Toxicogenetic monitoring in urban cities exposed to different airborne contaminants

Microparticles found in the air may be associated with organic matter that contains several compounds, such as Polycyclic Aromatic Hydrocarbons (PAHs) and nitro-PAHs, and may pose a significant risk to human health, possibly leading to DNA mutations and cancers. This study associated genotoxicity assays for evaluating human exposure with the atmospheric air of two urban areas in southern Brazil, that received different atmospheric contributions. Site 1 was under urban-industrial influence and the other was a non-industrial reference, Site 2. Organic extracts from the airborne particulate matter were tested for mutagenicity via the Salmonella/microsome assay and analyzed for PAH composition. Cells samples of people residing in these two cities were evaluated using the comet and micronucleus assay (MN).Concentrations of the individual PAHs ranged from 0.01ng/m3 (benzo[a]anthracene) to 5.08ng/m3 (benzo[ghi]perylene). As to mutagenicity analysis of airborne, Site 1 presented all the mutagenic responses, which varied from 3.2±1.22rev/m3 (TA98 no S9) to 32.6±2.05rev/m3 (TA98, S9), while Site 2 ranged from negative to minimal responses. Site 1 presented a high quantity of nitro and amino derivatives of PAHs, and peaked at 56.0±3.68rev/μg (YG1024 strain). The two groups presented very low DNA damage levels without intergroup difference. Although Site 1 presented high mutagenic responses in the air samples, high PAH levels, healthy people exposed to this environment did not show significative damage in their genetic material. However, the evaluation of different environmental and genetic damage in such population is necessary to monitor possible damages. © 2013 Elsevier Inc.

Detection of post-F1 fish hybrids in broodstock using molecular markers: Approaches for genetic management in aquaculture

Herein, we have developed molecular markers for nuclear genes to use in multiplex-PCR and PCR-RFLP, with the goal of characterising hybrid lines derived from crosses between pintado Pseudoplatystoma corruscans and cachara P. reticulatum. These markers, together with others described previously, were used to perform molecular identification analyses as genetic subsidies for Brazilian aquaculture. These analyses were performed due to the problems of high mortality in the offspring reported by the aquaculturist. From a total of 16 broodstock samples, 13 were genetically identified as hybrids; surprisingly, nine of these hybrids were found to be post-F1 lineages. These data show that the fertility of these animals can seriously affect the cultivated stocks, thus causing financial damage in this aquaculture system. The establishment of PCR-RFLP and multiplex-PCR as molecular techniques allows for both the correct management of these animals and the routine monitoring of production and trade of fish hybrids in aquaculture. Consequently, such tools will enable a sustainable development in the aquaculture industry. © 2012 Blackwell Publishing Ltd.

Genetic markers for the identification of hybrids among catfish species of the family Pimelodidae

Fish hybrids provide genetically manipulated products of excellent value for the commercial aquaculture industry. However, if handled or marketed incorrectly, they can cause great financial loss to producers as well as threaten the native species. Herein, molecular markers are established to identify hybrid lineages of pimelodids and characterize them in relation to their parental species, Pseudoplatystoma corruscans, Pseudoplatystoma reticulatum, Phractocephalus hemioliopterus and Leiarius marmoratus. The results show that the mitochondrial genes are useful for identification of the cross-direction through the characterization of the maternal lineage. The nuclear genes allow identification of the interspecific hybrids. Use of genetic markers can avoid misidentification of hybrids that occur in simple morphological analysis. Thus, the present results allow the routine monitoring of pimelodid hybrids for their correct management and trade in aquaculture. © 2013 Blackwell Verlag GmbH.

Habitat monitoring and genotoxicity in Ucides cordatus (Crustacea: Ucididae), as tools to manage a mangrove reserve in southeastern Brazil

In Brazil, the state of São Paulo contains both preserved areas (Juréia-Itatins Ecological Station) and extremely impacted ones (Cubatão Municipality). This study evaluated the concentrations of five metals (Cu, Cd, Cr, Pb, and Hg) in two mangroves with different levels of anthropogenic impact and the apparent genotoxicity to Ucides cordatus. Water and sediment samples were obtained, and metal concentrations were determined with an atomic absorption spectrophotometer. The genotoxic impact was quantified based on the number of micronucleated cells per 1,000 analyzed (MN‰), using hemolymph slides stained with Giemsa. Metal concentrations in water were below the detection limit, except for lead, although no significant difference was observed between the areas (P > 0.05). Sediment from Cubatão had higher concentrations of Cd, Pb, Cr, and Cu than sediment from Juréia-Itatins (P < 0.05), but no significant differences in metal concentrations were detected among depth strata of the sediment (P > 0.05). Crabs from Cubatão had a 2.6 times higher mean frequency of micronucleated cells (5.2 ± 1.8 MN‰) than those from Juréia-Itatins (2.0 ± 1.0 MN‰; P < 0.0001). The more-polluted conditions found in the mangrove sediments of Cubatão were reflected in the micronucleus assay, demonstrating their genotoxic effect; however, genetic damage should be attributed to a synergistic effect with other kinds of pollutants previously recorded in different environments of Cubatão. U. cordatus proved to be an excellent bioindicator of mangrove pollution. This study established, for the first time, the normal frequency of MN‰ in a population of this species within an ecological station. © 2013 Springer Science+Business Media Dordrecht.

Low levels of genetic diversity depicted from mitochondrial DNA sequences in a heavily exploited marine fish (Cynoscion acoupa, Sciaenidae) from the Northern coast of Brazil

The acoupa weakfish (Cynoscion acoupa - Sciaenidae) is a marine species of croaker with estuarine-dependent behavior, found in the western Atlantic from Panama to Argentina. It is one of the most exploited food fish on the northern coast of Brazil. In this study, DNA sequences were determined from the entire control region (D-loop) of the mitochondrial genome of 297 individuals collected during seven different months between December 2003 and August 2005 on the northern coast of Brazil (Amapá and Pará). Genetic variability expressed by haplotype (h = 0,892) and nucleotide (p = 0,003) diversities were low compared to other heavily exploited marine fish species from the western Atlantic and eastern Asia. AMOVA depicted a lack of genetic structuring among the samples from different years, indicating the presence of a single stock of C. acoupa within the sample area. The possible reasons for the low levels of genetic diversity are discussed. These results demonstrate a need for the monitoring of C. acoupa harvesting and the preservation of the estuaries within its geographic range, considering that this large fish depends on estuarine ecosystems during part of its life cycle.

Genetic Instability Persists in Non-Neoplastic Urothelial Cells from Patients with a History of Urothelial Cell Carcinoma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

History, structure, and genetic diversity of Brazilian Gir cattle

The Gir cattle breed (Bos indicus) is an important genetic resource for milk production throughout the tropics. The small number of Gir animals introduced in Brazil, rapid dissemination of this breed recently, and the intensification of selection practices could contribute to increase of inbreeding level and reduce genetic diversity in this population. The population was analyzed in terms of pedigree completeness level, inbreeding coefficient, coancestry, generation interval, effective population size, effective number of founders and ancestors, among others. Despite the low mean inbreeding (around 2%), minor problems were identified in the population structure of the Brazilian Gir cattle, e.g., trend of narrower bottlenecks in the pedigree in recent years. The effective population sizes based on inbreeding (94) or coancestry (165.9) as well as the effective number of ancestors (76) and founders (143) were relativity high. The major subdivision of the Gir breed was observed between 1993 and 2002 (dairy and dual-purpose herds, wide use of within-herd matings). In this period the level of inbreeding remained at a higher level, there was a small increase in coancestry and the number of equivalent subpopulations was approximately 6. After 2002, there was genetic exchange between subpopulations, reduction in the average inbreeding, pronounced increase in the average coancestry, and the number of equivalent subpopulations was about 2. Furthermore, it was found that the mean generation interval of the population tended to increase in recent years (around 9 years). About 23% of genetic diversity has been lost since the first generation of founders. Based on the effective population size, number of equivalent subpopulations, inbreeding, coancestry, and loss of genetic diversity, the Gir population is still highly structured, but there is ample room for artificial selection. The results regarding the effective number of founders and ancestors in the present population demonstrate the existence of bottlenecks in the pedigree and indicate the need for population structure monitoring. Nevertheless, the Brazilian Gir breed can perfectly face a breeding program with high selection intensity. (C) 2014 Elsevier B.V. All rights reserved.

Pregnancy Monitoring of In Vitro Produced Embryos in Buffaloes

In the present study, pregnancies obtained from 115 in vitro produced embryos were monitored by ultrasonography on days 30 and 60 after embryo transfer (ET), and at calving. Additionally, the health of newborns and recipients were also evaluated. On day 30 after ET, positive pregnancy was diagnosed in 50 animals (43.5%). A total of 8 fetal mortalities (16.0%) were verified from 30 days until calving, in which 2 occurred from 30 to 60 days after ET (4.0%), and 6 occurred from 60 days until calving (12.0%). In this last period of pregnancy, 3 pregnancy losses were due to abortion, and the other 3 were stillbirth. One additional animal was eliminated from the study, remaining 41 pregnancies. From these 41 pregnancies, a total of 20 female calves (48.8%) and 21 male calves (51.2%) were born. Pregnancies from female and male calves had a mean length of 309.8 and 310.9 days, respectively (range 300 to 328 days, and 297 to 320 days, respectively). Weight at calving was a mean of 31.4 and 33.8 kg for female and male calves, respectively. All calving occurred without intervention and dystocia was not observed in any case. No large offspring syndrome, hydramnios, hydroallantois, or umbilical cord anomalies were observed in calves. Delivery was normal in all recipients, and no puerperal infections, or retained placenta occurred. Suckling assistance was not necessary in any newborn. All genetic pedigree was confirmed later by DNA tests.

Genetic Identification of F1 and Post-F1 Serrasalmid Juvenile Hybrids in Brazilian Aquaculture

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A rigorous approach to facilitate and guarantee the correctness of the genetic testing management in human genome information systems

Abstract Background Recent medical and biological technology advances have stimulated the development of new testing systems that have been providing huge, varied amounts of molecular and clinical data. Growing data volumes pose significant challenges for information processing systems in research centers. Additionally, the routines of genomics laboratory are typically characterized by high parallelism in testing and constant procedure changes. Results This paper describes a formal approach to address this challenge through the implementation of a genetic testing management system applied to human genome laboratory. We introduced the Human Genome Research Center Information System (CEGH) in Brazil, a system that is able to support constant changes in human genome testing and can provide patients updated results based on the most recent and validated genetic knowledge. Our approach uses a common repository for process planning to ensure reusability, specification, instantiation, monitoring, and execution of processes, which are defined using a relational database and rigorous control flow specifications based on process algebra (ACP). The main difference between our approach and related works is that we were able to join two important aspects: 1) process scalability achieved through relational database implementation, and 2) correctness of processes using process algebra. Furthermore, the software allows end users to define genetic testing without requiring any knowledge about business process notation or process algebra. Conclusions This paper presents the CEGH information system that is a Laboratory Information Management System (LIMS) based on a formal framework to support genetic testing management for Mendelian disorder studies. We have proved the feasibility and showed usability benefits of a rigorous approach that is able to specify, validate, and perform genetic testing using easy end user interfaces.

Mechanism of resistance to tyrosine kinase inhibitors in philadelphia-positive acute lymphblastic leukaemia (all): from genetic alterations to impaired RNA editing

The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it represents the single most significant adverse prognostic marker. Despite imatinib has led to significant improvements in the treatment of patients with Ph+ ALL, in the majority of cases resistance developed quickly and disease progressed. Some mechanisms of resistance have been widely described but the full knowledge of contributing factors, driving both the disease and resistance, remains to be defined. The observation of rapid development of lymphoblastic leukemia in mice expressing altered Ikaros (Ik) isoforms represented the background of this study. Ikaros is a zinc finger transcription factor required for normal hemopoietic differentiation and proliferation, particularly in the lymphoid lineages. By means of alternative splicing, Ikaros encodes several proteins that differ in their abilities to bind to a consensus DNA-binding site. Shorter, DNA nonbinding isoforms exert a dominant negative effect, inhibiting the ability of longer heterodimer partners to bind DNA. The differential expression pattern of Ik isoforms in Ph+ ALL patients was analyzed in order to determine if molecular abnormalities involving the Ik gene could associate with resistance to imatinib and dasatinib. Bone marrow and peripheral blood samples from 46 adult patients (median age 55 yrs, 18-76) with Ph+ ALL at diagnosis and during treatment with imatinib (16 pts) or dasatinib (30 pts) were collected. We set up a fast, high-throughput method based on capillary electrophoresis technology to detect and quantify splice variants. 41% Ph+ ALL patients expressed high levels of the non DNA-binding dominant negative Ik6 isoform lacking critical N-terminal zinc-fingers which display abnormal subcellular compartmentalization pattern. Nuclear extracts from patients expressed Ik6 failed to bind DNA in mobility shift assay using a DNA probe containing an Ikaros-specific DNA binding sequence. In 59% Ph+ ALL patients there was the coexistence in the same PCR sample and at the same time of many splice variants corresponded to Ik1, Ik2, Ik4, Ik4A, Ik5A, Ik6, Ik6 and Ik8 isoforms. In these patients aberrant full-length Ikaros isoforms in Ph+ ALL characterized by a 60-bp insertion immediately downstream of exon 3 and a recurring 30-bp in-frame deletion at the end of exon 7 involving most frequently the Ik2, Ik4 isoforms were also identified. Both the insertion and deletion were due to the selection of alternative splice donor and acceptor sites. The molecular monitoring of minimal residual disease showed for the first time in vivo that the Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patient-derived leukaemia cells expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In order to mechanistically demonstrated whether in vitro the overexpression of Ik6 impairs the response to tyrosine kinase inhibitors (TKIs) and contributes to resistance, an imatinib-sensitive Ik6-negative Ph+ ALL cell line (SUP-B15) was transfected with the complete Ik6 DNA coding sequence. The expression of Ik6 strongly increased proliferation and inhibited apoptosis in TKI sensitive cells establishing a previously unknown link between specific molecular defects that involve the Ikaros gene and the resistance to TKIs in Ph+ ALL patients. Amplification and genomic sequence analysis of the exon splice junction regions showed the presence of 2 single nucleotide polymorphisms (SNPs): rs10251980 [A/G] in the exon2/3 splice junction and of rs10262731 [A/G] in the exon 7/8 splice junction in 50% and 36% of patients, respectively. A variant of the rs11329346 [-/C], in 16% of patients was also found. Other two different single nucleotide substitutions not recognized as SNP were observed. Some mutations were predicted by computational analyses (RESCUE approach) to alter cis-splicing elements. In conclusion, these findings demonstrated that the post-transcriptional regulation of alternative splicing of Ikaros gene is defective in the majority of Ph+ ALL patients treated with TKIs. The overexpression of Ik6 blocking B-cell differentiation could contribute to resistance opening a time frame, during which leukaemia cells acquire secondary transforming events that confer definitive resistance to imatinib and dasatinib.

Reproduction and artificial restocking of Acipenser naccarii (Bonaparte, 1836). Genetic and physiological characterization through microsatellite markers and radioimmunoassay of sexual steroids

The Adriatic sturgeon, Acipenser naccarii (Bonaparte, 1836), is a highly threatened species due to human activities, particularly overfishing and habitat destruction. Its peculiar ecology and biology (restricted areal and anadromy) makes this species particularly vulnerable. In March 2010 the IUCN has identified the Adriatic sturgeon as a critically endangered species according to the Red List of Threatened Species. Due to its rapid decline, starting from the 80s, at present there is no evidence of natural reproduction in wild environment, which makes the Adriatic sturgeon dependenton captive breeding programs that need to be improved in order to be effective for the survival of the species. For this purpose this study aims to characterize artificial restocking population of Adriatic sturgeon, with both genetic and physiological analysis in order to establish an efficient restocking program for future reproductions. The research is structured on two levels: First genetically, by analyzing 9 microsatellite loci. This gives information relatively about parent allocation and kinship between individuals that were sampled for this study. Hence to predict which reproduction events are the most optimal in terms of incrementing genetic diversity, by the estimation of multilocus pairwise band sharing coefficients. Second step, physiological analysis: testosterone (T) concentration levels in each individual were measured for sexing, without sacrificing the lives of the animals with the use of an invasive examination of the gonads. The combination of interdisciplinary analysis is important to obtain an overall picture in order to indicate the main broodstock participating in reproduction events and future optimal potential participants, in order to ensure a valid management for restocking program and their monitoring.

Orosomucoid (alpha1-acid glycoprotein) plasma concentration and genetic variants: effects on human immunodeficiency virus protease inhibitor clearance and cellular accumulation

BACKGROUND AND OBJECTIVE: Protease inhibitors are highly bound to orosomucoid (ORM) (alpha1-acid glycoprotein), an acute-phase plasma protein encoded by 2 polymorphic genes, which may modulate their disposition. Our objective was to determine the influence of ORM concentration and phenotype on indinavir, lopinavir, and nelfinavir apparent clearance (CL(app)) and cellular accumulation. Efavirenz, mainly bound to albumin, was included as a control drug. METHODS: Plasma and cells samples were collected from 434 human immunodeficiency virus-infected patients. Total plasma and cellular drug concentrations and ORM concentrations and phenotypes were determined. RESULTS: Indinavir CL(app) was strongly influenced by ORM concentration (n = 36) (r2 = 0.47 [P = .00004]), particularly in the presence of ritonavir (r2 = 0.54 [P = .004]). Lopinavir CL(app) was weakly influenced by ORM concentration (n = 81) (r2 = 0.18 [P = .0001]). For both drugs, the ORM1 S variant concentration mainly explained this influence (r2 = 0.55 [P = .00004] and r2 = 0.23 [P = .0002], respectively). Indinavir CL(app) was significantly higher in F1F1 individuals than in F1S and SS patients (41.3, 23.4, and 10.3 L/h [P = .0004] without ritonavir and 21.1, 13.2, and 10.1 L/h [P = .05] with ritonavir, respectively). Lopinavir cellular exposure was not influenced by ORM abundance and phenotype. Finally, ORM concentration or phenotype did not influence nelfinavir (n = 153) or efavirenz (n = 198) pharmacokinetics. CONCLUSION: ORM concentration and phenotype modulate indinavir pharmacokinetics and, to a lesser extent, lopinavir pharmacokinetics but without influencing their cellular exposure. This confounding influence of ORM should be taken into account for appropriate interpretation of therapeutic drug monitoring results. Further studies are needed to investigate whether the measure of unbound drug plasma concentration gives more meaningful information than total drug concentration for indinavir and lopinavir.

Multiplex strategy for multilocus sequence typing, fla typing, and genetic determination of antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates collected in Switzerland

We present an optimized multilocus sequence typing (MLST) scheme with universal primer sets for amplifying and sequencing the seven target genes of Campylobacter jejuni and Campylobacter coli. Typing was expanded by sequence determination of the genes flaA and flaB using optimized primer sets. This approach is compatible with the MLST and flaA schemes used in the PubMLST database and results in an additional typing method using the flaB gene sequence. An identification module based on the 16S rRNA and rpoB genes was included, as well as the genetic determination of macrolide and quinolone resistances based on mutations in the 23S rRNA and gyrA genes. Experimental procedures were simplified by multiplex PCR of the 13 target genes. This comprehensive approach was evaluated with C. jejuni and C. coli isolates collected in Switzerland. MLST of 329 strains resulted in 72 sequence types (STs) among the 186 C. jejuni strains and 39 STs for the 143 C. coli isolates. Fourteen (19%) of the C. jejuni and 20 (51%) of the C. coli STs had not been found previously. In total, 35% of the C. coli strains collected in Switzerland contained mutations conferring antibiotic resistance only to quinolone, 15% contained mutations conferring resistance only to macrolides, and 6% contained mutations conferring resistance to both classes of antibiotics. In C. jejuni, these values were 31% and 0% for quinolone and macrolide resistance, respectively. The rpoB sequence allowed phylogenetic differentiation between C. coli and C. jejuni, which was not possible by 16S rRNA gene analysis. An online Integrated Database Network System (SmartGene, Zug, Switzerland)-based platform for MLST data analysis specific to Campylobacter was implemented. This Web-based platform allowed automated allele and ST designation, as well as epidemiological analysis of data, thus streamlining and facilitating the analysis workflow. Data networking facilitates the exchange of information between collaborating centers. The described approach simplifies and improves the genotyping of Campylobacter, allowing cost- and time-efficient routine monitoring.

Genetic testing for glucokinase mutations in clinically selected patients with MODY: a worthwhile investment

The differential diagnosis for children with diabetes includes a group of monogenic diabetic disorders known as maturity-onset diabetes of the young (MODY). So far, six underlying gene defects have been identified. The most common subtypes are caused by mutations in the genes encoding the transcription factor HNF-1a (MODY 3) and the glycolytic enzyme glucokinase (GCK) (MODY 2). MODY 2 is the most benign form of diabetes as the threshold for glucose sensing is elevated resulting in mild, regulated hyperglycemia. MODY 2 may usually be treated with diet alone without risk of microvascular complications. Patients with MODY usually present as children or young adults. Genetic testing for MODY in diabetic subjects is often not performed because of the costs and its unavailability in Switzerland. We describe the impact of the genetic analysis for MODY 2 on diabetes management and treatment costs in a five-year-old girl. The patient and her diabetic mother were both found to have a heterozygous missense mutation (V203A) in the glucokinase gene. The five-year-old girl was started on insulin therapy for her diabetes but because her HbA1c remained between 5.8-6.4% (reference 4.1-5.7%) and her clinical presentation suggested MODY insulin was discontinued. She is now well controlled on a carbohydrate controlled diet regimen only. Omission of insulin treatment made regular blood glucose monitoring unnecessary and removed her risk of hypoglycemia. Costs for the genetic analysis were 500 Euro. At our centre costs for diabetes care of a patient with type 1 diabetes are approximately 2050 Euro/year compared to 410 Euro/year for the care of a patient with MODY 2. In addition, a diagnosis of MODY 2 may reassure patients and their families, as microvascular complications are uncommon. Thus there are both health and financial benefits in diagnosing MODY 2. We recommend genetic testing for MODY 2 in clinically selected patients even though this analysis is currently not available in Switzerland and costs are not necessarily covered by the health insurances.