A New Species of Riama from Ecuador Previously Referred to as Riama hyposticta (Boulenger, 1902) (Squamata: Gymnophthalmidae)

We describe Riama crypta, new species, from the western slopes of the Cordillera Occidental, Ecuador. This taxon was formerly referred to as Riama hyposticta, a rare species described on the basis of an adult male from northern Ecuador and here recorded from southwestern Colombia. The new species differs principally from Riama hyposticta by an incomplete superciliary series, formed just by the anteriormost superciliary scale (superciliary series complete in R. hyposticta, formed by five or six scales), no nasoloreal suture [= loreal absent] (complete (= loreal present] in R. hyposticta), distinct dorsolateral stripes at least anteriorly (scattered brown spots dorsally without dorsolateral stripes in R. hyposticta), and ventral coloration composed of small cream or brown spots or longitudinal stripes (dark brown with conspicuous transverse white bars and spots). Additionally, we document the presence of distal filiform appendages on the hemipenial lobes of both species.

Endophytic and entomopathogenic strains of Beauveria sp to control the bovine tick Rhipicephalus (Boophilus) microplus

Pathogenicity of strains of the entomopathogenic fungus Beauveria bassiana and endophytic strains of Beauveria sp against the bovine tick Rhipicephalus (Boophilus) microplus was tested in laboratory bioassays and under field conditions. Suspensions containing 10(5), 10(7) and 10(9) conidia/mL were prepared of each fungal strain for laboratory bioassays. The ticks were maintained at 28 degrees C, 90 +/- 5% relative humidity, and the following variables were evaluated: initial female weight, egg weight, hatching percentage, reproductive efficiency, and percentage control. For tests under field conditions, a Beauveria suspension containing 10(6) conidia/mL was sprayed on tick-infested cows. After 72 h, the ticks were collected to estimate mortality under field conditions. Laboratory bioassays showed a mortality of 20 to 50% of the ticks seven days after inoculation with 10(7) Beauveria conidia/mL. Under field conditions 10(6) Beauveria conidia/mL induced 18-32% mortality. All Beauveria strains were effective in biological control of R. (Boophilus) microplus under laboratory and field test conditions. This is the first demonstration that endophytic fungi can be used for biological control of the cattle tick; this could help reduce environmental contamination by diminishing the need for chemical acaricides. Two endophytic strains were isolated from maize leaves and characterized by molecular sequencing of 5.8S rDNA ITS1 and ITS2 and morphological analyses of conidia. We found that these two endophytic Beauveria isolates, designated B95 and B157, are close to Beauveria amorpha.

CHARACTERIZATION OF THE TREE COMPONENT IN A SEMIDECIDUOUS FOREST IN THE ESPINHACO RANGE: A SUBSIDY TO CONSERVATION

This study was conducted in the Private Reserve Mata do Jambreiro (912 ha), localized in the Iron Quadrangle, Minas Gerais, southeastern portion of the Espinhaco Range, which is predominantly covered by semideciduous seasonal montane forest. Three topographically and physiognomic similar areas located within a continuum forest fragment, distant by 1.3 to 1.5 km were sampled by the point-quadrat method. In each area, 30 points were marked. Individuals with a minimum perimeter at the breast height (PBH) of 15 cm were sampled, totaling 111 species belonging to 40 families. The most representative family was Fabaceae, with 14.29% of the total number of species. Low floristic similarity (5.3% to 34.4%) was observed between the areas, pointing out the importance of distribution of sample units in continuous fragments. Shannon diversity index (H') found was 4.22 and Pielou equability (J) 0.894. Soil analysis showed some differences in chemical composition between the three studied areas and was an important component for the interpretation of the floristic variation found. The low floristic similarity observed here for close areas justify the requirement of more detailed inventories by Brazilian Environmental Agencies for the legal authorization procedures prior to the establishment of new enterprising projects. Also, the professionals that conduct rapid inventories, mainly the Environmental Consultants, should give more attention to this kind of floristic variation and to the methods used to inventory complex forests.

In situ delivery of bone marrow cells and mesenchymal stem cells improves cardiovascular function in hypertensive rats submitted to myocardial infarction

This work aimed to evaluate cardiac morphology/function and histological changes induced by bone marrow cells (BMCs) and cultured mesenchymal stem cells (MSCs) injected at the myocardium of spontaneously hypertensive rats (SHR) submitted to surgical coronary occlusion. Female syngeneic adult SHR, submitted (MI) or not (C) to coronary occlusion, were treated 24 h later with in situ injections of normal medium (NM), or with MSCs (MSC) or BMCs (BM) from male rats. The animals were evaluated after 1 and 30 days by echocardiography, histology of heart sections and PCR for the Y chromosome. Improved ejection fraction and reduced left ventricle infarcted area were observed in MSC rats as compared to the other experimental groups. Treated groups had significantly reduced lesion tissue score, increased capillary density and normal (not-atrophied) myocytes, as compared to NM and C groups. The survival rate was higher in C, NM and MSC groups as compared to MI and BM groups. In situ injection of both MSCs and BMCs resulted in improved cardiac morphology, in a more physiological model of myocardial infarction represented by surgical coronary occlusion of spontaneously hypertensive rats. Only treatment with MSCs, however, ameliorated left ventricle dysfunction, suggesting a positive role of these cells in heart remodeling in infarcted hypertensive subjects.

The beneficial effects of exercise in rodents are preserved after detraining: a phenomenon unrelated to GLUT4 expression

Background: Although exercise training has well-known cardiorespiratory and metabolic benefits, low compliance with exercise training programs is a fact, and the harmful effects of physical detraining regarding these adaptations usually go unnoticed. We investigated the effects of exercise detraining on blood pressure, insulin sensitivity, and GLUT4 expression in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Methods: Studied animals were randomized into sedentary, trained (treadmill running/5 days a week, 60 min/day for 10 weeks), 1 week of detraining, and 2 weeks of detraining. Blood pressure (tail-cuff system), insulin sensitivity (kITT), and GLUT4 (Western blot) in heart, gastrocnemius and white fat tissue were measured. Results: Exercise training reduced blood pressure (19%), improved insulin sensitivity (24%), and increased GLUT4 in the heart (+34%); gastrocnemius (+36%) and fat (+22%) in SHR. In WKY no change in either blood pressure or insulin sensitivity were observed, but there was an increase in GLUT4 in the heart (+25%), gastrocnemius (+45%) and fat (+36%) induced by training. Both periods of detraining did not induce any change in neither blood pressure nor insulin sensitivity in SHR and WKY. One-week detraining reduced GLUT4 in SHR (heart: -28%; fat: -23%) and WKY (heart: -19%; fat: -22%); GLUT4 in the gastrocnemius was reduced after a 2-week detraining (SHR: -35%; WKY: -25%). There was a positive correlation between GLUT4 (gastrocnemius) and the maximal velocity in the exercise test (r = 0.60, p = 0.004). Conclusions: The study findings show that in detraining, despite reversion of the enhanced GLUT4 expression, cardiorespiratory and metabolic beneficial effects of exercise are preserved.

Cellular Renewal and Improvement of Local Cell Effector Activity in Peritoneal Cavity in Response to Infectious Stimuli

The peritoneal cavity (PerC) is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p.) inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (Mempty set) subsets, namely small peritoneal Mempty set (SPM) and large peritoneal Mempty set (LPM), in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for beta-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO) and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-gamma stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal Mempty set subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

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.

The case of thyroid hormones: how to learn physiology by solving a detective case

Lellis-Santos C, Giannocco G, Nunes MT. The case of thyroid hormones: how to learn physiology by solving a detective case. Adv Physiol Educ 35: 219-226, 2011; doi:10.1152/advan.00135.2010.Thyroid diseases are prevalent among endocrine disorders, and careful evaluation of patients' symptoms is a very important part in their diagnosis. Developing new pedagogical strategies, such as problem-based learning (PBL), is extremely important to stimulate and encourage medical and biomedical students to learn thyroid physiology and identify the signs and symptoms of thyroid dysfunction. The present study aimed to create a new pedagogical approach to build deep knowledge about hypo-/hyperthyroidism by proposing a hands-on activity based on a detective case, using alternative materials in place of laboratory animals. After receiving a description of a criminal story involving changes in thyroid hormone economy, students collected data from clues, such as body weight, mesenteric vascularization, visceral fat, heart and thyroid size, heart rate, and thyroid-stimulating hormone serum concentration to solve the case. Nevertheless, there was one missing clue for each panel of data. Four different materials were proposed to perform the same practical lesson. Animals, pictures, small stuffed toy rats, and illustrations were all effective to promote learning, and the detective case context was considered by students as inviting and stimulating. The activity can be easily performed independently of the institution's purchasing power. The practical lesson stimulated the scientific method of data collection and organization, discussion, and review of thyroid hormone actions to solve the case. Hence, this activity provides a new strategy and alternative materials to teach without animal euthanization.

Respiratory allergy to Blomia tropicalis: Immune response in four syngeneic mouse strains and assessment of a low allergen-dose, short-term experimental model

Background: The dust mite Blomia tropicalis is an important source of aeroallergens in tropical areas. Although a mouse model for B. tropicalis extract (BtE)-induced asthma has been described, no study comparing different mouse strains in this asthma model has been reported. The relevance and reproducibility of experimental animal models of allergy depends on the genetic background of the animal, the molecular composition of the allergen and the experimental protocol. Objectives: This work had two objectives. The first was to study the anti-B. tropicalis allergic responses in different mouse strains using a short-term model of respiratory allergy to BtE. This study included the comparison of the allergic responses elicited by BtE with those elicited by ovalbumin in mice of the strain that responded better to BtE sensitization. The second objective was to investigate whether the best responder mouse strain could be used in an experimental model of allergy employing relatively low BtE doses. Methods: Groups of mice of four different syngeneic strains were sensitized subcutaneously with 100 mu g of BtE on days 0 and 7 and challenged four times intranasally, at days 8, 10, 12, and 14, with 10 mu g of BtE. A/J mice, that were the best responders to BtE sensitization, were used to compare the B. tropicalis-specific asthma experimental model with the conventional experimental model of ovalbumin (OVA)-specific asthma. A/J mice were also sensitized with a lower dose of BtE. Results: Mice of all strains had lung inflammatory-cell infiltration and increased levels of anti-BtE IgE antibodies, but these responses were significantly more intense in A/J mice than in CBA/J, BALB/c or C57BL/6J mice. Immunization of A/J mice with BtE induced a more intense airway eosinophil influx, higher levels of total IgE, similar airway hyperreactivity to methacholine but less intense mucous production, and lower levels of specific IgE, IgG1 and IgG2 antibodies than sensitization with OVA. Finally, immunization with a relatively low BtE dose (10 mu g per subcutaneous injection per mouse) was able to sensitize A/J mice, which were the best responders to high-dose BtE immunization, for the development of allergy-associated immune and lung inflammatory responses. Conclusions: The described short-term model of BtE-induced allergic lung disease is reproducible in different syngeneic mouse strains, and mice of the A/J strain was the most responsive to it. In addition, it was shown that OVA and BtE induce quantitatively different immune responses in A/J mice and that the experimental model can be set up with low amounts of BtE.

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD(+) kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems.

Evaluation of Sphingolipids in Wistar Rats Treated to Prolonged and Single Oral Doses of Fumonisin B(1)

The objective of the present study was to evaluate sphingolipid levels (sphingosine-So and sphinganine-Sa) and to compare the Sa/So ratio in liver, serum and urine of Wistar rats after prolonged administration (21 days) of fumonisin B(1) (FB(1)). In parallel, the kinetics of sphingolipid elimination in urine was studied in animals receiving a single dose of FB(1). Prolonged exposure to FB(1) caused an increase in Sa levels in urine, serum and liver. The most marked effect on sphingolipid biosynthesis was observed in animals treated with the highest dose of FB(1). Animals receiving a single dose of FB(1) presented variations in Sa and So levels and in the Sa/So ratio.

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale

Background: Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus) microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection. Results: Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells. Conclusions: Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.

Aspergillus nidulans as a biological system to detect the genotoxic effects of mercury fumes on eukaryotes

Mercury (Hg) pollution is one of the most serious environmental problems. Due to public concern prompted by the symptoms displayed by people who consumed contaminated fish in Minamata, Japan in 1956, Hg pollution has since been kept under constant surveillance. However, despite considerable accumulation of knowledge on the noxious effects of ingested or inhaled Hg, especially for humans, there is virtually nothing known about the genotoxic effects of Hg. Because increased mitotic crossing over is assumed to be the first step leading to carcinogenesis, we used a sensitive short-term test (homozygotization index) to look for DNA alterations induced by Hg fumes. In one Aspergillus nidulans diploid strain (UT448//UT184), the effects of the Hg fumes appeared scattered all over the DNA, causing 3.05 times more recombination frequencies than the mean for other strains. Another diploid (Dp II- I//UT184) was little affected by Hg. This led us to hypothesize that a genetic factor present in the UT184 master strain genome, close to the nicB8 genetic marker, is responsible for this behavior. These findings corroborate our previous findings that the homozygotization index can be used as a bioassay for rapid and efficient assessment of ecotoxicological hazards.

Var transcription profiling of Plasmodium falciparum 3D7: assignment of cytoadherent phenotypes to dominant transcripts

Background: Cytoadherence of Plasmodium falciparum-infected red blood cells is mediated by var gene-encoded P. falciparum erythrocyte membrane protein-1 and host receptor preference depends in most cases on which of the 50-60 var genes per genome is expressed. Enrichment of phenotypically homogenous parasites by panning on receptor expressing cells is fundamental for the identification of the corresponding var transcript. Methods: P. falciparum 3D7 parasites were panned on several transfected CHO-cell lines and their var transcripts analysed by i) reverse transcription/PCR/cloning/sequencing using a universal DBL alpha specific oligonucleotide pair and ii) by reverse transcription followed by quantitative PCR using 57 different oligonucleotide pairs. Results: Each cytoadherence selected parasite line also adhered to untransfected CHO-745 cells and upregulation of the var gene PFD995/PFD1000c was consistently associated with cytoadherence to all but one CHO cell line. In addition, parasites panned on different CHO cell lines revealed candidate var genes which reproducibly associated to the respective cytoadherent phenotype. The transcription profile obtained by RT-PCR/cloning/sequencing differed significantly from that of RT-quantitative PCR. Conclusion: Transfected CHO cell lines are of limited use for the creation of monophenotypic cytoadherent parasite lines. Nevertheless, 3D7 parasites can be reproducibly selected for the transcription of different determined var genes without genetic manipulation. Most importantly, var transcription analysis by RT-PCR/cloning/sequencing may lead to erroneous interpretation of var transcription profiles.

Effect of oat bran on time to exhaustion, glycogen content and serum cytokine profile following exhaustive exercise

The aim of this study was to evaluate the effect of oat bran supplementation on time to exhaustion, glycogen stores and cytokines in rats submitted to training. The animals were divided into 3 groups: sedentary control group (C), an exercise group that received a control chow (EX) and an exercise group that received a chow supplemented with oat bran (EX-O). Exercised groups were submitted to an eight weeks swimming training protocol. In the last training session, the animals performed exercise to exhaustion, (e.g. incapable to continue the exercise). After the euthanasia of the animals, blood, muscle and hepatic tissue were collected. Plasma cytokines and corticosterone were evaluated. Glycogen concentrations was measured in the soleus and gastrocnemius muscles, and liver. Glycogen synthetase-alpha gene expression was evaluated in the soleus muscle. Statistical analysis was performed using a factorial ANOVA. Time to exhaustion of the EX-O group was 20% higher (515 +/- 3 minutes) when compared with EX group (425 +/- 3 minutes) (p = 0.034). For hepatic glycogen, the EX-O group had a 67% higher concentrations when compared with EX (p = 0.022). In the soleus muscle, EX-O group presented a 59.4% higher glycogen concentrations when compared with EX group (p = 0.021). TNF-alpha was decreased, IL-6, IL-10 and corticosterone increased after exercise, and EX-O presented lower levels of IL-6, IL-10 and corticosterone levels in comparison with EX group. It was concluded that the chow rich in oat bran increase muscle and hepatic glycogen concentrations. The higher glycogen storage may improve endurance performance during training and competitions, and a lower post-exercise inflammatory response can accelerate recovery.