Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

Background: Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS). Methods: In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in E. coli. Results: The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in E. coli showed that both were equally effective in terms of sensitivity and specificity. Conclusions: Our results therefore indicate that either of these proteins can be used in serological tests in Brazil.

Report of a chimeric origin of transposable elements in a bovine-coding gene

Despite the wide distribution of transposable elements (TEs) in mammalian genomes, part of their evolutionary significance remains to be discovered. Today there is a substantial amount of evidence showing that TEs are involved in the generation of new exons in different species. In the present study, we searched 22,805 genes and reported the occurrence of TE-cassettes in coding sequences of 542 cow genes using the RepeatMasker program. Despite the significant number (542) of genes with TE insertions in exons only 14 (2.6%) of them were translated into protein, which we characterized as chimeric genes. From these chimeric genes, only the FAST kinase domains 3 (FASTKD3) gene, present on chromosome BTA 20, is a functional gene and showed evidence of the exaptation event. The genome sequence analysis showed that the last exon coding sequence of bovine FASTKD3 is similar to 85% similar to the ART2A retrotransposon sequence. In addition, comparison among FASTKD3 proteins shows that the last exon is very divergent from those of Homo sapiens, Pan troglodytes and Canis familiares. We suggest that the gene structure of bovine FASTKD3 gene could have originated by several ectopic recombinations between TE copies. Additionally, the absence of TE sequences in all other species analyzed suggests that the TE insertion is clade-specific, mainly in the ruminant lineage.

Mutational analysis of genes p14ARF, p15INK4b, p16INK4a, and PTEN in human nervous system tumors

The cancer is one of the most common and severe problems in clinical medicine, and nervous system tumors represent about 2% of the types of cancer. The central role of the nervous system in the maintenance of vital activities and the functional consequences of the loss of neurons can explain how severe brain cancers are. The cell cycle is a highly complex process, with a wide number of regulatory proteins involved, and such proteins can suffer alterations that transform normal cells into malignant ones. The INK4 family members (CDK inhibitors) are the cell cycle regulators that block the progression of the cycle through the R point, causing an arrest in G1 stage. The p14ARF (alternative reading frame) gene is a tumor suppressor that inhibits p53 degradation during the progression of the cell cycle. The PTEN gene is related to the induction of growth suppression through cell cycle arrest, to apoptosis and to the inhibition of cell adhesion and migration. The purpose of the present study was to assess the mutational state of the genes p14ARF, p15INK4b, p16INK4a, and PTEN in 64 human nervous system tumor samples. Homozygous deletions were found in exon 2 of the p15INK4b gene and exon 3 of the p16INK4a gene in two schwannomas. Three samples showed a guanine deletion (63 codon) which led to a loss of heterozygosity in the p15 gene, and no alterations could be seen in the PTEN gene. Although the group of patients was heterogeneous, our results are in accordance with other different studies that indicate that homozygous deletion and loss of heterozygosity in the INK4 family members are frequently observed in nervous system tumors.

VIABILITY OF SPORULATED OOCYSTS OF NEOSPORA CANINUM AFTER EXPOSURE TO DIFFERENT PHYSICAL AND CHEMICAL TREATMENTS

The aim of the present study was to evaluate the viability of Neospora caninum sporulated oocysts after various chemical and physical treatments. Bioassays in gerbils and molecular techniques (PCR-RFLP) were used for identification of the oocysts shed by experimentally infected dogs. Sporulated oocysts were purified and divided into 11 treatment groups as follows: absolute ethanol for 1 hr; 20 C for 6 hr; 4 C for 6 hr; 60 C for 1 min; 100 C for 1 min; 10% formaldehyde for 1 hr; 10% ammonia for 1 hr; 2% iodine for 1 hr; 10% sodium hypochlorite for I hr; 70% ethanol for I hr; and one group was left untreated and kept as a positive control. All chemical treatments were performed at room temperature (37 C). A total of 33 gerbils, or 3 gerbils per treatment, were used for bioassays. After treatment, the oocysts were divided into aliquots of 1,000 oocysts and orally administered to gerbils. After 63 days, the gerbils were anesthetized and killed with 0.2 ml of T61; blood and tissue samples were collected for serological (IFAT and western blotting), molecular (real-time PCR), histopathology, and immunohistochemical tests. Treatments were considered effective only if all 5 detection techniques tested negative. High temperatures at 100 C for 1 min and 10% sodium hypochlorite for 1 hr were the only treatments that met this condition, effectively inactivating all oocysts.

Er : YAG laser, ultrasonic system, and curette produce different profiles on dentine root surfaces: An in vitro study

Objective: The aim of the present study was to compare the in vitro effects of the Er:YAG laser, an ultrasonic system, and manual curette on dentine root surface by roughness and micro-morphological analysis. Materials and Methods: Thirty-six flattened bovine roots were randomly assigned to one of the following groups: group 1 (n = 12): Er: YAG laser ( 2940 nm), 120 mJ/pulse, 10 Hz, 8.4 J/cm(2); group 2 ( n = 12): ultrasonic system; and group 3 ( n = 12): manual curette. The mean surface roughness (Ra) of each sample was measured using a profilometer before and after the treatments. The micro-morphology of the treated and untreated ( control) root surfaces was evaluated with scanning electron microscopy (SEM) at 50 x and 1000 x magnification. Results: Analysis with the profilometer showed that for equal times of instrumentation, the smoothest surfaces were produced by the Er: YAG laser and the ultrasonic system, followed by the curette ( p < 0.05). Morphological analyses demonstrated that treatment with the Er: YAG laser produced some areas with an irregular surface, craters, and ablation of the intertubular dentin. The smear layer was removed and dentine tubules were opened by both curettes and the ultrasonic system. The micro-morphology of the dentine root surface after ultrasonic treatment, however, demonstrated randomly distributed areas cratering. Conclusion: All instruments increased the roughness of the dentine root surface after treatment; however, the curette produced rougher surfaces than the other devices. SEM analysis revealed distinct root surface profiles produced by the three devices.

Adhesion of Endodontic Sealers to Human Root Dentine Submitted to Different Surface Treatments

Objective: To evaluate the adhesion of the endodontic sealers Epiphany, Apexit Plus, and AH Plus to root canal dentin submitted to different surface treatments, by using the push-out test. Methods: One hundred twenty-eight root cylinders obtained from maxillary canines were embedded in acrylic resin, had the canals prepared, and were randomly assigned to four groups (n = 32), according to root dentin treatment: (I) distilled water (control), (II) 17% EDTAC, (III) 1% NaOCl and (IV) Er:YAG laser with 16-Hz, 400-mJ input (240-mJ output) and 0.32-J/cm(2) energy density. Each group was divided into four subgroups (n = 8) filled with Epiphany (either dispensed from the automix syringe supplied by the manufacturer or prepared by hand mixing), Apexit Plus, or AH Plus. Data (MPa) were analyzed by ANOVA and Tukey's test. Results: A statistically significant difference (p < 0.01) was found among the root-canal sealers, except for the Epiphany subgroups, which had statistically similar results to each other (p > 0.01): AH Plus (4.77 +/- 0.85), Epiphany/hand mixed (3.06 +/- 1.34), Epiphany/automix syringe (2.68 +/- 1.35), and Apexit Plus (1.22 +/- 0.33). A significant difference (p < 0.01) was found among the dentin surface treatments. The highest adhesion values were obtained with AH Plus when root dentin was treated with Er: YAG laser and 17% EDTAC. Epiphany sealer presented the lowest adhesion values to root dentin treated with 17% EDTAC. Conclusions: The resin-based sealers had different adhesive behaviors, depending on the treatment of root canal walls. The mode of preparation of Epiphany (automix syringe or hand mixing) did not influence sealer adhesion to root dentin.

Ablation Rate and Morphology of Superficial and Deep Dentin Irradiated with Different Er:YAG Laser Energy Levels

Objective: In this study we evaluated the ablation rate of superficial and deep dentin irradiated with different Er:YAG laser energy levels, and observed the micromorphological aspects of the lased substrates with a scanning electron microscope (SEM). Background Data: Little is known about the effect of Er: YAG laser irradiation on different dentin depths. Materials and Methods: Sixty molar crowns were bisected, providing 120 specimens, which were randomly assigned into two groups ( superficial or deep dentin), and later into five subgroups (160, 200, 260, 300, or 360 mJ). Initial masses of the specimens were obtained. After laser irradiation, the final masses were obtained and mass losses were calculated followed by the preparation of specimens for SEM examination. Mass-loss values were subjected to two-way ANOVA and Fisher's least significant difference multiple-comparison tests (p < 0.05). Results: There was no difference between superficial and deep dentin. A significant and gradual increase in the mass-loss values was reached when energies were raised, regardless of the dentin depth. The energy level of 360 mJ showed the highest values and was statistically significantly different from the other energy levels. The SEM images showed that deep dentin was more selectively ablated, especially intertubular dentin, promoting tubule protrusion. At 360 mJ the micromorphological features were similar for both dentin depths. Conclusion: The ablation rate did not depend on the depth of the dentin, and an energy level lower than 360 mJ is recommended to ablate both superficial and deep dentin effectively without causing tissue damage.

Ultrastructural analysis of root canal dentine irradiated with 980-nm diode laser energy at different parameters

Objective: The purpose of this in vitro study was to investigate using the scanning electron microscope (SEM) the ultrastructural morphological changes of the radicular dentine surface after irradiation with 980-nm diode laser energy at different parameters and angles of incidence. Background Data: There have been limited reports on the effects of diode laser irradiation at 980 nm on radicular dentin morphology. Materials and Methods: Seventy-two maxillary canines were sectioned and roots were biomechanically prepared using K3 rotary instruments. The teeth were irrigated with 2 mL of distilled water between files and final irrigation was performed with 10 mL of distilled water. The teeth were then randomly divided into five groups (n = 8 each) according to their diode laser parameters: Group 1: no irradiation (control); group 2: 1.5 W/continuous wave (CW) emission (the manufacturer's parameters); group 3: 1.5 W/100 Hz; group 4: 3 W/CW; and group 5: 3 W/100 Hz. Laser energy was applied with helicoid movements (parallel to the canal walls) for 20 sec. Eight additional teeth for each group were endodontically prepared and split longitudinally and irradiation was applied perpendicularly to the root surface. Results: Statistical analysis showed no difference between the root canal thirds irradiated with the 980-nm diode laser, and similar results between the parameters 1.5 W/CW and 3 W/100 Hz (p > 0.05). Conclusion: When considering different output powers and delivery modes our results showed that changes varied from smear layer removal to dentine fusion.

Comparison of different models to estimate genetic parameters for carcass traits in a commercial broiler line

The effect of genetic and non-genetic factors for carcass, breast meat and leg weights, and yields of a commercial broiler line were investigated using the restricted maximum likelihood method, considering four different animal models, including or excluding maternal genetic effect with covariance between direct and maternal genetic effects, and maternal permanent environmental effect. The likelihood ratio test was used to determine the most adequate model for each trait. For carcass, breast, and leg weight, and for carcass and breast yield, maternal genetic and permanent environmental effects as well as the covariance between direct and maternal genetic effects were significant. The estimates of direct and maternal heritability were 0.17 and 0.04 for carcass weight, 0.26 and 0.06 for breast weight, 0.22 and 0.02 for leg weight, 0.32 and 0.02 for carcass yield, and 0.52 and 0.04 for breast yield, respectively. For leg yield, maternal permanent environmental effect was important, in addition to direct genetic effects. For that trait, direct heritability and maternal permanent environmental variance as a proportion of the phenotypic variance were 0.43 and 0.02, respectively. The results indicate that ignoring maternal effects in the models, even though they were of small magnitude (0.02 to 0.06), tended to overestimate direct genetic variance and heritability for all traits.

Preclinical Studies with Umbilical Cord Mesenchymal Stromal Cells in Different Animal Models for Muscular Dystrophy

Umbilical cord mesenchymal stromal cells (MSC) have been widely investigated for cell-based therapy studies as an alternative source to bone marrow transplantation. Umbilical cord tissue is a rich source of MSCs with potential to derivate at least muscle, cartilage, fat, and bone cells in vitro. The possibility to replace the defective muscle cells using cell therapy is a promising approach for the treatment of progressive muscular dystrophies (PMDs), independently of the specific gene mutation. Therefore, preclinical studies in different models of muscular dystrophies are of utmost importance. The main objective of the present study is to evaluate if umbilical cord MSCs have the potential to reach and differentiate into muscle cells in vivo in two animal models of PMDs. In order to address this question we injected (1) human umbilical cord tissue (hUCT) MSCs into the caudal vein of SJL mice; (2) hUCT and canine umbilical cord vein (cUCV) MSCs intra-arterially in GRMD dogs. Our results here reported support the safety of the procedure and indicate that the injected cells could engraft in the host muscle in both animal models but could not differentiate into muscle cells. These observations may provide important information aiming future therapy for muscular dystrophies.

Morphological and growth alterations on early development stages of Iridaea cordata (Rhodophyta) under different intensities of UVB radiation

This study describes the effects of different intensities of UVB radiation on growth and morphology of early development stages of Iridaea cordata in germlings, young gametophytes originated in the laboratory and young fronds collected in the Magellan Strait, Chile. The experiments were carried out during four weeks in controlled conditions of temperature and photoperiod and the results were compared with a control treatment (without UVB). All UVB irradiation treatments caused bleaching and decrease in growth rates of germlings. Additionally, initial upright fronds were not observed in any of the UVB treatments, where as those cultivated in UVB absence developed erect ones in the second week of culture. The young gametophytes exhibited morphological alteration (small number and size of basal ramifications, curling of tips, bleaching and necrosis) and decrease in growth when exposed to UVB radiation. Young fronds collected from the field showed mainly morphological alterations (curling of frond). Morphological alterations in young gametophytes and young fronds of I. cordata could be interpreted as a defense against UVB by reducing the area exposed to radiation. However, high level of UVB radiation can produce irreparable damage, such as necrosis, observed in young gametophytes originated in the laboratory. Finally, the UVB effects on early developmental stages of I. cordata depend on the UVB irradiance and time of exposition.

High Iodine Concentration Attenuates RET/PTC3 Oncogene Activation in Thyroid Follicular Cells

Background: Papillary thyroid carcinoma (PTC) is frequently associated with a RET gene rearrangement that generates a RET/PTC oncogene. RET/PTC is a fusion of the tyrosine kinase domain of RET to the 50 portion of a different gene. This fusion results in a constitutively active MAPK pathway, which plays a key role in PTC development. The RET/PTC3 fusion is primarily associated with radiation-related PTC. Epidemiological studies show a lower incidence of PTC in radiation-exposed regions that are associated with an iodine-rich diet. Since the influence of excess iodine on the development of thyroid cancer is still unclear, the aim of this study is to evaluate the effect of high iodine concentrations on RET/PTC3-activated thyroid cells. Methods: PTC3-5 cells, a rat thyroid cell lineage harboring doxycycline-inducible RET/PTC3, were treated with 10(-3) M NaI. Cell growth was analyzed by cell counting and the MTT assay. The expression and phosphorylation state of MAPK pathway-related (Braf, Erk, pErk, and pRet) and thyroid-specific (natrium-iodide symporter [Nis] and thyroid-stimulating hormone receptor [Tshr]) proteins were analyzed by Western blotting. Thyroid-specific gene expression was further analyzed by quantitative reverse transcription (RT)-polymerase chain reaction. Results: A significant inhibition of proliferation was observed, along with no significant variation in cell death rate, in the iodine-treated cells. Further, iodine treatment attenuated the loss of Nis and Tshr gene and protein expression induced by RET/PTC3 oncogene induction. Finally, iodine treatment reduced Ret and Erk phosphorylation, without altering Braf and Erk expression. Conclusion: Our results indicate an antioncogenic role for excess iodine during thyroid oncogenic activation. These findings contribute to a better understanding of the effect of iodine on thyroid follicular cells, particularly how it may play a protective role during RET/PTC3 oncogene activation.

The Spleen CD4(+) T Cell Response to Blood-Stage Plasmodium chabaudi Malaria Develops in Two Phases Characterized by Different Properties

The pivotal role of spleen CD4(+) T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4(+) T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4(+) T cells generated in CD1d(-/-) mice are I-A(b)-restricted (conventional cells), while their counterparts in I-Ab(-/-) mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4(+) T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4(+) T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-alpha and IFN-gamma production depend mostly on conventional CD4(+) T cells. IFN-gamma is produced simultaneously by non-conventional and conventional CD4(+) T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4(+) T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4(+) T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-gamma and parasite-specific antibody production. We concluded that conventional CD4(+) T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-conventional CD4(+) T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection.

Mycoflora and Co-Occurrence of Fumonisins and Aflatoxins in Freshly Harvested Corn in Different Regions of Brazil

Natural mycoflora and co-occurrence of fumonisins (FB(1), FB(2)) and aflatoxins (AFB(1), AFB(2), AFG(1) and AFG(2)) in freshly harvested corn grain samples from four regions of Brazil were investigated. Fusarium verticillioides was predominant in all samples. Analysis of fumonisins showed that 98% of the samples were contaminated with FB(1) and 74.5% with FB(1) + FB(2), with toxin levels ranging from 0.015 to 9.67 mu g/g for FB(1) and from 0.015 to 3.16 mu g/g for FB(2). Twenty-one (10.5%) samples were contaminated with AFB(1), seven (3.5%) with AFB(2) and only one (0.5%) with AFG(1) and AFG(2). Co-contamination with aflatoxins and fumonisins was observed in 7% of the samples. The highest contamination of fumonisins and aflatoxins was observed in Nova Odessa (SP) and Varzea Grande (MT), respectively. The lowest contamination of these mycotoxins was found in Varzea Grande and Nova Odessa, respectively.

Mycoplasma pneumoniae and/or Chlamydophila pneumoniae inoculation causing different aggravations in cholesterol-induced atherosclerosis in apoE KO male mice

Background: Chamydophila pneumoniae (CP) and/or Mycoplasma pneumoniae ( MP) are two bacteria detected in vulnerable atheromas. In this study we aimed to analyze whether CP and/or MP aggravates atherosclerosis induced by cholesterol-enriched diet in C57BL/6 apoE KO male mice. Thirty male apoE KO mice aged eight weeks fed by a diet containing 1% cholesterol until 32 weeks of age were divided into four groups: the first was inoculated with CP (n = 7), the second with MP (n = 12), the third with both CP + MP ( n = 5), and the fourth with saline (sham n = 6). The animals were re-inoculated at 36 weeks of age, and sacrificed at 40 weeks of age. Two ascending aorta and one aortic arch segments were sampled. In the most severely obstructed segment, vessel diameter, plaque height, percentage of luminal obstruction and the degree of adventitial inflammation were analyzed. The plaque area/intimal surface ratio was obtained by measuring all three segments. The adventitial inflammation was semiquantified (0 absent, 1 mild, 2 moderate, and 3 diffuse). Results: The mean and standard deviation of plaque height, % luminal obstruction, external diameter, the plaque area/intimal surface ratio and the adventitial inflammation values are the following for each group: MP (0.20 +/- 12 mm, 69 +/- 26%, 0.38 +/- 0.11 mm, 0.04 +/- 0.04 and 0.22 +/- 0.67), CP (0.23 +/- 0.08 mm, 90 +/- 26%, 0.37 +/- 0.08 mm, 0.04 +/- 0.03, and 0.44 +/- 0.53), MP + CP ( 18 +/- 0.08 mm, 84 +/- 4.0%, 0.35 +/- 0.25 mm, 0.03 +/- 0.03 and 1.33 +/- 0.82) and sham (0.08 +/- 0.09 mm, 42 +/- 46%, 0.30 +/- 0.10 mm, 0.02 +/- 0.03 and 0.71 +/- 0.76). A wider area of plaque/intimal surface was observed in MP + CP inoculated groups (p = 0.07 and 0.06) as well as an increased plaque height in CP (p = 0.01) in comparison with sham group. There was also an increased luminal obstruction (p = 0.047) in CP inoculated group in comparison to sham group. Adventitial inflammation in MP + CP inoculated group was higher than MP, CP and the sham groups (p = 0.02). Conclusion: Inoculation of CP, MP or both agents in C57BL/6 apoE KO male mice caused aggravation of experimental atherosclerosis induced by cholesterol-enriched diet, with distinct characteristics. CP inoculation increased the plaque height with positive vessel remodeling and co-inoculation of MP + CP caused the highest adventitial inflammation measures.