Diversity of Somatic Coliphages in Coastal Regions with Different Levels of Anthropogenic Activity in Sao Paulo State, Brazil

Bacteriophages are the most abundant and genetically diverse viruses on Earth, with complex ecology in both quantitative and qualitative terms. Somatic coliphages (SC) have been reported to be good indicators of fecal pollution in seawater. This study focused on determining the concentration of SC and their diversity by electron microscopy of seawater, plankton, and bivalve samples collected at three coastal regions in Sao Paulo, Brazil. The SC counts varied from < 1 to 3.4 x 103 PFU/100 ml in seawater (73 samples tested), from < 1 to 4.7 x 10(2) PFU/g in plankton (46 samples tested), and from < 1 to 2.2 x 10(1) PFU/g in bivalves (11 samples tested). In seawater samples, a relationship between the thermotolerant coliforms and Escherichia coli and SC was observed at the three regions (P = 0.0001) according to the anthropogenic activities present at each region. However, SC were found in plankton samples from three regions: Baixada Santista (17/20), Canal de Sao Sebastiao (6/14), and Ubatuba (3/12). In seawater samples collected from Baixada Santista, four morphotypes were observed: A1 (4.5%), B1 (50%), C1 (36.4%), and D1 (9.1%). One coliphage, Siphoviridae type T1, had the longest tail: between 939 and 995 nm. In plankton samples, Siphoviridae (65.8%), Podoviridae (15.8%), Microviridae (15.8%), and Myoviridae (2.6%) were found. In bivalves, only the morphotype B1 was observed. These SC were associated with enteric hosts: enterobacteria, E. coli, Proteus, Salmonella, and Yersinia. Baixada Santista is an area containing a high level of fecal pollution compared to those in the Canal de Sao Sebastiao and Ubatuba. This is the first report of coliphage diversity in seawater, plankton, and bivalve samples collected from Sao Paulo coastal regions. A better characterization of SC diversity in coastal environments will help with the management and evaluation of the microbiological risks for recreation, seafood cultivation, and consumption.

Network genealogy of 195-bp satellite DNA supports the superimposed hybridization hypothesis of Trypanosoma cruzi evolutionary pattern

Trypanosoma cruzi is highly diverse genetically and has been partitioned into six discrete typing units (DTUs), recently re-named T. cruzi I-VI. Although T. cruzi reproduces predominantly by binary division, accumulating evidence indicates that particular DTUs are the result of hybridization events. Two major scenarios for the origin of the hybrid lineages have been proposed. It is accepted widely that the most heterozygous TcV and TcVI DTUs are the result of genetic exchange between TcII and TcIII strains. On the other hand, the participation of a TcI parental in the current genome structure of these hybrid strains is a matter of debate. Here, sequences of the T. cruzi-specific 195-bp satellite DNA of TcI, TcII, Tat, TcV, and TcVI strains have been used for inferring network genealogies. The resulting genealogy showed a high degree of reticulation, which is consistent with more than one event of hybridization between the Tc DTUs. The data also strongly suggest that Tat is a hybrid with two distinct sets of satellite sequences, and that genetic exchange between TcI and TcII parentals occurred within the pedigree of the TcV and TcVI DTUs. Although satellite DNAs belong to the fast-evolving portion of eukaryotic genomes, in >100 satellite units of nine T. cruzi strains we found regions that display 100% identity. No DTU-specific consensus motifs were identified, inferring species-wide conservation. (C) 2010 Elsevier B.V. All rights reserved.

Induction of somatic embryogenesis in soybean: physicochemical factors influencing the development of somatic embryos

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

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere- (NT-B) and fibroblast- (NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p < 0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p < 0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.

Aberrant expression of E-cadherin and beta-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and beta-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n - 6) and control pregnancies (n - 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or beta-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total beta-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-beta-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/beta-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and beta-catenin proteins, along with defective beta-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

Chemically Assisted Enucleation Results in Higher G6PD Expression in Early Bovine Female Embryos Obtained by Somatic Cell Nuclear Transfer

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

A quantitative nature of somatic embryogenesis in soybean

The objectives of this research were to investigate the genetic parameters associated with the in vitro formation of somatic embryos in soybean and to determine the effect of light intensity on the embryogenic capability of F-1, F-2, and backcross (RC1P1 and RC1P2) progenies derived from crosses between embryogenic (IAS-5 and Embrapa-1) and nonembryogenic (Parana) cultivars. Immature cotyledons (4-6 mm in length) derived from the parental lines, F-1, F-2, RC1P1, and RC1P2 were grown for 90 d on the inductive N10 medium, after which the number of somatic embryos was recorded. Chi-square tests for goodness of fit showed that the genetic component of the somatic embryogenesis trait is controlled in a quantitative manner by approximately 10 genes. A normal distribution for somatic embryo formation in the F-2 generations was observed reinforcing the quantitative nature of the trait. Variation in light intensity (8-12 and 27-33 mu mol m(-2) s(-1)) had no effect on somatic embryo formation in the parental material tested.

Capacidade embriogênica da cultivar IAS-5 de soja

O objetivo deste trabalho foi estudar o controle genético da formação de embriões somáticos da cultivar IAS-5 de soja. O experimento foi conduzido em casa de vegetação, cultivando-se quatro plantas por vaso, sob fotoperíodo de 14 horas e temperatura em torno de 28°C. Efetuaram-se cruzamentos entre os parentais não-embriogênicos (cultivares IAC-6, Paraná e IAC-15) e embriogênico (cultivar IAS-5) e retrocruzamentos para obtenção das gerações F1, F2,RC1P1 e RC1P2. Cotilédones imaturos, com 4-6 mm de comprimento, derivados dos parentais das gerações F1, F2, RC1P1 e RC1P2 foram cultivados em placas de Petri contendo meio N10, por um período de 90 dias, em câmara de crescimento. Os embriões somáticos derivados da indução foram contados, e os números, usados para obtenção dos parâmetros genéticos. Os resultados obtidos mostraram que o caráter capacidade de produção de embriões somáticos da cultivar IAS-5 é de natureza quantitativa e controlado por, aproximadamente, 20 genes.

Development of globular embryos from the hybridization between 'Pêra Rio' sweet orange and 'Poncã' mandarin

Objetivou-se estudar a influência de diversas concentrações do meio MT, sacarose, vitaminas, carvão ativado e ácido giberélico no cultivo de embriões imaturos oriundos do cruzamento entre laranjeira 'Pêra Rio' x tangerineira 'PONCÃ' . Os embriões foram excisados sob condições assépticas e inoculados em 15 mL do meio de cultura MT, de acordo com cada experimento a seguir: 1) concentrações do meio de cultura MT (0%, 50%, 100%, 150% e 200%) combinados com 0, 30, 60 e 90 g.L-1 de sacarose; 2) concentrações de vitaminas do meio MT (0%, 50%, 100%, 150% e 200%) combinados com 0, 30, 60 e 90 g.L-1 de sacarose e; 3) concentrações de carvão ativado (0; 0.5; 1; 1.5 e 2 g.L-1) combinados com GA3 (0; 0.01; 0.1; 1 e 10 mg.L-1). Após a inoculação, os embriões foram mantidos por 90 dias em sala de crescimento à temperatura de 27±1ºC, fotoperíodo de 16 horas e irradiância de 32 µmol.m-2.s-1. A utilização de 50% e 100% do meio MT associado a 60 e 90 g.L-1 de sacarose, respectivamente, acrescido de 0.01 mg.L-1 de GA3, proporcionou melhor desenvolvimento de embriões globulares. Não houve necessidade da adição de carvão ativado e vitaminas no meio MT para o cultivo de embriões globulares.

Piezoelectric biosensors for real-time monitoring of hybridization and detection of hepatitis C virus

The piezoelectric quartz crystal resonators modified with oligonucleotide probes were used for detection of hepatitis C virus (HCV) in serum. The gold electrodes on either rough or smooth surface crystals were modified with a self-assembled monolayer of cystamine. After activation with glutaraldehyde, either avidin or streptavidin were immobilized and used for attachment of biotinylated DNA probes (four different sequences). Piezoelectric biosensors were used in a flow-through setup for direct monitoring of DNA resulting from the reverse transcriptase-linked polymerase chain reaction (RT-PCR) amplification of the original viral RNA. The samples of patients with hepatitis C were analyzed and the results were compared with the standard RT-PCR procedure (Amplicor test kit of Roche, microwell format with spectrophotometric evaluation). The piezoelectric hybridization assay was completed in 10 min and the same sensing surface was suitable for repeated use. (C) 2004 Elsevier B.V. All rights reserved.

Indicator-free electrochemical DNA hybridization biosensor

A new electrochemical hybridization biosensor protocol without an external indicator is described. The biosensor format involves the immobilization of inosine-substituted (guanine-free) probe onto the carbon paste transducer, and a direct chronopotentiometric detection of the duplex formation by the appearance of the guanine oxidation peak of the target. Such a use of the intrinsic DNA electrochemical response for monitoring hybridization events offers several advantages (over the common use of external indicators), including the appearance of a new peak, a Aat background, or simplicity. A 4 min short hybridization period allows a detection limit around 120 ng/ml. Performance characteristics of the sensor are described along with future prospects. (C) 1998 Elsevier B.V. B.V. All rights reserved.

Genotoxic effects of (-)-cubebin in somatic cells of mice

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Genotoxicity assessment of the antimalarial compound artesunate in somatic cells of mice

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)