Leaf application of silicic acid to upland rice and corn

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Inflammatory and Cell-Mediated Immune Responses in the Respiratory Tract of Chickens to Infection with Avian Infectious Bronchitis Virus

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

Molecular investigation of zoonotic genotypes of Giardia intestinalis isolates in humans, dogs and cats, sheep, goats and cattle in Araçatuba (Sãoo Paulo State, Brazil) by the analysis of beta-giardin gene fragments

In the period from July 2009 to October 2010, fecal samples from 61 animals and 154 humans from the municipality of Aracatuba (São Paulo State, Brazil) were studied. Fecal samples from animals were collected in the Municipal Animal Shelter and the Veterinary Hospital of the Universidade Estadual Paulista. Human fecal specimens were collected in playschools in the outskirts of the city by the private network of clinical analysis laboratories of the municipal. Diagnosis was done by optical microscopy using the Faust and Hoffmann, Pons and Janer techniques. The genotypes of Giardia intestinalis were characterized by PCR-RFLP and confirmed by sequencing the ß-giardin gene. Human specimens were positive in 25.3% (39/154) of the cases with 26.8% (36/134) of the specimens from children and 15% (3/20) from adults being positive. The frequency of G. intestinalis among the animals was 23.0% (14/61). A total of 32 isolates of G. intestinalis obtained from human feces and six from dogs and cats were characteristic of the A genotype (AI and AII/AIII). The results of this study in respect to frequency of giardiasis are similar to reported in most studies in Brazil. The prevalence observed in animal populations conforms to worldwide infection rates. G. intestinalis genotypes considered zoonotic were detected in both pets and humans from the city of Aractuba, suggesting a possible zoonotic transmission of the parasite in the northwestern region of São Paulo State. The absence of these genotypes in farm animals may imply that they are not involved in the chain of transmission to humans in this region.

Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro

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

Agronomic efficiency and grain quality of upland rice cultivars as a function of nitrogen topdressing

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

Effect of the particle size of maize, rice, and sorghum in extruded diets for dogs on starch gelatinization, digestibility, and the fecal concentration of fermentation products

The influence of rice, maize, and sorghum raw material particle size in extruded dry dog food on the digestibility of nutrients and energy and the fecal concentration of fermentation products was investigated. Three diets with similar nutrient compositions were formulated, each with 1 starch source. Before incorporation into diets, the cereals were ground into 3 different particle sizes (approximately 300, 450, and 600 mu m); therefore, a total of 9 diets were in a 3 x 3 factorial arrangement (3 cereals and 3 particle sizes). Fifty-four beagle dogs (12.0 +/- 0.1 kg BW) were randomly assigned to the diets, with 6 dogs per diet. The digestibility was measured with the chromium oxide method. The data were evaluated with ANOVA considering the carbohydrate source, grinding effect, and interactions. The means were compared with the Tukey test and polynomial contrasts (P < 0.05). With the same grinding procedure, rice was reduced to smaller particles than other cereals. The cereal mean geometric diameter (MGD) was directly related to starch gelatini-zation (SG) during extrusion. For rice diets, the MGD and SG did not change nutrient digestibility (P > 0.05); only GE digestibility was reduced at the largest MGD (P < 0.01). For maize and sorghum diets, the total tract apparent nutrient digestibility was reduced for foods with greater MGD and less SG (P < 0.01). A linear reduction in nutrient digestibility according to cereal particle size was observed for sorghum (r(2) < 0.72; P < 0.01). Higher concentrations of fecal total shortchain fatty acids (SCFA) were observed for sorghum diets (P < 0.05) than for other diets. The rice diets led to the production of feces with less lactate (P < 0.05). The increase in raw material MGD did not influence fecal SCFA for rice diets, but for the dogs fed maize and sorghum foods, an increase in propionate and butyrate concentrations were observed as MGD increased (P < 0.05). In conclusion, for dogs fed different particle sizes of the cereal starches in the extruded diets, the digestibility and fecal characteristics were affected, and this effect was ingredient dependent.

Preparation and characterization of monomodal grapevine virus a capsid protein

Grapevine virus A (GVA), a flexible filament of approximately 800 nm in length is composed of capsid subunits that spontaneously assembles around a positive sense genomic RNA. In addition to encapsidation, plant viruses capsid proteins (CPs) participate in other processes throughout infection and GVA CP is involved in cell-to-cell translocation of the virus. A protocol was developed to obtain low-molecular weight GVA-CP that is not prone to aggregation and spontaneous assembly and this was characterized by circular dichroism and dynamic light scattering. These results indicate the suitably of GVA-CP for X-ray crystallographic and NMR studies that should lead to the elucidation of the first three-dimensional structure of a flexible filamentous virus from the Betaflexiviridae family.

Genetic variation of germination cold tolerance in Japanese rice germplasm

Low temperatures at the initial stages of rice development prevent fast germination and seedling establishment and may cause significant productivity losses. In order to develop rice cultivars exhibiting cold tolerance, it is necessary to investigate genetic resources, providing basic knowledge to allow the introduction of genes involved in low temperature germination ability from accessions into elite cultivars. Japanese rice accessions were evaluated at the germination under two conditions: 13 degrees C for 28 days (cold stress) and 28 degrees C for seven days (optimal temperature). The traits studied were coleoptile and radicle length under optimal temperature, coleoptile and radicle length under cold and percentage of the reduction in coleptile and radicle length due to low temperature. Among the accessions studied, genetic variation for traits related to germination under low temperatures was observed and accessions exhibiting adequate performance for all investigated traits were identified. The use of multivariate analysis allowed the identification of the genotypes displaying cold tolerance by smaller reductions in coleoptile and radicle lenght in the presence of cold and high vigour, by higher coleoptile and radicle growth under cold.

Diversity of propanil-degrading bacteria isolated from rice rhizosphere and their potential for plant growth promotion

The herbicide propanil has long been used in rice production in southern Brazil. Bacteria isolated from contaminated soils in Massaranduba, Santa Catarina, Brazil, were found to be able to grow in the presence of propanil, using this compound as a carbon source. Thirty strains were identified as Pseudomonas (86.7%), Serratia (10.0%), and Acinetobacter (3.3%), based on phylogenetic analysis of 16S rDNA. Little genetic diversity was found within species, more than 95% homology, suggesting that there is selective pressure to metabolize propanil in the microbial community. Two strains of Pseudomonas (AF7 and AF1) were selected in bioreactor containing chemotactic growth medium, with the highest degradation activity of propanil exhibited by strain AF7, followed by AF1 (60 and 40%, respectively). These strains when encapsulated in alginate exhibited a high survival rate and were able to colonize the rice root surfaces. Inoculation with Pseudomonas strains AF7 and AF1 significantly improved the plant height of rice. Most of the Pseudomonas strains produced indoleacetic acid, soluble mineral phosphate, and fixed nitrogen. These bacterial strains could potentially be used for the bioremediation of propanil-contaminated soils and the promotion of plant growth.

Genetic transformation of Citrus sinensis with Citrus tristeza virus (CTV) derived sequences and reaction of transgenic lines to CTV infection

Transgenic Citrus sinensis (L.) Osb. plants, cvs. Valencia and Hamlin, expressing Citrus tristeza virus (CTV) derived sequences were obtained by genetic transformation. The gene constructs were pCTV-CP containing the 25 kDa major capsid protein gene (CTV-CP), pCTV-dsCP containing the same CTV-CP gene in an intron-spliced hairpin construct, and pCTV-CS containing a 559 nt conserved region of the CTV genome. The transgenic lines were identified by PCR and the transgene integration was confirmed by Southern blot. Transgene mRNA could be detected in most transgenic lines containing pCTV-CP or pCTV-CS transgene. The mRNA of pCTV-dsCP transgene was almost undetectable, with very light bands in most analyzed plants. The transgene transcription appears to be closely linked to the type of gene construct. The virus challenge assays reveals that all transgenic lines were infected. However, it was possible to identify propagated clones of transgenic plants of both cultivars studied with a low virus titer, with values similar to the non-inoculated plants (negative control). These results suggested that the transgenic plants present some level of resistance to virus replication. The higher number of clones with low virus titer and where mRNA could not be detected or was presented in a very light band was found for pCTV-dsCP-derived transgenic lines.

Virulence attributes and genetic variability of oral Candida albicans and Candida tropicalis isolates

The wide spectrum of candidiasis and its clinical importance encourage the research with the purpose of clarifying the mechanisms of pathogenicity and identification of virulence factors of Candida sp. Therefore, the aim of this study was to verify the adhesion capacity, protease activity and genotypic diversity of oral C. albicans and C. tropicalis isolates. The adhesion ability to the extracellular matrix glycoproteins laminin and fibronectin was evaluated using the ELISA technique. The research of proteases was carried out in agar plate containing bovine albumin and through a quantitative method in buffer solution containing haemoglobin. Intra and interspecies polymorphisms was verified through random amplified polymorphic DNA (RAPD) technique. All C. albicans and C. tropicalis isolates binded to immobilised laminin and fibronectin. Ca33 and Ct13 isolates had relative adhesion index significantly higher than the other isolates for both glycoproteins (P < 0.001). Protease activity was observed in all isolates of C. albicans using either the semi-quantitative or quantitative assay. The protease activity of C. tropicalis was better detected through the quantitative assay. The genotypic diversity by RAPD revealed a heterogeneous population in both species. Nevertheless, C. tropicalis presented higher genetic variability than C. albicans strains.

Hepatitis B virus infection in Haemodialysis Centres from Santa Catarina State, Southern Brazil. Predictive risk factors for infection and molecular epidemiology

Abstract Background Patients under haemodialysis are considered at high risk to acquire hepatitis B virus (HBV) infection. Since few data are reported from Brazil, our aim was to assess the frequency and risk factors for HBV infection in haemodialysis patients from 22 Dialysis Centres from Santa Catarina State, south of Brazil. Methods This study includes 813 patients, 149 haemodialysis workers and 772 healthy controls matched by sex and age. Serum samples were assayed for HBV markers and viraemia was detected by nested PCR. HBV was genotyped by partial S gene sequencing. Univariate and multivariate statistical analyses with stepwise logistic regression analysis were carried out to analyse the relationship between HBV infection and the characteristics of patients and their Dialysis Units. Results Frequency of HBV infection was 10.0%, 2.7% and 2.7% among patients, haemodialysis workers and controls, respectively. Amidst patients, the most frequent HBV genotypes were A (30.6%), D (57.1%) and F (12.2%). Univariate analysis showed association between HBV infection and total time in haemodialysis, type of dialysis equipment, hygiene and sterilization of equipment, number of times reusing the dialysis lines and filters, number of patients per care-worker and current HCV infection. The logistic regression model showed that total time in haemodialysis, number of times of reusing the dialysis lines and filters, and number of patients per worker were significantly related to HBV infection. Conclusions Frequency of HBV infection among haemodialysis patients at Santa Catarina state is very high. The most frequent HBV genotypes were A, D and F. The risk for a patient to become HBV positive increase 1.47 times each month of haemodialysis; 1.96 times if the dialysis unit reuses the lines and filters ≥ 10 times compared with haemodialysis units which reuse < 10 times; 3.42 times if the number of patients per worker is more than five. Sequence similarity among the HBV S gene from isolates of different patients pointed out to nosocomial transmission.

Identification of Aspergillus flavus isolates as potential biocontrol agents of aflatoxin contamination in crops

Aspergillus flavus, a haploid organism found worldwide in a variety of crops, including maize, cottonseed, almond, pistachio, and peanut, causes substantial and recurrent worldwide economic liabilities. This filamentous fungus produces aflatoxins (AFLs) B1 and B2, which are among the most carcinogenic compounds from nature, acutely hepatotoxic and immunosuppressive. Recent efforts to reduce AFL contamination in crops have focused on the use of nonaflatoxigenic A. flavus strains as biological control agents. Such agents are applied to soil to competitively exclude native AFL strains from crops and thereby reduce AFL contamination. Because the possibility of genetic recombination in A. flavus could influence the stability of biocontrol strains with the production of novel AFL phenotypes, this article assesses the diversity of vegetative compatibility reactions in isolates of A. flavus to identify heterokaryon self-incompatible (HSI) strains among nonaflatoxigenic isolates, which would be used as biological controls of AFL contamination in crops. Nitrate nonutilizing (nit) mutants were recovered from 25 A. flavus isolates, and based on vegetative complementation between nit mutants and on the microscopic examination of the number of hyphal fusions, five nonaflatoxigenic (6, 7, 9 to 11) and two nontoxigenic (8 and 12) isolates of A. flavus were phenotypically characterized as HSI. Because the number of hyphal fusions is reduced in HSI strains, impairing both heterokaryon formation and the genetic exchanges with aflatoxigenic strains, the HSI isolates characterized here, especially isolates 8 and 12, are potential agents for reducing AFL contamination in crops

Molecular basis oh herpes simplex virus entry into the cell and retargeting of the viral tropism for the design of oncolytic herpesviruses

Herpes simplex virus 1 (HSV-1) infects oral epitelial cells, then spreads to the nerve endings and estabilishes latency in sensory ganglia, from where it may, or may not reactivate. Diseases caused by virus reactivation include mild diseases such as muco-cutaneous lesions, and more severe, and even life-threatening encephalitis, or systemic infections affecting diverse organs. Herpes simplex virus represents the most comprehensive example of virus receptor interaction in Herpesviridae family, and the prototype virus encoding multipartite entry genes. In fact, it encodes 11-12 glycoproteins and a number of additional membrane proteins: five of these proteins play key roles in virus entry into subsceptible cells. Thus, glycoprotein B (gB) and glycoprotein C (gC) interact with heparan sulfate proteoglycan to enable initial attachment to cell surfaces. In the next step, in the entry cascade, gD binds a specific surface receptor such as nectin1 or HVEM. The interaction of glycoprotein D with the receptor alters the conformation of gD to enable the activation of gB, glycoprotein H, and glycoprotein L, a trio of glycoproteins that execute the fusion of the viral envelope with the plasma membrane. In this thesis, I described two distinct projects: I. The retargeting of viral tropism for the design of oncolytic Herpesviruses: • capable of infecting cells through the human epitelial growth factor receptor 2 (HER2), overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis; • detargeted from its natural receptors, HVEM and nectin1. To this end, we inserted a ligand to HER2 in gD. Because HER2 has no natural ligand, the selected ligand was a single chain antibody (scFv) derived from MAb4D5 (monoclonal antibody to HER2), herein designated scHER2. All recombinant viruses were targeted to HER2 receptor, but only two viruses (R-LM113 and R-LM249) were completely detargeted from HVEM and nectin1. To engineer R-LM113, we removed a large portion at the N-terminus of gD (from aa 6 to aa 38) and inserted scHER2 sequence plus 9-aa serine-glycine flexible linker at position 39. On the other hand, to engineer R-LM249, we replaced the Ig-folded core of gD (from aa 61 to aa 218) with scHER2 flanked by Ser-Gly linkers. In summary, these results provide evidence that: i. gD can tolerate an insert almost as big as gD itself; ii. the Ig-like domain of gD can be removed; iii. the large portion at the N-terminus of gD (from aa 6 to aa 38) can be removed without loss of key function; iv. R-LM113 and R-LM249 recombinants are ready to be assayed in animal models of mammary and ovary tumour. This finding and the avaibility of a large number of scFv greatly increase the collection of potential receptors to which HSV can be redirected. II. The production and purification of recombinant truncated form of the heterodimer gHgL. We cloned a stable insect cell line expressing a soluble form of gH in complex with gL under the control of a metalloprotein inducible promoter and purified the heterodimer by means of ONE-STrEP-tag system by IBA. With respect to biological function, the purified heterodimer is capable: • of reacting to antibodies that recognize conformation dependent epitopes and neutralize virion infectivity; • of binding a variety cells at cell surface. No doubt, the availability of biological active purified gHgL heterodimer, in sufficient quantities, will speed up the efforts to solve its crystal structure and makes it feasible to identify more clearly whether gHgL has a cellular partner, and what is the role of this interaction on virus entry.

Role of αvβ3 – Integrin and TLR2 in the innate response to Herpes Simplex Virus infection and Delivery of retargeted oncolytic Herpes Simplex via carrier cells

In the first part of my thesis I studied the mechanism of initiation of the innate response to HSV-1. Innate immune response is the first line of defense set up by the cell to counteract pathogens infection and it is elicited by the activation of a number of membrane or intracellular receptors and sensors, collectively indicated as PRRs, Patter Recognition Receptors. We reported that the HSV pathogen-associated molecular patterns (PAMP) that activate Toll-like receptor 2 (TLR2) and lead to the initiation of innate response are the virion glycoproteins gH/gL and gB, which constitute the conserved fusion core apparatus across the Herpesvirus. Specifically gH/gL is sufficient to initiate a signaling cascade which leads to NF-κB activation. Then, by gain and loss-of-function approaches, we found that αvβ3-integrin is a sensor of and plays a crucial role in the innate defense against HSV-1. We showed that αvβ3-integrin signals through a pathway that concurs with TLR2, affects activation/induction of interferons type 1, NF-κB, and a polarized set of cytokines and receptors. Thus, we demonstrated that gH/gL is sufficient to induce IFN1 and NF-κB via this pathway. From these data, we proposed that αvβ3-integrin is considered a class of non-TLR pattern recognition receptors. In the second part of my thesis I studied the capacity of human mesenchymal stromal cells isolated by fetal membranes (FM-hMSCs) to be used as carrier cells for the delivery of retargeted R-LM249 virus. The use of systemically administrated carrier cells to deliver oncolytic viruses to tumoral targets is a promising strategy in oncolytic virotherapy. We observed that FM-hMSCs can be infected by R-LM249 and we optimized the infection condition; then we demonstrate that stromal cells sustain the replication of retargeted R-LM249 and spread it to target tumoral cells. From these preliminary data FM-hMSCs resulted suitable to be used as carrier cells