Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi-quantitative and quantitative RT-PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene α-galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish-green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.

Polymorphisms in candidate genes and their association with carcass traits and meat quality in Nellore cattle

The objective of this work was to estimate the allele polymorphism frequencies of genes in Nellore cattle and associate them with meat quality and carcass traits. Six hundred males were genotyped for the following polymorphisms: DGAT1 (VNTR with 18 nucleotides at the promoter region); ANK1, a new polymorphism, identified and mapped here at the gene regulatory region NW_001494427.3; TCAP (AY428575.1:g.346G>A); and MYOG (NW_001501985:g.511G>C). In the association study, phenotype data of hot carcass weight, ribeye area, backfat thickness, percentage of intramuscular fat, shear force, myofibrillar fragmentation index, meat color (L*, a*, b*), and cooking losses were used. Allele B from the ANK1 gene was associated with greater redness (a*). Alleles 5R, 6R, and 7R from the DGAT1 VNTR gene were associated with increased intramuscular fat, reduced cooking losses and increased ribeye area, respectively. The single nucleotide polymorphism (SNP) of the TCAP gene was not polymorphic, and MYOG alleles were not associated with any of the evaluated characteristics. These results indicate that ANK1 and DGAT1 genes can be used in the selection of Nellore cattle for carcass and meat quality.

Frequencies of candidate genes and associations with carcass and meat traits in Nellore and crossbred cattle

Abstract: The objective of this work was to estimate allelic frequencies of the polymorphisms IGF2/MboII (G > T) of the insulin-like growth factor 2 (IGF2) gene, DQ499531.1:g.134A > T of the pro-melanin-concentrating hormone (PMCH) gene, and DQ667048.1:g.3290G > T of the RARrelated orphan receptor C (RORC) gene in beef cattle of different genetic groups, and to evaluate the associations between these polymorphisms and traits related to carcass composition and meat quality. Data on carcass and meat quality of 499 animals was used: of 313 Nellore (Bos indicus) and of 186 Nellore crossed with different taurine (Bos taurus) breeds. For the IGF2/MboII polymorphism, the frequencies found for the G allele were 0.231 and 0.631 for Nellore and crossed breeds, respectively. For the DQ499531.1:g.134A > T polymorphism, the allelic frequencies of A were 0.850 for Nellore and 0.905 for crossed breeds. For the DQ667048.1:g.3290G > T polymorphism, the allelic frequencies of G were 0.797 and 0.460 for Nellore and crossed breeds, respectively. The evaluated single nucleotide polymorphisms (SNPs) are not significantly associated with carcass and meat traits (rib eye area, back fat thickness, shear force, total lipids, and myofibrillar fragmentation index), suggesting little utility of the analyzed polymorphisms of the IGF2, PMHC, and RORC genes as selection markers in the studied cattle populations.

Bioanalytical method for the quantification of the monastrol derivative anticancer candidate lasom 65 in pre-clinical pharmacokinetic investigations

A simple HPLC/UV method was developed for the determination of the anticancer candidate LaSOM 65 in rat plasma. Samples were cleaned by protein precipitation with acetonitrile (recovery > 95%), after which they were subjected to chromatography under the isocratic elution of an acetonitrile:water (45:55, ν/ν) solution with detection at 303 nm. The method was linear (r² > 0.98) over the concentration range (0.05-2 µg mL-1) with intra- and inter-day precision ranging from 9.6% to 13.6% and 4.3% to 5.4%, respectively. The accuracy of the method ranged from 85% to 113.6%, and it showed sufficient sensitivity to determine pharmacokinetic parameters of LaSOM 65 after intravenous administration to Wistar rats.

Distinct features of Pepper yellow mosaic virus isolates from tomato and sweetpepper

Determination of virus diversity in the field is vital to support a sustainable breeding program for virus resistance of horticultural crops. The present study aimed to characterize four field potyvirus isolates found naturally infecting sweet pepper (Capsicum annuum) (Sa66 and Sa115) and tomato (Lycopersicon esculentum) (IAC3 and Sa21) plants. Their biological characteristics revealed differences among the isolates in their ability to infect distinct Capsicum spp. and tomato genotypes, and in the severity of symptoms caused by these isolates compared to the infection caused by an isolate of Pepper yellow mosaic virus (PepYMV). Absence of cross-reaction was found among the studied isolates with antiserum against Potato virus Y (PVY). However, all isolates reacted, at different intensities, with antiserum against PepYMV. All isolates showed high identity percentage (97 to 99%) of the amino acid sequence of the coat protein with PepYMV (accession AF348610) and low (69 to 80%) with other potyvirus species. The comparison of the 3' untranslated region also confirmed this finding with 97 to 98% identity with PepYMV, and of 47 to 71% with other potyviruses. The results showed that PepYMV isolates were easily differentiated from PVY by serology and that the host response of each isolate could be variable. In addition, the nucleotide sequence of the coat protein and 3' untranslated region was highly conserved among the isolates.

Quantitative analysis of two important epidemiological features of the common bean: Phaeoisariopsis griseola pathosystem

This work quantifies two important epidemiological features of the bean (Phaseolus vulgaris)/Phaeoisariopsis griseola pathosystem. The first is the effect of the number of nights of leaf wetness on infection efficiency. Infection efficiency was below 10% when inoculated leaflets were exposed to less than two nights of leaf wetness. Optimum infection efficiencies were obtained after three to four nights of leaf wetness, at about 50%. Further nights of leaf wetness did not increase the infection efficiency. The second feature quantified is the relative rate of leaflet defoliation for varying levels of angular leaf spot severity. It increased with disease severity according to a logarithm-like curve, and a relative rate of 0.23 day-1 was estimated for a severity of 18%. The implications of these results on the disease epidemiology are discussed.

Determination of copper at wide range concentrations using instrumental features of high-resolution continuum source flame atomic absorption spectrometry

This work describes a method to determine Cu at wide range concentrations in a single run without need of further dilutions employing high-resolution continuum source flame atomic absorption spectrometry. Different atomic lines for Cu at 324.754 nm, 327.396 nm, 222.570 nm, 249.215 nm and 224.426 nm were evaluated and main figures of merit established. Absorbance measurements at 324.754 nm, 249.215 nm and 224.426 nm allows the determination of Cu in the 0.07 - 5.0 mg L-1, 5.0 - 100 mg L-1 and 100 - 800 mg L-1 concentration intervals respectively with linear correlation coefficients better than 0.998. Limits of detection were 21 µg L-1, 310 µg L-1 and 1400 µg L-1 for 324.754 nm, 249.215 nm and 224.426 nm, respectively and relative standard deviations (n = 12) were £ 2.7%. The proposed method was applied to water samples spiked with Cu and the results were in agreement at a 95% of confidence level (paired t-test) with those obtained by line-source flame atomic absorption spectrometry.

Histopathological features of infections caused by Fusarium oxysporum and F. solani in purple passionfruit plants (Passiflora edulis Sims)

The purple passionfruit plant, Passifloraedulis Sims, ranks second in fruit exportation in Colombia, and its main destination is the European market. However, its production is affected by several diseases, including fusariosis. This paper presents the histopathological features of different tissues affected by the pathogens Fusarium oxysporum and Fusarium solani. Both microorganisms produce similar responses on the plant: colonization of xylem vessels by hyphae and microconidia, hypertrophy and hyperplasia of the cambium, xylem and phloem; destruction of xylem fibers and amyloplasts in parenchymatous cells; and production of gels by the plant. However, there are differences in the colonization mechanism, F. solani penetrates and is concentrated especially at the collar zone, while F. oxysporum penetrates the roots and moves through the vascular system to colonize the plant.

Comparison of nuclear grade and immunohistochemical features in situ and invasive components of ductal carcinoma of breast

PURPOSE:To compare the prognostic and predictive features between in situ and invasive components of ductal breast carcinomas. METHODS:We selected 146 consecutive breast samples with ductal carcinoma in situ (DCIS) associated with adjacent invasive breast carcinoma (IBC). We evaluated nuclear grade and immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6), and epidermal growth factor receptor (EGFR) in both components, in situ and invasive, and the Ki-67 percentage of cells in the invasive part. The DCIS and IBC were classified in molecular surrogate types determined by the immunohistochemical profile as luminal (RE/PR-positive/ HER2-negative), triple-positive (RE/RP/HER2-positive), HER2-enriched (ER/PR-negative/HER2-positive), and triple-negative (RE/RP/HER2-negative). Discrimination between luminal A and luminal B was not performed due to statistical purposes. Correlations between the categories in the two groups were made using the Spearman correlation method. RESULTS:There was a significant correlation between nuclear grade (p<0.0001), expression of RE/RP (p<0.0001), overexpression of HER2 (p<0.0001), expression of EGFR (p<0.0001), and molecular profile (p<0.0001) between components in situ and IBC. CK 5/6 showed different distribution in DCIS and IBC, presenting a significant association with the triple-negative phenotype in IBC, but a negative association among DCIS. CONCLUSIONS: Our results suggest that classical prognostic and predictive features of IBC are already determined in the preinvasive stage of the disease. However the role of CK5/6 in invasive carcinoma may be different from the precursor lesions.

Aspectos clinicopatológicos de 620 casos neurológicos de cinomose em cães: Clinicopathological features in 620 neurological cases of canine distemper

Os protocolos de 5.361 necropsias de cães realizadas no Laboratório de Patologia Veterinária da Universidade Federal de Santa Maria de 1965 a 2006 foram revisados à procura de casos de cinomose. Seiscentos e oitenta e três casos (12,7%) da doença foram encontrados, dos quais 620 apresentavam sinais neurológicos. Desses 620, os seguintes dados foram recuperados para cada caso: idade, sinais clínicos, achados histopatológicos e presença ou não de doença concomitante. Faixas etárias foram classificadas como filhotes (até 1 ano), adultos (de 1 a 9 anos) e idosos (10 anos de idade ou mais). Lesões histológicas foram observadas em 565 (91,1%) dos 620 casos com sinais neurológicos de cinomose e em 554 desses casos a idade foi registrada no protocolo com a seguinte distribuição por faixa etária: 45,9% de filhotes, 51,4% de adultos e 2,7% de idosos. Os sinais neurológicos compreendiam um largo espectro de distúrbios motores, posturais e do comportamento, que podiam ocorrer juntos ou individualmente. Os sinais clínicos mais freqüentes foram mioclonia (38,4%), incooordenação motora (25,0%), convulsões (18,5%) e paraplegia (13,4%). Em 98,4% dos 565 cães com alterações histopatológicas no encéfalo, foram observadas desmielinização, encefalite não-supurativa ou uma combinação dessas duas lesões. Corpúsculos de inclusão foram observados em diferentes células de 343 dos 565 cães com alterações histopatológicas no encéfalo. Em 170 (49,6%) o tipo celular com inclusão não foi mencionado no protocolo; nos restantes, as inclusões foram vistas em astrócitos (94,8% dos casos), neurônios (3,5%), oligodendrócitos (1,1%) e células do epêndima (0,6%). Levando em consideração o tipo de lesões e as faixas etárias, casos com desmielinização e encefalite não-supurativa ocorreram em 40,0% dos filhotes, 51,2% dos adultos e 72,7% dos cães idosos. Somente desmielinização foi descrita em 48,4% dos filhotes, 41,3% dos adultos e 35,7% dos cães idosos. Somente encefalite não-supurativa foi descrita em 11,6% dos filhotes, 7,5% dos adultos e 7,1% dos cães idosos.

Development and evaluation of a recombinant DNA vaccine candidate expressing porcine circovirus 2 structural protein

Porcine circovirus 2 (PCV2) is generally associated with the porcine circovirosis syndrome, which is considered an important disease of swine and has potentially serious economic impact on the swine industry worldwide. This article describes the construction of a recombinant plasmid expressing the PCV2 structural protein and the evaluation of cellular and humoral immune responses produced by this recombinant vaccine in BALB/c mice. The vaccine candidate was obtained and analyzed in vivo, in an effort to determine the ability to induce a specific immune response in mice. DNA was extracted from a Brazilian PCV2 isolate and the gene coding for Cap protein was amplified by PCR and inserted into an expression plasmid. Groups of BALB/c mice were inoculated intra-muscularly and intradermally in a 15-day interval, with 100 µg and 50 µg of the vaccine construct, respectively. Another group was inoculated intramuscularly with 100 µg of empty plasmid, corresponding to the control group. Seroconversion and cellular response in BALB/c mice were compared and used for vaccine evaluation. Seroconversion was analyzed by ELISA. After a series of 3 immunizations the spleen cells of the immunized animals were used to perform lymphocyte proliferation assays. Seroconversion to PCV2 was detected by ELISA in the animals inoculated with the vaccine construct when compared with control groups. Lymphocyte proliferation assays showed a stronger cell proliferation in the inoculated animals compared with the control group. Thus, the vaccine candidate construct demonstrated to be able to induce both humoral and cellular responses in inoculated mice.

Morphological features and vascularization study of caprine cyclic corpus luteum

Corpus luteum is a temporary endocrine gland that regulates either the estrous cycle and pregnancy. It presents extreme dependency on the adequate blood supply. This work aims to evaluate goat corpus luteum (CL) vascular density (VD) over the estrous cycle. For that purpose, 20 females were submitted to estrus synchronization/ovulation treatment using a medroxyprogesterone intra-vaginal sponge as well as intramuscular (IM) application of cloprostenol and equine chorionic gonadotrophine (eCG). After sponge removal, estrus was identified at about 72hs. Once treatment was over, female goats were then subdivided into 4 groups (n=5 each) and slaughtered on days 2, 12, 16 and 22 after ovulation (p.o). Ovaries were collected, withdrawn and weighted. CL and ovaries had size and area recorded. Blood samples were collected and the plasma progesterone (P4) was measured through RIA commercial kits. The VD was 24.42±6.66, 36.26±5.61, 8.59±2.2 and 3.97±1.12 vessels/mm² for days 2, 12, 16 and 22 p.o, respectively. Progesterone plasma concentrations were 0.49±0.08, 2.63±0.66, 0.61±0.14 and 0.22±0.04ng/ml for days 2, 12, 16 e 22 p.o, respectively. Studied parameters were affected by the estrous cycle phase. Values greater than 12 p.o were observed. In the present work we observed that ovulation occurred predominantly in the right ovary (70% of the animals), which in turn presented bigger measures than the contra lateral one. There is a meaningful relationship between the weight and size of the ovary and these of CL (r=0.87, r=0.70, respectively, p<0.05). It is possible to conclude that morphology of goat's ovaries and plasma progesterone concentration changed according to estrous cycle stages. We propose these parameters can be used as indicators of CL functional activity.

Gross anatomical features of the oropharyngeal cavity of the ostrich (Struthio camelus)

Most descriptions of the ostrich oropharynx and oesophagus are superficial and supply little meaningful morphological data. The aim of this investigation is describe the ostrich oropharingeal cavity, in order to supply the deficiency of macroscopic data about this important animal. Five heads of 12 to 14-month-old ostriches of either sex were anatomically dissected to expose the oropharynx. The ostrich oropharynx was "bell-shaped" composed by the maxillary and mandibular ramphoteca. The roof and floor presented two distinct regions different in colour of the mucosa. The rostral region was pale pink contrasting to creamy-pink coloured caudal region. The median longitudinal ridge extended rostrally from the apex of the choana to the tip of the beak in the roof and it is clearly more prominent and rigid than the homolog in the floor that appeared thin and stretched rostrally, continuing caudally surrounding the tongue and the laryngeal mound eventually merging with the oesophageal mucosa. The floor was formed by the interramal region, tongue and laryngeal mound containing shield-shaped glottis. It can be concluded that the present study, in addition to confirming the basic features of the oropharynx previously described for the ostrich, clarified the contradictory information presented in the literature and also provided new, unreported morphological data, some of which may be important when studying nutrition and health in these birds.

Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

Eimeria spp. from Japanese quails (Coturnix japonica): new characteristic features and diagnostic tools

The Japanese quail Coturnix japonica originated from North Africa, Europe and Asia, is used worldwide as an experimental animal and model for aviculture. The current paper characterizes Eimeria bateri, Eimeria tsunodai and Eimeria uzura recovered from C. japonica. Based on the fact that quails have a global distribution, as are their coccidia, the findings of this study should provide the means for diagnosis of those Eimeria spp. in other regions and continents. Eimeria bateri showed the greatest intensity of infection and shed oocysts from the fourth day after infection; in contrast, E. tsunodai and E. uzura shed oocysts from the fifth day after infection. The three species shared a high degree of similarity and were all polymorphic. Yet, the application of line regressions, histograms and ANOVA provided means for the identification of these species. Finally, the algorithm was very efficient since verified that resultant values were not superimposed.