Rapid detection of bovine coronavirus by a semi-nested RT-PCR

Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

Perfil mineral de vacas mestiças Girolanda no período de transição em sistema semi-intensivo em duas estações do ano

Neste estudo foi avaliado o perfil metabólico de vacas leiteiras no período de transição durante o verão e o inverno. Foram utilizados 31 animais pluríparos mestiços girolando em cada estação, totalizando 62 vacas. No verão permaneciam em pasto com suplementação de silagem de milho e concentrado. No inverno, a exigência nutricional era suprida apenas com silagem de milho e concentrado. Foram feitas um total de 11 coletas de sangue de cada animal segundo o seguinte protocolo: quatro coletas pré-parto espaçadas semanalmente, no dia do parto e com 2, 5, 10, 15, 21 e 30 dias pós-parto. Foram avaliadas as concentrações de cálcio, fósforo e magnésio. Todos os analitos variaram em função do estatus fisiológico. As concentrações médias de cálcio e magnésio foram maiores no verão do que no inverno. Em ambas as estações, as menores concentrações médias de cálcio ocorreram próximas ao parto, sendo que 75% dos animais no inverno e 35,48% dos animais no verão estavam hipocalcêmicos. Apenas no décimo dia as concentrações de cálcio voltaram aos níveis do pré-parto, demonstrando que esse tempo é necessário para a adaptação da nova condição de lactante. As concentrações de magnésio foram menores no pós-parto do que no pré-parto nas duas estações, demonstrando a necessidade desse mineral para a produção de leite. As concentrações médias de magnésio permaneceram sempre dentro dos valores de referência, porém 19% dos animais aos dois dias pós-parto no inverno e 7% dos animais aos 10 dias pós-parto no verão tinham concentrações inferiores a esses limites. As concentrações de fósforo sofreram grande variação ao longo do peri-parto, porém sempre em níveis elevados, sendo que 49,8% dos animais no inverno e 37,3% no verão tinham níveis de fósforo acima dos valores de referência

Weed community and growth under the canopy of trees adapted to the brazilian semi-arid region

The objectives of this work were to evaluate the floristic composition and dry biomass of weeds under the canopy of seven perennial species adapted to the Semi-Arid region of Brazil, and correlate these characteristics with growth traits of the perennial species. The following perennial species were evaluated in two experiments (E1 and E2): mesquite (Prosopis juliflora), jucá (Caesalpinia ferrea), white popinac (Leucaena leucocephala), mofumbo (Combretum leprosum), neem (Azadirachata indica), sabiá (Mimosa caesalpiniaefolia) and tamarind (Tamarindus indica). In E1, the seven species were evaluated in a random block design with four replicates and nine plants per plot. In E2, evaluation comprised four species (mesquite, jucá, white popinac, and tamarind) in a random block design with eight replicates and nine plants per plot. A circle with an area of 1.77 m² was established around the trunk of each plant, two years after they were transplanted to the permanent location. The weeds collected within this circle were cut even with the ground, classified and weighed. At this time, plant height, and crown and stem diameters were evaluated in all trees of each plot. In E1 there were no differences between tree species as to weed frequency under their canopies; however, weed growth was smaller under the canopy of sabiá trees. Mesquite and sabiá had the greatest plant height and crown diameter means, but only sabiá had the greatest stem diameter. In E2, the perennial species were not different with regard to weed frequency and growth under their canopies, but mesquite had the greatest growth, as measured by plant height (with significant results for jucá as well) and crown and stem diameter.

Structure and dynamics of a woody plant community of a tropical semi-deciduous seasonal forest in the "Estação Ecológica do Panga", municipality of Uberlândia, Minas Gerais, Brazil

Studies of plant community dynamics are essential in understanding the demographic patterns of species since changes in demographic rates can affect the floristic composition and future structure. The purpose of the present study was to analyze the changes in the community structure and floristic composition of woody plants in a tropical semi-deciduous forest in Uberlândia in central Brazil, during a 10-years period. The data were collected in 1989 and in 2000 in 50 quadrats (10 m x 10 m) where all trees with a minimum circumference at breast height of 10 cm were sampled. In 1989, 93 species and 1103 individuals were registered. Over a period of 10 years, seven new species were added to the community, although eight disappeared. The main change that occurred during this period in the floristic composition was the replacement of savannah species occurring in forest gaps by those from the forest understory.

Comportamento fenológico de três espécies de Jatropha (Euphorbiaceae) da Caatinga, semi-árido do Brasil

O comportamento fenológico vegetativo e reprodutivo de Jatropha mollissima (Pohl) Baill., Jatropha mutabilis (Pohl) Baill. e Jatropha ribifolia (Pohl) Baill. foi comparado em uma área de caatinga hiperxerófila arbustiva-arbórea do nordeste brasileiro, no período de julho de 2005 a junho de 2007. Adicionalmente, investigou-se a correlação entre as fenofases e as variáveis abióticas (temperatura, umidade relativa, precipitação e fotoperíodo), a capacidade de armazenamento de água e o padrão de distribuição espacial. Todas as espécies apresentaram alta capacidade de armazenamento de água e comportamento fenológico contínuo e irregular com ausência de flores, frutos, queda foliar e brotamento nos meses mais frios e úmidos do ano. Apenas a floração de J. ribifolia apresentou correlação significativa com a precipitação. As três espécies apresentaram populações com distribuição agregada, favorecida principalmente devido ao processo de dispersão de sementes por autocoria. Os resultados obtidos indicam que a alta capacidade de armazenamento de água pelas espécies garante a ocorrência dos eventos fenológicos mesmo na ausência de chuvas.

The genus Stachybotrys (anamorphic fungi) in the semi-arid region of Brazil

(The genus Stachybotrys (anamorphic fungi) in the semi-arid region of Brazil). Stachybotrys is characterized by macronematous, mononematous, unbranched or branched conidiophores, with discrete terminal and phialidic conidiogenous cells, and aseptate, reniform, ellipsoidal to spherical, smooth or verrucose conidia, which are produced in a slimy mass. Eight species have been reported from Brazil, occurring in the soil, air and leaf litter. During investigation of conidial fungi on decaying leaf litter in semi-arid areas of Brazil nine species were found: S. bisbyi (Sriniv.) G.L. Barron, S. chartarum (Ehrenb.) S. Hughes, S. globosa P. C. Misra & S. K. Srivast., S. kampalensis Hansf., S. longispora Matsush., S. nephrospora Hansf., S. nilagirica Subram., Stachybotrys parvispora S. Hughes and S. verrucispora Matsush. Stachybotrys nilagirica is a new record from Brazil. Descriptions, comments, geographic distribution and illustrations are presented for above mentioned species. A key for all species recorded in semi-arid region of Brazil is presented.

Photosynthetic responses of four tropical tree species grown under gap and understorey conditions in a semi-deciduous forest

Leaf CO2 assimilation (A) as a function of photosynthetic photon flux density (Q) or intercellular CO2 concentration (Ci) and chlorophyll fluorescence measurements were carried out on four tropical woody species growing in forest gap and understorey (Bauhinia forficata Link. and Guazuma ulmifolia Lam. as pioneers, and Hymenaea courbaril L. and Esenbeckia leiocarpa Engl. as non-pioneers). Chlorophyll fluorescence indicated similar acclimation capacities of photochemical apparatus to contrasting light environments irrespective to plant species. Maximum CO2 assimilation and quantum yield derived from A/Q curves indicated higher photosynthetic capacity in pioneer than in non-pioneer species in forest gap. However, the differences among species did not show a straightforward relation with their successional status regarding data derived from A/Q curves under understorey conditions. Both successional groups are able to sustain positive carbon balance under contrasting natural light availabilities, modifying photochemical and biochemical photosynthetic traits with similar phenotypic plasticity capacity.

Floristic diversity of two crystalline rocky outcrops in the Brazilian northeast semi-arid region

Floristic composition and structure of vegetation were studied in two rocky outcrop areas in the semi-arid region of northeastern Brazil. From April 2007 to September 2008, 18 monthly field trips were carried out. Vascular plants were randomly collected throughout the outcrop areas. For structural analysis, 30 plots of 1 × 1 m were set in the vegetation islands. The checklist presented combines 211 species (69 families and 168 genera), although only 56 species were collected in the plots. Fabaceae (18 spp.; 8.5%), Asteraceae (17 spp.; 8%), Orchidaceae (13 spp.; 6.1%), Euphorbiaceae (13 spp.; 6.1%), Bromeliaceae (10 spp.; 4.7%), and Poaceae (eight spp.; 3.8%) are the richest families. Overall, 1,792 shrub and herbaceous specimens were counted in the plots. The Shannon-Wiener (H) diversity index values were 2.572 and 2.547 nats individual-1. The species that presented the highest absolute abundance values (number of plants) had low frequencies in the plots and vice-versa. The biological spectrum had a high proportion of phanerophytes and therophytes, followed by cryptophytes, chamaephytes, and hemicryptophytes. The studied flora shares floristic components similar to other rocky outcrop areas of the semi-arid region in northeastern Brazil, including in relation to dominant groups in the vegetation structure.

Sistemas de polinização e de reprodução de três espécies de Jatropha (Euphorbiaceae) na Caatinga, semi-árido do Brasil

São apresentados a biologia floral, a dinâmica de produção de néctar, visitantes florais e sistemas reprodutivos de Jatropha ribifolia (Pohl) Baill. (Euphorbiaceae) e uma comparação dos dados obtidos para J. mollissima (Pohl) Baill. e J. mutabilis (Pohl) Baill. O estudo foi desenvolvido em uma área de caatinga hiperxerófila, na Estação Biológica de Canudos, Bahia, Brasil, de maio de 2005 a junho de 2007. As flores das três espécies estão organizadas em dicásios protogínicos. Em J. ribifolia as flores estaminadas e pistiladas duram cerca de 48 horas e a abertura é diurna, enquanto em J. mollissima e J. mutabilis as estaminadas duram entre 12 e 15 horas e as pistiladas entre 36 e 48 horas e a abertura é crepuscular. A produção de néctar, a viabilidade polínica e a receptividade estigmática iniciaram-se logo após a abertura total das flores e se sobrepuseram até sua senescência. A produção de néctar variou ao longo do dia e as flores pistiladas produziram maiores volumes que as estaminadas. A viabilidade polínica e a receptividade estigmática foram observadas nas três espécies durante toda a vida da flor. Houve diferença significativa entre os tratamentos dos sistemas reprodutivos para J. mollissima (KW = 59,796), J. mutabilis (KW = 59,058) e J. ribifolia (KW = 63,660). As três espécies produziram frutos por geitonogamia manual e xenogamia manual e apenas J. ribifolia produziu frutos por geitonogamia espontânea. As abelhas Apis mellifera, Xylocopa frontalis e X. grisescens e os beija-flores Chlorostilbon lucidus e Anopetia gounellei são os polinizadores potenciais de J. mollissima e J. mutabilis. Já para J. ribifolia, os potenciais polinizadores são A. mellifera e X. grisescens. A utilização de uma gama variada de vetores de pólen permite o fluxo polínico e a formação de frutos nas três espécies. Por sua vez, o sequenciamento no período da floração e diferenças nas dimensões das flores e nos horários da antese ajudam a manter o isolamento reprodutivo das três espécies e evitam a perda de pólen interespecífico, que poderia ser alta devido à partilha de alguns dos polinizadores.

Effect of race, genetic population structure, and genetic models in two-locus association studies: clustering of functional renin-angiotensin system gene variants in hypertension association studies

Previous genetic association studies have overlooked the potential for biased results when analyzing different population structures in ethnically diverse populations. The purpose of the present study was to quantify this bias in two-locus association studies conducted on an admixtured urban population. We studied the genetic structure distribution of angiotensin-converting enzyme insertion/deletion (ACE I/D) and angiotensinogen methionine/threonine (M/T) polymorphisms in 382 subjects from three subgroups in a highly admixtured urban population. Group I included 150 white subjects; group II, 142 mulatto subjects, and group III, 90 black subjects. We conducted sample size simulation studies using these data in different genetic models of gene action and interaction and used genetic distance calculation algorithms to help determine the population structure for the studied loci. Our results showed a statistically different population structure distribution of both ACE I/D (P = 0.02, OR = 1.56, 95% CI = 1.05-2.33 for the D allele, white versus black subgroup) and angiotensinogen M/T polymorphism (P = 0.007, OR = 1.71, 95% CI = 1.14-2.58 for the T allele, white versus black subgroup). Different sample sizes are predicted to be determinant of the power to detect a given genotypic association with a particular phenotype when conducting two-locus association studies in admixtured populations. In addition, the postulated genetic model is also a major determinant of the power to detect any association in a given sample size. The present simulation study helped to demonstrate the complex interrelation among ethnicity, power of the association, and the postulated genetic model of action of a particular allele in the context of clustering studies. This information is essential for the correct planning and interpretation of future association studies conducted on this population.

Category fluency test: effects of age, gender and education on total scores, clustering and switching in Brazilian Portuguese-speaking subjects

Verbal fluency tests are used as a measure of executive functions and language, and can also be used to evaluate semantic memory. We analyzed the influence of education, gender and age on scores in a verbal fluency test using the animal category, and on number of categories, clustering and switching. We examined 257 healthy participants (152 females and 105 males) with a mean age of 49.42 years (SD = 15.75) and having a mean educational level of 5.58 (SD = 4.25) years. We asked them to name as many animals as they could. Analysis of variance was performed to determine the effect of demographic variables. No significant effect of gender was observed for any of the measures. However, age seemed to influence the number of category changes, as expected for a sensitive frontal measure, after being controlled for the effect of education. Educational level had a statistically significant effect on all measures, except for clustering. Subject performance (mean number of animals named) according to schooling was: illiterates, 12.1; 1 to 4 years, 12.3; 5 to 8 years, 14.0; 9 to 11 years, 16.7, and more than 11 years, 17.8. We observed a decrease in performance in these five educational groups over time (more items recalled during the first 15 s, followed by a progressive reduction until the fourth interval). We conclude that education had the greatest effect on the category fluency test in this Brazilian sample. Therefore, we must take care in evaluating performance in lower educational subjects.

A semi-automatic computerized method to measure baroreflex-mediated heart rate responses that reduces interobserver variability

Arterial baroreflex sensitivity estimated by pharmacological impulse stimuli depends on intrinsic signal variability and usually a subjective choice of blood pressure (BP) and heart rate (HR) values. We propose a semi-automatic method to estimate cardiovascular reflex sensitivity to bolus infusions of phenylephrine and nitroprusside. Beat-to-beat BP and HR time series for male Wistar rats (N = 13) were obtained from the digitized signal (sample frequency = 2 kHz) and analyzed by the proposed method (PRM) developed in Matlab language. In the PRM, time series were low-pass filtered with zero-phase distortion (3rd order Butterworth used in the forward and reverse direction) and presented graphically, and parameters were selected interactively. Differences between basal mean values and peak BP (deltaBP) and HR (deltaHR) values after drug infusions were used to calculate baroreflex sensitivity indexes, defined as the deltaHR/deltaBP ratio. The PRM was compared to the method traditionally (TDM) employed by seven independent observers using files for reflex bradycardia (N = 43) and tachycardia (N = 61). Agreement was assessed by Bland and Altman plots. Dispersion among users, measured as the standard deviation, was higher for TDM for reflex bradycardia (0.60 ± 0.46 vs 0.21 ± 0.26 bpm/mmHg for PRM, P < 0.001) and tachycardia (0.83 ± 0.62 vs 0.28 ± 0.28 bpm/mmHg for PRM, P < 0.001). The advantage of the present method is related to its objectivity, since the routine automatically calculates the desired parameters according to previous software instructions. This is an objective, robust and easy-to-use tool for cardiovascular reflex studies.

The performance of semi-quantitative differential PCR is similar to that of real-time PCR for the detection of the MYCN gene in neuroblastomas

Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR) for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time PCR. Similar results were obtained for 100 (99%) samples, no significant difference was detected between the median log10 MYCN copy number (1.53 by SQ-PCR versus 1.55 by absolute real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). In the comparison of SQ-PCR and relative real-time PCR, SQ-PCR versus relative real-time PCR concordant results were found in 100 (99%) samples, no significant difference was found in median log10 MYCN copy number (1.53 by SQ-PCR versus 1.27 by relative real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). These findings indicate that the performance of SQ-PCR was comparable to that of real-time PCR for the amplification and quantification of MYCN copy number. Thus, SQ-PCR can be reliably used as an alternative assay in laboratories without facilities for real-time PCR.

Differential gene expression profiles of hepatocellular carcinomas associated or not with viral infection

Chronic hepatitis B (HBV) and C (HCV) virus infections are the most important factors associated with hepatocellular carcinoma (HCC), but tumor prognosis remains poor due to the lack of diagnostic biomarkers. In order to identify novel diagnostic markers and therapeutic targets, the gene expression profile associated with viral and non-viral HCC was assessed in 9 tumor samples by oligo-microarrays. The differentially expressed genes were examined using a z-score and KEGG pathway for the search of ontological biological processes. We selected a non-redundant set of 15 genes with the lowest P value for clustering samples into three groups using the non-supervised algorithm k-means. Fisher’s linear discriminant analysis was then applied in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Different transcriptional levels of genes were identified in HCC of different etiologies and from different HCC samples. When comparing HBV-HCC vs HCV-HCC, HBV-HCC/HCV-HCC vs non-viral (NV)-HCC, HBC-HCC vs NV-HCC, and HCV-HCC vs NV-HCC of the 58 non-redundant differentially expressed genes, only 6 genes (IKBKβ, CREBBP, WNT10B, PRDX6, ITGAV, and IFNAR1) were found to be associated with hepatic carcinogenesis. By combining trios, classifiers could be generated, which correctly classified 100% of the samples. This expression profiling may provide a useful tool for research into the pathophysiology of HCC. A detailed understanding of how these distinct genes are involved in molecular pathways is of fundamental importance to the development of effective HCC chemoprevention and treatment.

Clustering of psychiatric and somatic illnesses in the general population: multimorbidity and socioeconomic correlates

The distribution of psychiatric disorders and of chronic medical illnesses was studied in a population-based sample to determine whether these conditions co-occur in the same individual. A representative sample (N = 1464) of adults living in households was assessed by the Composite International Diagnostic Interview, version 1.1, as part of the São Paulo Epidemiological Catchment Area Study. The association of sociodemographic variables and psychological symptoms regarding medical illness multimorbidity (8 lifetime somatic conditions) and psychiatric multimorbidity (15 lifetime psychiatric disorders) was determined by negative binomial regression. A total of 1785 chronic medical conditions and 1163 psychiatric conditions were detected in the population concentrated in 34.1 and 20% of respondents, respectively. Subjects reporting more psychiatric disorders had more medical illnesses. Characteristics such as age range (35-59 years, risk ratio (RR) = 1.3, and more than 60 years, RR = 1.7), being separated (RR = 1.2), being a student (protective effect, RR = 0.7), being of low educational level (RR = 1.2) and being psychologically distressed (RR = 1.1) were determinants of medical conditions. Age (35-59 years, RR = 1.2, and more than 60 years, RR = 0.5), being retired (RR = 2.5), and being psychologically distressed (females, RR = 1.5, and males, RR = 1.4) were determinants of psychiatric disorders. In conclusion, psychological distress and some sociodemographic features such as age, marital status, occupational status, educational level, and gender are associated with psychiatric and medical multimorbidity. The distribution of both types of morbidity suggests the need of integrating mental health into general clinical settings.