Molecular dynamics and protein frustration analysis of human fused in Sarcoma protein variants in Amyotrophic Lateral Sclerosis type 6: An In Silico approach.

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disorder. The disease is characterized by degeneration of upper and lower motor neurons, leading to death usually within five years after the onset of symptoms. While most cases are sporadic, 5%-10% of cases can be associated with familial inheritance, including ALS type 6, which is associated with mutations in the Fused in Sarcoma (FUS) gene. This work aimed to evaluate how the most frequent ALS-related mutations in FUS, R521C, R521H, and P525L affect the protein structure and function. We used prediction algorithms to analyze the effects of the non-synonymous single nucleotide polymorphisms and performed evolutionary conservation analysis, protein frustration analysis, and molecular dynamics simulations. Most of the prediction algorithms classified the three mutations as deleterious. All three mutations were predicted to reduce protein stability, especially the mutation R521C, which was also predicted to increase chaperone binding tendency. The protein frustration analysis showed an increase in frustration in the interactions involving the mutated residue 521C. Evolutionary conservation analysis showed that residues 521 and 525 of human FUS are highly conserved sites. The molecular dynamics results indicate that protein stability could be compromised in all three mutations. They also affected the exposed surface area and protein compactness. The analyzed mutations also displayed high flexibility in most residues in all variants, most notably in the interaction site with the nuclear import protein of FUS.

Neospora caninum DNA distribution in tissues of gerbils as experimental models of chronic neosporosis / [Distribuição do DNA de Neospora caninum em tecidos de gerbilos como modelos experimentais de neosporose crônica]

Neospora caninum is the main etiologic agent of neosporosis in domestic animals and its pathogenesis comprises two characteristic phases: acute and chronic. Rodents are used as experimental models to mimic acute and chronic bovine neosporosis. In this study, we inoculated a total of 27 female gerbils, with different doses of N. caninum tachyzoites aiming to induce chronic disease. DNA was extracted from different organs of each animal after spontaneous death or euthanasia. Encephalic tissues were submitted to a highly sensitive real time PCR aiming to detect chronically infected animals. All the other samples were submitted to standard PCR. A total of 11 gerbils died due to acute neosporosis, as confirmed by N. caninum DNA detection in organs. 5x103 tachyzoites/mL of N. caninum was the dosage of antigen that can induce chronic infection in gerbils. In the encephalon sections of some animals that showed clinical signs of persistent infection, we found 70% positive for the anterior encephalon section, suggesting this area as preferential for cyst formation. Therefore, we determined the doses of tachyzoites that cause acute or chronic infection and detection of positive tissues, preferably, systemic organs during acute and encephalon in chronic phases.(AU)

Neospora caninum é o principal agente etiológico da neosporose em animais domésticos, e sua patogênese compreende duas fases características: aguda e crônica. Roedores são usados como modelos experimentais para simular neosporose bovina aguda e crônica. Neste estudo, foi inoculado um total de 27 gerbilos, fêmeas, com diferentes doses de taquizoítos de N. caninum, visando induzir doença crônica. O DNA foi extraído de diferentes órgãos de cada animal após a morte espontânea ou a eutanásia. Os tecidos encefálicos foram submetidos à PCR em tempo real de alta sensibilidade para detecção de animais com infecção crônica. Todas as outras amostras foram submetidas à PCR padrão. Um total de 11 gerbilos morreu devido à neosporose aguda, como confirmado pela detecção de DNA de N. caninum nos órgãos. A dosagem de antígeno que pode induzir infecção crônica foi de 5x103 taquizoítos/mL de N. caninum. Em seções do encéfalo de alguns animais, que apresentaram sinais clínicos de infecção persistente, encontraram-se 70% de positividade para a seção do encéfalo anterior, sugerindo essa área como preferencial para a formação de cisto. Assim, foram determinadas,, em gerbilos, as dosagens de taquizoítos capazes de induzir infecção crônica ou aguda, bem como foram detectados tecidos positivos, preferencialmente, em órgãos sistêmicos, na fase aguda, e no encéfalo, na crônica.(AU)

Development and characterization of nanostructured lipid carrier-based gels for the transdermal delivery of donepezil.

Donepezil is one of the main compounds used in the therapy of Alzheimer's disease. Oral administration of this drug presents many drawbacks, resulting in treatment non-adherence among patients. Thus, the development of transdermal formulations for donepezil delivery is important. The aim of this study was to prepare and to evaluate nanostructured lipid carrier-based gels (NLC gel) able to improve the skin delivery of donepezil free base (DPB). The components of nanostructured lipid carriers (NLCs) were selected after evaluating their enhancing effects using in vitro DPB skin delivery assays. DPB-loaded NLC were prepared by a microemulsion technique, by employing stearic acid as a solid lipid, oleic acid as a liquid lipid, lecithin as a surfactant, and sodium taurodeoxycholate as a co-surfactant. The DPB-NLC dispersions were characterized morphologically using atomic force microscopy and physicochemically using dynamic light scattering and surface charge measurements. These data along, with the encapsulation studies, indicated that uniformly nano-sized particles with high drug encapsulation were fabricated. In vitro skin permeation assays were performed, and the results indicated that drug skin permeation from DPB-NLC gel was increased, not only by the enhancing effect of their components, but the lipid nanocarriers also presented an additional enhancing effect to increase drug flux across the skin. Therefore, DPB-NLC gel is an interesting formulation for the enhanced treatment of Alzheimer's disease.

In silico analysis and molecular dynamics simulation of human superoxide dismutase 3 (SOD3) genetic variants.

Oxidative stress is a major factor in aging processes. Superoxide dismutase 3 (SOD3) plays a key role in the protection of extracellular oxidative stress. Missense mutations in SOD3 have been described to be associated with the occurrence of pulmonary, cardiovascular, and neoplastic diseases. This study aims to analyze the effects of missense mutations on the SOD3 structure and function by modeling a complete SOD3 structure as well as analyzing the differences between the wild-types and mutants using computational simulations. Here, ten algorithms were used to predict the structural and functional effects of missense mutations. A complete model of SOD3 protein was made by ab initio and comparative modeling using the Rosetta algorithm and validated by PROCHECK, Verify 3D, QMEAN, and ProSa. Molecular dynamics (MD) simulations were performed and analyzed using the GROMACS package. The deleterious potential of the A58T and R231G mutants was not predicted by the majority of the used algorithms. The analyzed mutations were predicted as destabilizing by at least one algorithm. The MD analyses indicated that protein flexibility may be increased by all of the analyzed mutations, while the protein-ligand stability may be decreased. They also suggested that the variants A91T and R231G increase the overall dimensions of SOD3 and decrease its accessible surface area. Our findings, therefore, indicated that the analyzed mutations could affect the protein structure and its ability to interact with other molecules, which may be related to the functional impairment of SOD3 upon A58T and R231G mutations, as well as their involvement in pathologies.

Kinetics of acid-base parameters in conventional hemodialysis

Details about the acid-base changes in hemodialysis are scarce in the literature but are potentially relevant to adequate management of patients. We addressed the acid-base kinetics during hemodialysis and throughout the interdialytic period in a cross-sectional study of adults undergoing conventional hemodialysis. Samples for blood gas analysis were obtained from the arterial limb of the arteriovenous fistula before the first session of the week (HD1), immediately at the end of HD1, and on sequential collections at 15, 30, 45, 60, and 120 min post-HD1. Additional blood samples were collected after ∼20 h following the end of the first dialysis and immediately prior to the initiation of the second dialysis of the week. Thirty adult patients were analyzed (55±15 years, 50% men, 23% diabetic; dialysis vintage 69±53 months). Mean serum bicarbonate levels increased at the end of HD1 (22.3±2.7 mEq/L vs 17.5±2.3 mEq/L, P<0.001) and remained stable until 20 h after the end of the session. The mean values of pCO2 before HD1 were below reference and at 60 and 120 min post-HD1 were significantly lower than at the start (31.3±2.7 mmHg and 30.9±3.7 mmHg vs 34.3±4.1 mmHg, P=0.041 and P=0.010, respectively). The only point of collection in which mean values of pCO2 were above 35 mmHg was 20 h post-dialysis. Serum bicarbonate levels remained stable for at least 20 h after the dialysis sessions, a finding that may have therapeutic implications. During dialysis, the respiratory response for correction of metabolic acidosis (i.e., pCO2 elevation) was impaired.

Kinetics of acid-base parameters in conventional hemodialysis.

Details about the acid-base changes in hemodialysis are scarce in the literature but are potentially relevant to adequate management of patients. We addressed the acid-base kinetics during hemodialysis and throughout the interdialytic period in a cross-sectional study of adults undergoing conventional hemodialysis. Samples for blood gas analysis were obtained from the arterial limb of the arteriovenous fistula before the first session of the week (HD1), immediately at the end of HD1, and on sequential collections at 15, 30, 45, 60, and 120 min post-HD1. Additional blood samples were collected after ∼20 h following the end of the first dialysis and immediately prior to the initiation of the second dialysis of the week. Thirty adult patients were analyzed (55±15 years, 50% men, 23% diabetic; dialysis vintage 69±53 months). Mean serum bicarbonate levels increased at the end of HD1 (22.3±2.7 mEq/L vs 17.5±2.3 mEq/L, P<0.001) and remained stable until 20 h after the end of the session. The mean values of pCO2 before HD1 were below reference and at 60 and 120 min post-HD1 were significantly lower than at the start (31.3±2.7 mmHg and 30.9±3.7 mmHg vs 34.3±4.1 mmHg, P=0.041 and P=0.010, respectively). The only point of collection in which mean values of pCO2 were above 35 mmHg was 20 h post-dialysis. Serum bicarbonate levels remained stable for at least 20 h after the dialysis sessions, a finding that may have therapeutic implications. During dialysis, the respiratory response for correction of metabolic acidosis (i.e., pCO2 elevation) was impaired.

Diversity of Anopheles spp. (Diptera: Culicidae) in an Amazonian Urban Area.

The genus Anopheles encompasses several species considered as vectors of human infecting Plasmodium. Environmental changes are responsible for behavior changes in these vectors and therefore the pattern of malaria transmission. To better understand the dynamics of malaria transmission, this study aimed at identify the species of adult anophelines found in a malaria endemic urban area of the Amazon region, Mâncio Lima, located in the Acre State Brazil. Using Shannon-type light traps installed at 11 collection points near fish ponds, a total of 116 anophelines were collected belonging to nine species. Anopheles darlingi Root 1926 and An. albitarsis s.l. Lynch-Arribalzaga 1878 were the most abundant and predominant species. Despite the low number of captured adult anophelines, the occurrence of An. darlingi throughout all urban area and the presence of secondary vectors reinforce the need of a permanent and continuous entomological surveillance.

Effects of season on boar semen parameters and antioxidant enzymes in the south subtropical region in Brazil.

Although boar semen productivity is affected by seasonality, its effects are not equal among different regions which raise concerns regarding the profitability of boar stud farms. Therefore, the goals of this study were (i) to evaluate the seasonal effect on semen production in a commercial boar stud farm located in a subtropical climate region and (ii) to verify whether the activities of superoxide dismutase and glutathione peroxidase in spermatozoa and seminal plasma were associated with seminal traits of fresh and cooled semen. Nine boars were collected twice per season, and routine seminal parameter analyses were performed together with superoxide dismutase and glutathione peroxidase activities in seminal plasma and spermatozoa. Despite a reduction in sperm concentration in spring and summer, most seminal parameters were constant year-round. Temperature-humidity index was higher in the summer compared to spring, autumn and winter (p < .05). Superoxide dismutase activity in spermatozoa was increased in summer compared to autumn and winter (p < .05). The activities of both enzymes in seminal plasma and spermatozoa glutathione peroxidase remained unaltered throughout the seasons. In conclusion, seasonality showed little influence in overall boar seminal parameters despite microclimatic differences among seasons, and spermatozoa collected during summer increased superoxide dismutase activity.

Infrared thermography as a noninvasive method to assess scrotal insulation on sperm production in beef bulls.

This study evaluated the thermoregulation and spermatogenic changes by scrotal temperature gradient using infrared thermography in testicular compromised bulls. Bulls were insulated (n = 6) for 72 hr and control animals (n = 3) remained without insulation during all the experimental period. Seminal evaluation was performed prior, at insult removal and once per week for 13 consecutive weeks. Mean temperature gradient in insulated animals was lower at the time of insulation removal compared to the week prior and after the insult (p < .05). Two weeks after insult, sperm motility was lower in insulated compared to control animals (p < .01) and spermatozoa total defects were higher in insulated compared to control animals (p < .05). Two and seven weeks after insult, the major defects were higher in insulated compared to control animals (p < .05). Scrotal temperature gradient showed a positive correlation with sperm mass motion (p < .01) and a negative correlation with ocular globe temperature (p < .01) in insulated animals. The infrared thermography can be used to evaluate ocular globe temperature in bulls; however, it is only effective to detect changes in scrotal temperature gradient at the insult removal.

The involvement of growth hormone in equine oocyte maturation, receptor localization and steroid production by cumulus-oocyte complexes in vitro.

The objectives of this study were to evaluate the effects of equine growth hormone (eGH) on nuclear and cytoplasmic maturation of equine oocytes in vitro, steroid production by cumulus cells, and expression and subcellular localization of eGH-receptors (eGH-R) on equine ovarian follicles. Cumulus-oocyte complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries. The COCs were morphologically evaluated and randomly allocated to be cultured in either a control maturation medium or supplemented with 400 ng/mL eGH, for 30 h at 38.5°C in air with 5% CO2. The COCs were stained with 10 µg/mL propidium iodide and 10 µg/mL fluorescein isothiocyanate-labeled Lens culinaris agglutinin. Chromatin configuration and distribution of cortical granules were assessed via confocal microscopy. Compared to control, COCs incubated with eGH had: more oocytes that reached metaphase II (35/72, 48.6% vs. 60/89, 67.4%, respectively; P=0.02); greater concentrations of testosterone (0.21 ± 0.04 vs. 0.06 ± 0.01 ng/mL; P=0.01), progesterone (0.05 ± 0.01 vs. 0.02 ± 0.00 ng/mL; P=0.04), and oestradiol (76.80 ± 14.26 vs. 39.58 ± 8.87 pg/mL; P=0.05) in the culture medium, but no significant differences in concentration of androstenedione. Based on Real Time RT-PCR analyses, expression of the eGH-R gene was greater in cumulus cells and COCs at the start than at the end of in vitro maturation. Positive immunostaining for eGH-R was present in cumulus cells, the oocytes and granulosa cells. In conclusion, addition of eGH to maturation medium increased rates of cytoplasmic maturation and had an important role in equine oocyte maturation, perhaps mediated by the presence of eGH-R in ovarian follicles.

Influence of equine growth hormone, insulin-like growth factor-I and its interaction with gonadotropins on in vitro maturation and cytoskeleton morphology in equine oocytes.

In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E2), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles <30 mm in diameter and matured for 30 h at 38.5°C in air with 5% CO2. In experiment 1, selected cumulus-oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 µg/ml E2, 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-α-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO3-iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E2, gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P < 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E2, gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.

Elemental distribution images in prostate samples by X-ray fluorescence microtomography.

An X-ray transmission microtomography (CT) system combined with an X-ray fluorescence microtomography (XRFµCT) system was implemented in the Brazilian Synchrotron Light Laboratory (LNLS), in order to determine the elemental distribution in prostate samples aiming at establishing a correlation between the concentration of some elements and the characteristics and pathology of the tissues. The CT images were reconstructed using a filtered-back projection algorithm and the XRFµCT images were reconstructed using a filtered-back projection algorithm with absorption corrections.

Biological tissues analysis by XRF microtomography.

The main of this work is to determine the elemental distribution in breast and prostate tissue samples in order to verify the concentration of some elements correlated with characteristics and pathology of each tissue observed by the X-ray transmission microtomography (microCT). The experiments were performed at the X-ray fluorescence beamline of the Brazilian Synchrotron Light Laboratory. The microCT images were reconstructed using a filtered-back-projection algorithm and the XRF microtomographies were reconstructed using a filtered-back-projection algorithm with absorption corrections.

Elemental distribution mapping on breast tissue samples.

The main of this work is to determine the elemental and absorption distribution map in breast tissue samples by X-ray fluorescence and X-ray transmission microtomography. The experiments were performed at the X-ray fluorescence beamline (D09B-XRF) in the Brazilian Synchrotron Light Source (LNLS), Campinas, Brazil. A quasi-monochromatic beam produced by a multilayer monochromator was used as an incident beam. The sample was placed on a high precision goniometer and translation stages that allow rotating as well as translating it perpendicularly to the beam. The fluorescence photons were collected with an energy dispersive HPGe detector placed at 90 degrees to the incident beam, while transmitted photons were detected with a fast Na(Tl) scintillation counter () placed behind the sample on the beam direction. All the tomographic images were reconstructed using a filtered-back projection algorithm. In the breast tissue samples, it was possible to determine the distribution of Zn, Cu and Fe in healthy tissue and tumor.

Diffraction-enhanced imaging microradiography applied in breast samples.

The diffraction-enhanced imaging (DEI) is a powerful tool to observe tumors and other diseases in breast tissue and provide more precise diagnostics. In this work DEI was used to analyze breast tissues details that have poor attenuation contrast. An X-ray imaging system with DEI techniques was developed using synchrotron radiation. The DEI experiment was performed in D10A-XRD2 beamline at the Brazilian Synchrotron--LNLS. The pre-monochromator, upstream of the beamline was adjusted to 10.7 keV. The samples were positioned between two channel-cut Si(333) in non-dispersive geometry mounted in a double axes diffractometer. A direct conversion water-cooled CCD camera of 1242 pixel x 1152 pixel of 25 microm x 25 microm each was used as a two-dimensional detector in scanning mode. The DEI system could show details in low attenuation tissues based on the contrast imaging obtained by attenuation, refraction gradient and ultra-small angle scatter characteristics. In this work the capacity to observe different types of structures and details in breast tissues were investigated.

[Chagas' disease in a urban population of the health district of Rio Verde, Mato Grosso do Sul State, Brazil]. / Doença de Chagas na população urbana do distrito sanitário de Rio Verde, Mato Grosso do Sul, Brasil.

In order to evaluate some aspects of the Chagas' disease on urban population of the 12 municipalities of the sanitarian district of Rio Verde, Mato Grosso do Sul state, on 1998 we carried out a serological survey investigating anti-T. cruzi IgG by indirect immunofluorescence on blood samples collected on filter paper of 14,709 resident persons, confirmed by indirect hemagglutination and ELISA in serum. The parasitaemia was evaluated by indirect xenodiagnosis on 134 chagasic patients and the morbidity evaluated by anamnesis, physical exam and electrocardiogram in 191 pairs of chagasic/non-chagasic patients. The seropositivity was 1.83% (0.93% in autochtonous, 5.01% in alochtonous), xenodiagnosis was positive in 17.2% of the individuals (12.3% in autochtonous, 20.8% in alochtonous) and 24.6% of the seropositive patients presented chronic chagasic cardiopathy (19.1% in autochtonous, 27.8 in alochtonous). The analysis of the dates showed that alochtonous population is the main responsible for presence of the infection and morbidity of Chagas disease in the studied area.

Necrotizing enterocolitis in neonates with congenital heart disease: risk factors and outcomes.

OBJECTIVE: Necrotizing enterocolitis (NEC) is primarily a disease of the premature infant. Among children born at term, however, congenital heart disease may be an important predisposing factor for this condition. To determine risk factors for NEC in patients with congenital heart disease, we conducted a case-control study of neonates with cardiac disease admitted to the cardiac intensive care unit at our center during the 4-year period from January 1995 to December 1998. METHODS: Cardiac diagnosis and age at admission were analyzed for association with NEC among the 643-patient inception cohort. Demographic, preoperative, and operative variables were recorded retrospectively in 21 neonates with congenital heart disease who developed NEC and 70 control neonates matched by diagnosis and age at admission. Using parametric and nonparametric analysis, cases and controls were compared with respect to previously identified risk factors for NEC. RESULTS: Among the entire cohort of 643 neonates with heart disease admitted to the cardiac intensive care unit, diagnoses of hypoplastic left heart syndrome (odds ratio [OR] = 3.8 [1.6-9.1]) and truncus arteriosus or aortopulmonary window (OR = 6.3 [1.7-23.6]) were independently associated with development of NEC by multivariable analysis. In the case-control analysis, earlier gestational age at birth (36.7 +/- 2. 7 weeks vs 38.1 +/- 2.3 weeks), prematurity (OR = 3.9 [1.2-12.5]), highest dose of prostaglandin >0.05 microg/kg/minute (OR = 3.9 [1. 2-12.5]), and episodes of low cardiac output (meeting specific laboratory criteria) or clinical shock (OR = 6.5 [1.8-23.5]) correlated with the development of NEC. Earlier gestational age and episodes of low output were the only factors that remained significantly associated with NEC by multivariable analysis. Although there was no difference in hospital mortality between patients with and without NEC, mean hospital stay was significantly longer in those who developed NEC (36 +/- 22 days vs 19 +/- 14 days). CONCLUSIONS: The risk of NEC in neonates with congenital heart disease is substantial. Factors associated with an elevated risk of NEC in infants with heart disease include premature birth, hypoplastic left heart syndrome, truncus arteriosus, and episodes of poor systemic perfusion or shock. Heightened suspicion is warranted in newborns with these risk factors.

Effects of sweet taste stimulation on growth and sucking in preterm infants.

OBJECTIVE: To evaluate the effect of sweet taste stimulation in augmenting the reported growth-enhancing effects of nonnutritive sucking in preterm infants who are gavage-fed. DESIGN: Random assignment of preterm infants to receive stimulation by one of three methods during each feeding until totally orally fed. SETTING: Hospital intensive-care and infant transitional units. PATIENTS: Eligibility criteria included body weight greater than or equal to 1,250 g, gestational age younger than 34 weeks, growth parameters appropriate for gestational age, tolerating at least 100 kcals/kg/day by gavage feeding with evidence of weight gain, and no clinical evidence of health complications. Data are presented for 42 infants who completed 14 days of treatment. INTERVENTIONS: Exposure to a sweet pacifier, a latex pacifier, or maternal heartbeat sounds during gavage feedings. MAIN OUTCOME MEASURES: Growth, time to total oral feeding, and sucking responses. RESULTS: No significant differences in sucking measures were noted among treatment groups. Differences in progression time to total oral feedings and weight gain favored the sweet-pacifier group but were not statistically significant. CONCLUSIONS: Oral stimulation of gavage-fed, preterm infants during a 2-week hospitalization was not sufficient to elicit a significant improvement in growth efficiency, progression to total oral feedings, or sucking maturation. Additional studies may show a beneficial effect of chemosensory stimulation in preterm infants.