SELECTION OF TRICHOGRAMMA PRETIOSUM LINEAGES FOR CONTROL OF GRAPHOLITA MOLESTA IN PEACH

Grapholita molesta (Lepidoptera: Tortricidae) is one of the main pests of peach trees in Brazil, causing fruit losses of 3-5%. Among possible biological control agents, Trichogramma pretiosum (Hymenoptera: Trichogrammatidae) has been found in peach orchards. Our objectives were to study the rearing of T pretiosum in eggs of G. molesta and Anagasta kuehniella (Lepidoptera: Pyralidae), and select lineages of this parasitoid that have the potential to control G. molesta. Selection of best lineages was made from 5 populations of T pretiosum collected from organically-cultivated peach orchards. The study was done under controlled temperature (25 +/- 2 degrees C), relative humidity (70 +/- 10%) and 14:10 h (light:dark) photoperiod conditions. Grapholita molesta eggs were found to be adequate hosts for the development of T pretiosum, and the parameters for number of parasitized eggs, percent parasitized eggs, and sex ratio were similar to those for A. kuehniella eggs. The highest rate of parasitism of G. molesta eggs occurred in eggs with up to 48 h of embryonic development. Among the lineages of T pretiosum that were collected, HO8, PO8, PEL, and L3M showed the best biological performance and are therefore indicated for semi-field and field studies for biological control of oriental fruit moth.

LOCAL HEAD LOSS OF NON-COAXIAL EMITTERS INSERTED IN POLYETHYLENE PIPE

The design of a lateral line for drip irrigation requires accurate evaluation of head losses in not only the pipe but in the emitters as well. A procedure was developed to determine localized head losses within the emitters by the formulation of a mathematical model that accounts for the obstruction caused by the insertion point. These localized losses can be significant when compared with tire total head losses within the system due to the large number of emitters typically installed along the lateral line. Air experiment was carried out by altering flow characteristics to create Reynolds numbers (R) from 7,480 to 32,597 to provide turbulent flow and a maximum velocity of 2.0 m s(-1). The geometry of the emitter was determined by an optical projector and sensor An equation was formulated to facilitate the localized head loss calculation using the geometric characteristics of the emitter (emitter length, obstruction ratio, and contraction coefficient). The mathematical model was tested using laboratory measurements on four emitters. The local head loss was accurately estimated for the Uniram (difference of +13.6%) and Drip Net (difference of +7.7%) emitters, while appreciable deviations were found for the Twin Plus (-21.8%) and Tiran (+50%) emitters. The head loss estimated by the model was sensitive to the variations in the obstruction area of the emitter However, the variations in the local head loss did not result in significant variations in the maximum length of the lateral lines. In general, for all the analyzed emitters, a 50% increase in the local head loss for the emitters resulted in less than an 8% reduction in the maximum lateral length.

DEVELOPMENT OF A NOVEL SET OF MICROSATELLITE MARKERS FOR CASTOR BEAN, RICINUS COMMUNIS (EUPHORBIACEAE)

Premise of study: Microsatellite primers were developed for castor bean (Ricinus communis L.) to investigate genetic diversity and population structure, and to provide support to germplasm management. Methods and Results: Eleven microsatellite loci were isolated using an enrichment cloning protocol and used to characterize castor bean germplasm from the collection at the Instituto Agronomico de Campinas (IAC). In a survey of 76 castor bean accessions, the investigated loci displayed polymorphism ranging from two to five alleles. Conclusions: The information derived from microsatellite markers led to significant gains in conserved allelic richness and provides support to the implementation of several molecular breeding strategies for castor bean.

DEVELOPMENT OF MICROSATELLITE MARKERS FOR AULONEMIA ARISTULATA (POACEAE) AND CROSS-AMPLIFICATION IN OTHER BAMBOO SPECIES

Premise of the study: Microsatellite primers were developed for Aulonemia aristulata, an endangered species of economic interest, to further describe its genetic variability and population structure. We also tested cross-amplification in 18 other bamboo species. Methods and Results: Using an enrichment genomic library, 13 microsatellite loci were isolated and characterized in A. aristulata. Seven of these loci were polymorphic. Twelve markers were cross-amplified in at least ten of the tested bamboo species. Conclusions: These markers will be useful for studies on the genetic diversity and structure of A. aristulata, which are important for future conservation, management and breeding programs of this species.

MicroRNA transcriptome profiles during swine skeletal muscle development

Background: MicroRNA (miR) are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts. To evaluate the role of miR in skeletal muscle of swine, global microRNA abundance was measured at specific developmental stages including proliferating satellite cells, three stages of fetal growth, day-old neonate, and the adult. Results: Twelve potential novel miR were detected that did not match previously reported sequences. In addition, a number of miR previously reported to be expressed in mammalian muscle were detected, having a variety of abundance patterns through muscle development. Muscle-specific miR-206 was nearly absent in proliferating satellite cells in culture, but was the highest abundant miR at other time points evaluated. In addition, miR-1 was moderately abundant throughout developmental stages with highest abundance in the adult. In contrast, miR-133 was moderately abundant in adult muscle and either not detectable or lowly abundant throughout fetal and neonate development. Changes in abundance of ubiquitously expressed miR were also observed. MiR-432 abundance was highest at the earliest stage of fetal development tested (60 day-old fetus) and decreased throughout development to the adult. Conversely, miR-24 and miR-27 exhibited greatest abundance in proliferating satellite cells and the adult, while abundance of miR-368, miR-376, and miR-423-5p was greatest in the neonate. Conclusion: These data present a complete set of transcriptome profiles to evaluate miR abundance at specific stages of skeletal muscle growth in swine. Identification of these miR provides an initial group of miR that may play a vital role in muscle development and growth.

Plasmodium vivax circumsporozoite genotypes: a limited variation or new subspecies with major biological consequences?

Background: Plasmodium vivax circumsporozoite variants have been identified in several geographical areas. The real implication of the genetic variation in this region of the P. vivax genome has been questioned for a long time. Although previous studies have observed significant association between VK210 and the Duffy blood group, we present here that evidences of this variation are limited to the CSP central portion. Methods: The phylogenetic analyses were accomplished starting from the amplification of conserved domains of 18 SSU RNAr and Cyt B. The antibodies responses against the CSP peptides, MSP-1, AMA-1 and DBP were detected by ELISA, in plasma samples of individuals infected with two P. vivax CS genotypes: VK210 and P. vivax-like. Results: These analyses of the two markers demonstrate high similarity among the P. vivax CS genotypes and surprisingly showed diversity equal to zero between VK210 and P. vivax-like, positioning these CS genotypes in the same clade. A high frequency IgG antibody against the N- and C-terminal regions of the P. vivax CSP was found as compared to the immune response to the R- and V-repetitive regions (p = 0.0005, Fisher's Exact test). This difference was more pronounced when the P. vivax-like variant was present in the infection (p = 0.003, Fisher's Exact test). A high frequency of antibody response against MSP-1 and AMA-1 peptides was observed for all P. vivax CS genotypes in comparison to the same frequency for DBP. Conclusions: This results target that the differences among the P. vivax CS variants are restrict to the central repeated region of the protein, mostly nucleotide variation with important serological consequences.

Seasonality Role on the Phenolics from Cultivated Baccharis dracunculifolia

Baccharis dracunculifolia is the source of Brazilian green propolis (BGP). Considering the broad spectrum of biological activities attributed to green proplis, B. dracunculifolia has a great potential for the development of new cosmetic and pharmaceutical products. In this work, the cultivation of 10 different populations of native B. dracunculifolia had been undertaken aiming to determine the role of seasonality on its phenolic compounds. For this purpose, fruits of this plant were collected from populations of 10 different regions, and 100 individuals of each population were cultivated in an experimental area of 1800 m(2). With respect to cultivation, the yields of dry plant, essential oil and crude extract were measured monthly resulting in mean values of 399 +/- 80 g, 0.6 +/- 0.1% and 20 +/- 4%, respectively. The HPLC analysis allowed detecting seven phenolic compounds: caffeic acid, ferulic acid, aromadendrin-4'-methyl ether (AME), isosakuranetin, artepillin C, baccharin and 2-dimethyl-6-carboxyethenyl-2H-1-benzopyran acid, which were the major ones throughout the 1-year monthly analysis. Caffeic acid was detected in all cultivated populations with mean of 4.0%. AME displayed the wide variation in relation to other compounds showing means values of 0.65 +/- 0.13% at last quarter. Isosakuranetin and artepillin C showed increasing concentrations with values between 0% and 1.4% and 0% and 1.09%, respectively. The obtained results allow suggesting that the best time for harvesting this plant, in order to obtain good qualitative and quantitative results for these phenolic compounds, is between December and April.

A Novel Organotellurium Compound (RT-01) as a New Antileishmanial Agent

Leishmaniasis is a neglected disease and endemic in developing countries. A lack of adequate and definitive chemotherapeutic agents to fight against this infection has led to the investigation of numerous compounds. The aim of this study was to investigate the effect of RT-01, an organotellurane compound presenting biological activities, in 2 experimental systems against Leishmania amazonensis. The in vitro system consisted of promastigotes and amastigotes forms of the parasite, and the in vivo system consisted of L. amazonensis infected BALB/c mice, an extremely susceptible mouse strain. The compound proved to be toxic against promastigotes and amastigotes. The study also showed that treatment with RT-01 produces an effect similar to that treatment with the reference antimonial drug, Glucantime, in L. amazonensis infected mice. The best results were obtained following RT-01 intralesional administration (720 mu g/kg/day); mice showed significant delay in the development of cutaneous lesions and decreased numbers of parasites obtained from the lesions. Significant differences in tissue pathology consisted mainly of no expressive accumulation of inflammatory cells and well-preserved structures in the skin tissue of RT-01-treated mice compared with expressive infiltration of infected cells replacing the skin tissue in lesions of untreated mice. These findings highlight the fact that the apparent potency of organotellurane compounds, together with their relatively simple structure, may represent a new avenue for the development of novel drugs to combat parasitic diseases.

The development of an immobilized enzyme reactor containing glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi: the effect of species' specific differences on the immobilization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD+ from NADH. The production of NADH stimulated by D-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K-m) values determined for D-glyceraldehyde-3-phosphate and NAD(+) were K-m = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.

Ferromagnetic resonance for the quantification of superparamagnetic iron oxide nanoparticles in biological materials

The aim of the present work is the presentation of a quantification methodology for the control of the amount of superparamagnetic iron oxide nanoparticles (SPIONs) administered in biological materials by means of the ferromagnetic resonance technique (FMR) applied to studies both in vivo and in vitro. The in vivo study consisted in the analysis of the elimination and biodistribution kinetics of SPIONs after intravenous administration in Wistar rats. The results were corroborated by X-ray fluorescence. For the in vitro study, a quantitative analysis of the concentration of SPIONs bound to the specific AC133 monoclonal antibodies was carried out in order to detect the expression of the antigenic epitopes (CD133) in stem cells from human umbilical cord blood. In both studies FMR has proven to be an efficient technique for the SPIONs quantification per volume unit (in vivo) or per labeled cell (in vitro).

Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region

Background: Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region. Methodology/Principal Findings: Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a. Conclusions/Significance: The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.

HTLV-1 Tax Specific CD8+ T Cells Express Low Levels of Tim-3 in HTLV-1 Infection: Implications for Progression to Neurological Complications

The T cell immunoglobulin mucin 3 (Tim-3) receptor is highly expressed on HIV-1-specific T cells, rendering them partially ""exhausted'' and unable to contribute to the effective immune mediated control of viral replication. To elucidate novel mechanisms contributing to the HTLV-1 neurological complex and its classic neurological presentation called HAM/TSP (HTLV-1 associated myelopathy/tropical spastic paraparesis), we investigated the expression of the Tim-3 receptor on CD8(+) T cells from a cohort of HTLV-1 seropositive asymptomatic and symptomatic patients. Patients diagnosed with HAM/TSP down-regulated Tim-3 expression on both CD8(+) and CD4(+) T cells compared to asymptomatic patients and HTLV-1 seronegative controls. HTLV-1 Tax-specific, HLA-A*02 restricted CD8(+) T cells among HAM/TSP individuals expressed markedly lower levels of Tim-3. We observed Tax expressing cells in both Tim-3(+) and Tim-3(-) fractions. Taken together, these data indicate that there is a systematic downregulation of Tim-3 levels on T cells in HTLV-1 infection, sustaining a profoundly highly active population of potentially pathogenic T cells that may allow for the development of HTLV-1 complications.

Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor Fc gamma RIIB expression on B cells

Background: Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA) on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period. Results: Maternal immunization with OVA showed increased levels of Fc gamma RIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-gamma-secreting T cells and IL-4 and IL-12-secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specifc IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced Fc gamma RIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers. Conclusion: Maternal immunization upregulates the inhibitory Fc gamma RIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.

Adipose Tissue-Derived Stem Cells from Humans and Mice Differ in Proliferative Capacity and Genome Stability in Long-Term Cultures

Adipose tissue-derived stem cells (ASCs) are among the more attractive adult stem cell options for potential therapeutic applications. Here, we studied and compared the basic biological characteristics of ASCs isolated from humans (hASCs) and mice (mASCs) and maintained in identical culture conditions, which must be examined prior to considering further potential clinical applications. hASCs and mASCs were compared for immunophenotype, differentiation potential, cell growth characteristics, senescence, nuclear morphology, and DNA content. Although both strains of ASCs displayed a similar immunophenotype, the percentage of CD73(+) cells was markedly lower and CD31(+) was higher in mASC than in hASC cultures. The mean population doubling time was 98.08 +/- 6.15 h for hASCs and 52.58 +/- 3.74 h for mASCs. The frequency of nuclear aberrations was noticeably lower in hASCs than in mASCs regardless of the passage number. Moreover, as the cells went through several in vitro passages, mASCs showed changes in DNA content and cell cycle kinetics (frequency of hypodiploid, G0/G1, G2/M, and hyperdiploid cells), whereas all of these parameters remained constant in hASCs. Collectively, these results suggest that mASCs display higher proliferative capacity and are more unstable than hASCs in long-term cultures. These results underscore the need to consider specificities among model systems that may influence outcomes when designing potential human applications.

Development and mechanical testing of a short intramedullary nail for fixation of femoral rotational osteotomy in cerebral palsy patients

Background: Rotational osteotomy is frequently indicated to correct excessive femoral anteversion in cerebral palsy patients. Angled blade plate is the standard fixation device used when performed in the proximal femur, but extensile exposure is required for plate accommodation. The authors developed a short locked intramedullary nail to be applied percutaneously in the fixation of femoral rotational osteotomies in children with cerebral palsy and evaluated its mechanical properties. Methods: The study was divided into three stages. In the first part, a prototype was designed and made based on radiographic measurements of the femoral medullary canal of ten-year-old patients. In the second, synthetic femoral models based on rapid-prototyping of 3D reconstructed images of patients with cerebral palsy were obtained and were employed to adjust the nail prototype to the morphological changes observed in this disease. In the third, rotational osteotomies were simulated using synthetic femoral models stabilized by the nail and by the AO-ASIF fixed-angle blade plate. Mechanical testing was done comparing both devices in bending-compression and torsion. Results: The authors observed proper adaptation of the nail to normal and morphologically altered femoral models, and during the simulated osteotomies. Stiffness in bending-compression was significantly higher in the group fixed by the plate (388.97 +/- 57.25 N/mm) than in that fixed by the nail (268.26 +/- 38.51 N/mm) as torsional relative stiffness was significantly higher in the group fixed by the plate (1.07 +/- 0.36 Nm/degrees) than by the nail (0.35 +/- 0.13 Nm/degrees). Conclusions: Although the device presented adequate design and dimension to fit into the pediatric femur, mechanical tests indicated that the nail was less stable than the blade plate in bending-compression and torsion. This may be a beneficial property, and it can be attributed to the more flexible fixation found in intramedullary devices.