally described by Field et al. (4), Carson et al. (1), and Ufnar et al. (22), respectively.Giardia duodenalis tpi nucleotide sequences amplified directly from urban stream waters were included after phylogenetic analysis into two well-defined clusters of assemblages A and B. These results were supported by high bootstrap values, as indicated in Fig. ​Fig.1.1. The level of cysts recovered from these samples ranged from 10,400 to 62,000 cysts/liter; however, mean percent recoveries varied from 20% to 50%, which suggests that the urban stream may harbor and receive much higher loads of cysts.Open in a separate windowFIG. 1.Phylogenetic tree of G. duodenalis assemblages A (VPW2, VPW3, and VPW7) and B (VPW1, VPW4, VPW5, and VPW6) from urban stream samples forming two clusters in a neighbor-joining analysis of tpi nucleotide sequences. Only bootstrap values >80% are shown in the tree.Three genomic regions used for detection and characterization of HAV revealed the predominance of HAV strains belonging to subgenotype IA, the most frequent genotype associated with human disease worldwide (9). A neighbor-joining tree constructed from the alignment of nucleotide sequences from urban stream samples and sequences of HAV strains from case patients (unpublished data) was used to investigate the relationship between genotypes present in environmental and clinical samples. The comparative analysis indicated a high degree of identity (98 to 99%) between nucleotide sequences from the urban stream and the strains from sporadic HAV cases. The phylogenetic analysis grouped all of these sequences into two unique clades within subgenotype IA, strongly supported by significant bootstrap values (Fig. 2A, B, and C).Open in a separate windowFIG. 2.Phylogenetic analysis of the HAV 5′ NTR (A; 284 nucleotides [nt]), VP1 amino terminus (B; 172 nt), and complete VP1 (C; 820 nt) regions. Nucleotide sequences of HAV reference strains are designated by their GenBank accession number, including the name of the country of origin, except for Venezuelan isolates, which are shown in bold. S, isolates derived from human sporadic cases; W, urban stream isolates. Phylogenetic analysis was performed by neighbor joining, and phylogenetic distances were calculated by the Kimura two-parameter test. Bootstrap values ≥90% are shown in the trees. Letters in bold indicate the subtype.Three reliable published assays for detection of human-specific markers of fecal pollution identified and confirmed the predominant point source of water pollution. Sequence analysis of three randomly selected PCR products from each marker revealed ≥99% sequence identity with published sequences (GenBank) derived from different geographical areas, thus indicating the validity and specificity of the molecular markers as reliable indicators of human sewage pollution in Venezuela.The results of this research demonstrate that the molecular assays applied for detection and characterization of sewage-borne pathogens in surface waters may have practical applications for epidemiological investigations on distribution of predominant human-specific genotypes circulating in urban populations. Previous studies identified the most predominant waterborne gastroenteritis viruses circulating in Metropolitan Caracas (16).The molecular-based monitoring approach for rapid and precise identification of sewage-borne pathogens and sewage markers in surface waters has important implications for sewage-related health issues that require special attention in Venezuela and South America. Deficient sewerage coverage and lack of municipal wastewater treatment, commonly associated with informal settlements around densely populated urban areas, are responsible for many of the environmental degradation and public health problems that occur in these countries. The precise identification of human pathogens in the environment offers an appropriate and alternative approach for initial assessment of risks of exposure to waterborne pathogens. Current bacterial indicators of fecal pollution (fecal coliforms, Escherichia coli, and enterococci) do not allow identification of the relative sources of impacts (i.e., sewage, urban runoff, and agricultural waste) on surface waters. Thus, the molecular detection of sewage-borne pathogens and sewage markers in surface waters may be more effective than the bacterial indicator approach for forecasting pathogen distribution and for managing and reducing risks associated with inappropriate sewage disposal into natural waters in Venezuela and South America.     

Proteomic analysis in mammary glands of rat offspring exposed in utero to bisphenol A

Bisphenol A (BPA) is a ubiquitous environmental contaminant with established endocrine disruptor properties. The objective of our study was to determine the effects of prenatal exposure to BPA on the rat mammary gland proteome in postnatal rats as a first step toward the investigation of translational biomarkers of susceptibility in the human population. Pregnant rats were treated orally with 0, 25 or 250 µg BPA/kg body weight from days 10 to 21 post-conception. Female offspring were euthanized at 21 and 50 days, and mammary glands were collected. Proteomic analysis was conducted using 2-DE, followed by a combination of MALDI-TOF–TOF and LC–MS/MS, which led to the identification of 21 differentially abundant proteins including vimentin, SPARC and 14–3–3. Western blot analysis of key downstream signaling proteins demonstrated increased phospho-AKT, c-Raf, phospho-ERKs-1 and 2, but decreased TGF-β in mammary glands of 50 day old rats exposed prenatally to BPA. Our studies indicate for the first time that key proteins involved in signaling pathways such as cellular proliferation are regulated at the protein level by BPA. This data is expected to aid in the understanding of how BPA may be influencing the susceptibility of the mammary gland to cancer transformation.     

A pseudohitchhiking model of X vs. autosomal diversity

We study levels of X-linked vs. autosomal diversity using a model developed to analyze the hitchhiking effect. Repeated bouts of hitchhiking are thought to lower X-linked diversity for two reasons: first, because sojourn times of beneficial mutations are shorter on the X, and second, because adaptive substitutions may be more frequent on the X. We investigate whether each of these effects does, in fact, cause reduced X-linked diversity under hitchhiking. We study the strength of the hitchhiking effect on the X vs. autosomes when there is no recombination and under two different recombination schemes. When recombination occurs in both sexes, X-linked vs. autosomal diversity is reduced by hitchhiking under a broad range of conditions, but when there is no recombination in males, as in Drosophila, the required conditions are considerably more restrictive.     

A reduced protein model with accurate native-structure identification ability

A protein model that is simple enough to be used in protein-folding simulations but accurate enough to identify a protein native fold is described. Its geometry consists of describing the residues by one, two, or three pseudoatoms, depending on the residue size. Its energy is given by a pairwise, knowledge-based potential obtained for all the pseudoatoms as a function of their relative distance. The pseudoatomic potential is also a function of the primary chain separation and residue order. The model is tested by gapless threading on a large, representative set of known protein and decoy structures obtained from the "Decoys 'R' Us" database. It is also tested by threading on gapped decoys generated for proteins with many homologs. The gapless threading tests show near 98% native-structure recognition as the lowest energy structure and almost 100% as one of the three lowest energy structures for over 2200 test proteins. In decoy threading tests, the model recognized the majority of the native structures. It is also able to recognize native structures among gapped decoys, in spite of close structural similarities. The results indicate that the pseudoatomic model has native recognition ability similar to comparable atomic-based models but much better than equivalent residue-based models.