Differential gene expression in dairy cows under negative energy balance and ketosis: A systematic review and meta-analysis.

Development of ketosis in high-producing dairy cows contributes to several animal health issues and highlights the need for a better understanding of the genetic basis of metabolic diseases. To evaluate the pattern of differential gene expression in the liver of cows under negative energy balance (NEB), and under subclinical and clinical ketosis, a meta-analysis of gene expression and genome-wide association studies results was performed. An initial systematic review identified 118 articles based on the key words "cow," "liver," "negative energy balance," "ketosis," "expression," "qPCR," "microarray," "proteomic," "RNA-Seq," and "GWAS." After further screening for only peer-reviewed and pertinent articles for gene expression during NEB and clinical and subclinical ketosis (considering plasma levels of ß-hydroxybutyrate), 20 articles were included in the analysis. From the systematic review, 430 significant SNPs identified by genome-wide association studies (GWAS) were assigned to genes reported in gene expression studies by considering chromosome and base pair positions in the ARS-UCD 1.2 bovine assembly. Venn diagrams were created to integrate the data obtained in the systematic review, and Gene Ontology enrichment analysis was carried out using official gene names. A QTL enrichment analysis was also performed to identify potential positional candidate loci. Twenty-four significant SNPs were located within the coordinates of differentially expressed genes located on chromosomes 2, 3, 6, 9, 11, 14, 27, and 29. Three significant metabolic pathways were associated with NEB and subclinical and clinical ketosis. In addition, 2 important genes, PPARA (peroxisome proliferator activated receptor alpha) and ACACA (acetyl-coenzyme A carboxylase α), were identified, which were differentially expressed in the 3 metabolic conditions. The PPARA gene is involved in the regulation of lipid metabolism and fatty liver disease and the ACACA gene encodes an enzyme that catalyzes the carboxylation of acetyl-coenzyme A to malonyl-coenzyme A, which is a rate-limiting step in fatty acid synthesis. Gene network analysis revealed co-expression interactions among 34 genes associated with functions involving fatty acid transport and fatty acid metabolism. For the annotated QTL, 9 QTL were identified for ketosis. The genes FN1 (fibronectin 1) and PTK2 (protein tyrosine kinase 2), which are mainly involved in cell adhesion and formation of extracellular matrix constituents, were enriched for QTL previously associated with the trait "ketosis" on chromosome 2 and for the trait "milk iron content" on chromosome 14, respectively. This integration of gene expression and GWAS data provides an additional understanding of the genetic background of NEB and subclinical and clinical ketosis in dairy cattle. Thus, it is a useful approach to identify biological mechanisms underlying these metabolic conditions in dairy cattle.

The use of genetic markers in the molecular epidemiology of histoplasmosis: a systematic review.

Histoplasmosis is a systemic mycosis caused by Histoplasma capsulatum, a dimorphic fungal pathogen that can infect both humans and animals. This disease has worldwide distribution and affects mainly immunocompromised individuals. In the environment, H. capsulatum grows as mold but undergoes a morphologic transition to the yeast morphotype under special conditions. Molecular techniques are important tools to conduct epidemiologic investigations for fungal detection, identification of infection sources, and determination of different fungal genotypes associated to a particular disease symptom. In this study, we performed a systematic review in the PubMed database to improve the understanding about the molecular epidemiology of histoplasmosis. This search was restricted to English and Spanish articles. We included a combination of specific keywords: molecular typing [OR] genetic diversity [OR] polymorphism [AND] H. capsulatum; molecular epidemiology [AND] histoplasmosis; and molecular epidemiology [AND] Histoplasma. In addition, we used the specific terms: histoplasmosis [AND] outbreaks. Non-English or non-Spanish articles, dead links, and duplicate results were excluded from the review. The results reached show that the main methods used for molecular typing of H. capsulatum were: restriction fragment length polymorphism, random amplified polymorphic DNA, microsatellites polymorphism, sequencing of internal transcribed spacers region, and multilocus sequence typing. Different genetic profiles were identified among H. capsulatum isolates, which can be grouped according to their source, geographical origin, and clinical manifestations.

El análisis estadístico en la Farmacopea Europea: diseño completamente aleatorizado en un bioensayo de factor VIII / Statistical analysis in the European Pharmacopoeia: completely randomised design in a factor VIII bioassay

La Farmacopea Europea proporciona una guía para diseñar los bioensayos que deben realizarse deacuerdo a las monografías publicadas, así como para el análisis o interpretación de sus resultados.Uno de estos bioensayos es el ensayo cromogénico de la actividad del factor VIII de coagulaciónfrente a un estándar de concentración conocida mediante un diseño cero común (3h) con 3 dosis depreparación, completamente aleatorizado, aplicando un modelo de razón de pendientes.En este trabajo, hemos realizado e interpretado un análisis de varianza, y, finalmente, hemos calculadola potencia estimada de la preparación problema, con su correspondiente intervalo de confianza al95%.Para ello, hemos contado con el software CombiStats v4.0, suministrado por el European Directoratefor the Quality of Medicines & HealthCare (EDQM) y utilizado rutinariamente por las AgenciasEuropeas de Evaluación, así como con el programa Microsoft Office Excel 2003. Los resultadosobtenidos han sido similares en ambos casos.Este trabajo demuestra la importancia de las ciencias matemáticas, y, en particular, de laestadística, en la optimización de los resultados obtenidos en las investigaciones científicas(AU)

The European Pharmacopoeia provides the guidance for the design f bioassays prescribed in theEuropean Pharmacopoeia monographs and for the analysis or interpretation of its results.One of these bioassays is the chromogenic assay of human coagulation Factor VIII activity comparedwith a standard of known concentration according to a completely randomized “common zero (3h) –design” with 3 doses per preparation and using the slope-ratio model.In this paper we have implemented and interpreted an analysis of variance and, finally, we havecalculated the estimated potency of the preparation problem, with its corresponding confidenceinterval 95%, using the CombiStats v4.0 software, provided by the European Directorate for theQuality of Medicines & HealthCare (EDQM) and used for European Medicines Evaluation Agenciesas well as the software Microsoft Office Excel 2003. The results obtained have been similar.This paper shows the importance of mathematical sciences and, in particular, statistics in theoptimization of the results of scientific research(AU)

Genetic diversity of Histoplasma capsulatum strains isolated from soil, animals, and clinical specimens in Rio de Janeiro State, Brazil, by a PCR-based random amplified polymorphic DNA assay.

Little is known about the genetic strain diversity and geographical range of Histoplasma capsulatum isolated in Rio de Janeiro State, Brazil. We characterized 13 environmental, 7 animal, and 28 clinical H. capsulatum isolates by using a PCR-based random amplified polymorphic DNA (RAPD) assay. DNA fingerprinting of these soil, animal, and clinical specimens was performed with four primers (1253, 1281, D-9355, and D-10513) and generated amplicons with considerable polymorphism. Although all of the isolates exhibited more than 80% genetic relatedness, they could be clustered into four to six genotypes for each primer. The RAPD profiles of H. capsulatum isolated from Rio de Janeiro State could be distinguished from those of the U.S. strains included in this study (Downs, G222B, G-186B, and FLS1) by showing less than 70% similarity to each primer. The genetic polymorphisms between H. capsulatum strains isolated from animals and soil obtained in the same geographic areas were 100% similar, suggesting that an environmental microniche could be acting as a source of infection for animals and the local human population.

Interpretation and clinical correlation of serological tests in paracoccidioidomycosis.

In order to correlate the findings of two serological tests, double immunodiffusion (IDD) and immunoblotting (IB), with the clinical diagnosis and follow-up of paracoccidioidomycosis (PCM), 325 serum samples from PCM patients were tested at the beginning of specific therapy and after its completion. Group I included 245 PCM patients at the onset of symptoms without treatment. In 221 cases (90.2%) the IDD showed positive reactions and in 24 (9.8%) the results were negative. Of the 24 IDD negative samples, 23 were investigated by IB and were positive. Group II included 80 PCM patients under follow-up after treatment. There were four cases of relapse in which the IDD and IB tests were positive (100%). Among the 76 cases with inactive mycotic infection, the IDD was negative in 71.2% and positive in 28.8%; the IB was positive in all cases (100%). The control group (Group III) included 27 samples from patients with other mycoses, tuberculosis and from healthy individuals. All showed negative IDD tests but positive reactions with IB, which could be abolished by serum dilutions without altering the PCM reactivity. Therefore, the utilization of the IB, an immunoenzymatic method for the diagnosis of PCM, raised the sensitivity to 100%.

Strain characterization of Candida parapsilosis fungemia by molecular typing methods.

The present study used two molecular typing methods to investigate a cluster of eight cases of Candida parapsilosis fungemia in a hospital in Rio de Janeiro, Brazil. Candida parapsilosis is an important opportunistic pathogen that is frequently involved in outbreaks of nosocomial fungemia. Identification of a common source of infection and determination of genetic relatedness among the strains involved in outbreaks are important for infection control. Candida parapsilosis strains were isolated from the bloodstream of patients housed in an intensive-care unit (n=5) and in individual rooms (n=3). An additional strain of Candida parapsilosis was isolated from a hyperalimentation infusion flask, which was implicated by molecular typing to be the source of infection. All strains were identified using morphological and biochemical methods. The genetic relationship between patients' strains and the hyperalimentation infusion strain was assessed by electrophoretic karyotype (EK) analysis and random amplification of polymorphic DNA (RAPD). Both methods resulted in patterns that allowed differentiation of the isolates. Candida parapsilosis fungemia, in three of the eight patients, resulted from a common source of infection, as demonstrated by molecular typing methods. Image analysis of EK patterns indicated that these strains were closest to Candida parapsilosis Group II, a grouping that is a less frequent clinical isolate than the major Group I strains.

Comparison of MB-check and Löwenstein-Jensen media for recovery of mycobacteria.

We have compared the rate of recovery of mycobacteria with the MB-Check culture system (liquid phase) and the Löwenstein-Jensen (LJ) medium in 2,907 clinical specimens obtained from 830 patients submitted for mycobacterial culture during 1-year period. Direct smear examination was carried out by auramine-rhodamine staining. All primary isolates from the culture media were confirmed by Ziehl-Neelsen staining and identified by acridinium-ester-labeled DNA probes specific for Mycobaterium tuberculosis complex. A total of 214 isolates were of the M. tuberculosis complex (88 patients) and 54 of "potentially pathogenic environmental mycobacteria" (45 patients). A total of 117 (54.7%) samples were smear-positive and the remaining 97 (45.3%) were smear-negative. There was a significant difference in the percentage of positive cultures obtained by the MB-Check method (99.1%) as compared with the LJ medium (73.8%) (P < 0.05). This difference, however, occurred almost exclusively at the expense of the 97 smear-negative samples (positive cultures 97.95% by the MB-Check method vs. 42.3% by the LJ culture, P < 0.05). The number of patients diagnosed of tuberculosis by the MB-Check was significantly higher as compared with LJ medium (88 [100%] vs. 77 [87.5%], P < 0.05). In 11 (12.5%) patients, the diagnosis was only established by the MB-Check system. In smear-positive samples, the mean (+/-SD) detection time for M. tuberculosis complex was 14.8 +/- 8 days with MB-Check and 19.9 +/- 7 days with LJ medium. The corresponding figures in smear-negative samples were 22.8 +/- 3 days and 27.8 +/- 6 days, respectively. DNA probes directly applied to MB-Check liquid medium showed a sensitivity of 98.8% and specificity of 100%. These results indicate that the MB-Check system is more efficient for the recovery of mycobacteria than LJ medium.