Testing advances in molecular discrimination among Chinook salmon life histories: evidence from a blind test.

The application of DNA-based markers toward the task of discriminating among alternate salmon runs has evolved in accordance with ongoing genomic developments and increasingly has enabled resolution of which genetic markers associate with important life-history differences. Accurate and efficient identification of the most likely origin for salmon encountered during ocean fisheries, or at salvage from fresh water diversion and monitoring facilities, has far-reaching consequences for improving measures for management, restoration and conservation. Near-real-time provision of high-resolution identity information enables prompt response to changes in encounter rates. We thus continue to develop new tools to provide the greatest statistical power for run identification. As a proof of concept for genetic identification improvements, we conducted simulation and blind tests for 623 known-origin Chinook salmon (Oncorhynchus tshawytscha) to compare and contrast the accuracy of different population sampling baselines and microsatellite loci panels. This test included 35 microsatellite loci (1266 alleles), some known to be associated with specific coding regions of functional significance, such as the circadian rhythm cryptochrome genes, and others not known to be associated with any functional importance. The identification of fall run with unprecedented accuracy was demonstrated. Overall, the top performing panel and baseline (HMSC21) were predicted to have a success rate of 98%, but the blind-test success rate was 84%. Findings for bias or non-bias are discussed to target primary areas for further research and resolution.

Germline mutations in retinoma patients: relevance to low-penetrance and low-expressivity molecular basis.

PURPOSE: To study phenotype-genotype correlation in patients who have retinoma, which is a benign tumor resembling the post irradiation regression pattern of retinoblastoma (RB). METHODS: We selected patients who had retinoma and positive family history for RB and patients who had retinoma in one eye and either retinoma or RB in the other eye. The study included 22 patients with available DNA: 18 from 11 families and four sporadic cases. DNA was extracted from peripheral blood leukocytes. The RB1 gene was screened by DHPLC and direct sequencing of the promoter and all the exons. RESULTS: We identified 17 occurrences of 11 distinct germline mutations in two sporadic and in 15 familial cases (nine families). The 11 identified mutations were located in exons 1, 10,11,13,14, and 19 to 23. Four of the identified mutations were not previously reported, including g.64407delT, g.153236A>T, g.156743delTCTG, and g.162078delA. Eight out the 11 mutations were truncating and three were nontruncating (missense). There was no correlation between the type of mutation and the number of tumor foci per eye (RB or retinomas). Highly heterogeneous intrafamilial expressivity was observed. CONCLUSIONS: To our knowledge, this study is the largest series of mutations of consecutive retinoma patients. The present data suggest that the type of inherited mutations underlying retinoma is undistinguishable from RB related ones, i.e., largely dominated by truncating mutants. This finding is in contrast with the RB1 genotypic spectrum of mutations associated with low-penetrance RB, i.e., nontruncating mutants. The molecular mechanism underlying low-penetrance and attenuated expressivity (retinomas) appeared to be distinct.

Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.

Oxidative stress activation of miR-125b is part of the molecular switch for Hailey-Hailey disease manifestation.

Hailey-Hailey disease (HHD) is an autosomal dominant disorder characterized by suprabasal cutaneous cell separation (acantholysis) leading to the development of erosive and oozing skin lesion. Micro RNAs (miRNAs) are endogenous post-transcriptional modulators of gene expression with critical functions in health and disease. Here, we evaluated whether the expression of specific miRNAs may play a role in the pathogenesis of HHD. Here, we report that miRNAs are expressed in a non-random manner in Hailey-Hailey patients. miR-125b appeared a promising candidate for playing a role in HHD manifestation. Both Notch1 and p63 are part of a regulatory signalling whose function is essential for the control of keratinocyte proliferation and differentiation and of note, the expression of both Notch1 and p63 is downregulated in HHD-derived keratinocytes. We found that both Notch1 and p63 expression is strongly suppressed by miR-125b expression. Additionally, we found that miR-125b expression is increased by an oxidative stress-dependent mechanism. Our data suggest that oxidative stress-mediated induction of miR-125b plays a specific role in the pathogenesis of HHD by regulating the expression of factors playing an important role in keratinocyte proliferation and differentiation.

Molecular epidemiology of human respiratory syncytial virus in Uruguay: 1985-2001 - A review

The variability of the G glycoprotein from human respiratory syncytial viruses (HRSV) (groups A and B) isolated during 17 consecutive epidemics in Montevideo, Uruguay have been analyzed. Several annual epidemics were studied, where strains from groups A and B circulated together throughout the epidemics with predominance of one of them. Usually, group A predominates, but in some epidemics group B is more frequently detected. To analyse the antigenic diversity of the strains, extracts of cells infected with different viruses of group A were tested with a panel of anti-G monoclonal antibodies (MAbs). The genetic variability of both groups was analyzed by sequencing the C-terminal third of the G protein gene. The sequences obtained together with previously published sequences were used to perform phylogenetic analyses. The data from Uruguayan isolates, together with those from the rest of the world provide information regarding worldwide strain circulation. Phylogenetic analyses of HRSV from groups A and B show a model of evolution analogous to the one proposed for influenza B viruses providing information that would be beneficial for future immunization programs and to design safe vaccines.

A follow up study of cytomegalovirus infection in a group of Turkish renal transplant recipients using molecular assays

The clinical value of an in-house cytomegalovirus nested polymerase chain reaction (CMV-PCR) and a commercial molecular assay hybrid capture CMV DNA assay (HCA) was evaluated in monitoring a group of renal transplant patients for six months follow up. In this study, the sensitivity, specificity, positive predictive value, and negative predictive value of nested CMV DNA PCR assay and HCA at the beginning of the study were 70, 42.9, 46.7, 66.7, and 60, 78.6, 66.7, and 73.3% respectively. After six months, they were 80, 66.7, 80, 66.7 for CMV PCR and 73.3, 88.9, 91.7, 66.7% for HCA respectively. These results indicate that in monitoring and predicting CMV infections in renal transplant recipients, not only qualitative but also quantitative assays must be used together in order to decide the preemptive strategies.

Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue.

Background: The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months. Methods: RNA from fresh frozen (FF) and FFPE tumor samples of 82 patients were used for quality control, and independent FFPE tissues of 342 postmenopausal participants of BIG 1-98 with ER-positive cancer were analyzed by measuring prospectively selected genes and computing scores representing the functions of the estrogen receptor (eight genes, ER_8), the progesterone receptor (five genes, PGR_5), Her2 (two genes, HER2_2), and proliferation (ten genes, PRO_10) by quantitative reverse transcription PCR (qRT-PCR) on TaqMan Low Density Arrays. Molecular scores were computed for each category and ER_8, PGR_5, HER2_2, and PRO_10 scores were combined into a RISK_25 score. Results: Pearson correlation coefficients between FF- and FFPE-derived scores were at least 0.94 and high concordance was observed between molecular scores and immunohistochemical data. The HER2_2, PGR_ 5, PRO_10 and RISK_25 scores were significant predictors of disease free-survival (DFS) in univariate Cox proportional hazard regression. PRO_10 and RISK_25 scores predicted DFS in patients with histological grade II breast cancer and in lymph node positive disease. The PRO_10 and PGR_ 5 scores were independent predictors of DFS in multivariate Cox regression models incorporating clinical risk indicators; PRO_10 outperformed Ki-67 labeling index in multivariate Cox proportional hazard analyses. Conclusions: Scores representing the endocrine responsiveness and proliferation status of breast cancers were developed from gene expression analyses based on RNA derived from FFPE tissues. The validation of the molecular scores with tumor samples of participants of the BIG 1-98 trial demonstrates that such scores can serve as independent prognostic factors to estimate disease free survival (DFS) in postmenopausal patients with estrogen receptor positive breast cancer.

Molecular characterization of human T-cell lymphotropic virus coinfecting human immunodeficiency virus 1 infected patients in the Amazon region of Brazil

The present work evaluated the epidemiology of human immunodeficiency virus 1/human T-cell lymphotropic virus (HIV-1/HTLV) coinfection in patients living in Belém (state of Pará) and Macapá (state of Amapá), two cities located in the Amazon region of Brazil. A total of 169 blood samples were collected. The sera were tested by enzyme-linked immunosorbent assay to determine the presence of antibodies anti-HTLV-1/2. Confirmation of infection and discrimination of HTLV types and subtypes was performed using a nested polymerase chain reaction targeting the pX and 5' LTR regions, followed by restriction fragment length polymorphism and sequencing analysis. The presence of anti-HTLV1/2 was detected in six patients from Belém. The amplification of the pX region followed by RFLP analysis, demonstrated the presence of HTLV-1 and HTLV-2 infections among two and four patients, respectively. Sequencing HTLV-1 5' LTR indicated that the virus is a member of the Cosmopolitan Group, Transcontinental subgroup. HTLV-2 strains isolated revealed a molecular profile of subtype HTLV-2c. These results are a reflex of the epidemiological features of HIV-1/HTLV-1/2 coinfection in the North region of Brazil, which is distinct from other Brazilian regions, as reported by previous studies.

Molecular imaging by micro-CT: specific E-selectin imaging.

The primary goal of this study was to design a fluorescent E-selectin-targeted iodine-containing liposome for specific E-selectin imaging with the use of micro-CT. The secondary goal was to correlate the results of micro-CT imaging with other imaging techniques with cellular resolution, i.e., confocal and intravital microscopy. E-selectin-targeted liposomes were tested on endothelial cells in culture and in vivo in HT-29 tumor-bearing mice (n = 12). The liposomes contained iodine (as micro-CT contrast medium) and fluorophore (as optical contrast medium) for confocal and intravital microscopy. Optical imaging methods were used to confirm at the cellular level, the observations made with micro-CT. An ischemia-reperfusion model was used to trigger neovessel formation for intravital imaging. The E-selectin-targeted liposomes were avidly taken up by activated endothelial cells, whereas nontargeted liposomes were not. Direct binding of the E-selectin-targeted liposomes was proved by intravital microscopy, where bright spots clearly appeared on the activated vessels. Micro-CT imaging also demonstrated accumulation of the targeted lipsomes into subcutaneous tumor by an increase of 32 +/- 8 HU. Hence, internalization by activated endothelial cells was rapid and mediated by E-selectin. We conclude that micro-CT associated with specific molecular contrast agent is able to detect specific molecular markers on activated vessel walls in vivo.

Susceptibility of clinical isolates of multiresistant Pseudomonas aeruginosa to a hospital disinfectant and molecular typing

The aim of this study was to evaluate the susceptibility of 35 resistant Pseudomonas aeruginosa clinical isolates to a quaternary ammonium hospital disinfectant. The methodology was the AOAC Use-Dilution Test, with disinfectant at its use-concentration. In addition, the chromosomal DNA profile of the isolates were determined by macro-restriction pulsed field gel electrophoresis (PFGE) method aiming to verify the relatedness among them and the behavior of isolates from the same group regarding the susceptibility to the disinfectant. Seventy one percent of the isolates were multiresistant to antibiotics and 43% showed a reduced susceptibility to the disinfectant. The PFGE methodology detected 18 major clonal groups. We found isolates with reduced susceptibility to the disinfectant and we think that these are worrying data that should be further investigated including different organisms and chemical agents in order to demonstrate that microorganisms can be destroyed by biocide as necessary. We also found strains of the same clonal groups showing different susceptibility to the disinfectant. This is an interesting observation considering that only few works are available about this subject. PFGE profile seems not to be a reliable marker for resistance to disinfectants.

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

Application of molecular techniques in the study of Staphylococcus aureus clonal evolution - A Review

Staphylococcus aureus is an important agent of healthcare-associated and community-acquired infections. A major characteristic of this microorganism is the ability to develop resistance to antimicrobial agents. Several molecular techniques have been applied for the characterization of S. aureus in epidemiological studies. In the present review, we discuss the application of molecular techniques for typing S. aureus strains and describe the nomenclature and evolution of epidemic clones of this important pathogen.

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.

Molecular trees of trypanosomes incongruent with fossil records of hosts

Molecular trees of trypanosomes have confirmed conventionally accepted genera, but often produce topologies that are incongruent with knowledge of the evolution, systematics, and biogeography of hosts and vectors. These distorted topologies result largely from incorrect assumptions about molecular clocks. A host-based phylogenetic tree could serve as a broad outline against which the reasonability of molecular phylogenies could be evaluated. The host-based tree of trypanosomes presented here supports the " invertebrate first " hypothesis of trypansosome evolution, supports the monophyly of Trypanosomatidae, and indicates the digenetic lifestyle arose three times. An area cladogram of Leishmania supports origination in the Palaearctic during the Palaeocene.