Resumo em Inglês:

Dermatophagoides farinae (Der f), one of the main species of house dust mites, produces more than 30 allergens. A recently identified allergen belonging to the alpha-tubulin protein family, Der f 33, has not been characterized in detail. In this study, we used bioinformatics tools to construct the secondary and tertiary structures and predict the B and T cell epitopes of Der f 33. First, protein attribution, protein patterns, and physicochemical properties were predicted. Then, a reasonable tertiary structure was constructed by homology modeling. In addition, six B cell epitopes (amino acid positions 34–45, 63–67, 103–108, 224–230, 308–316, and 365–377) and four T cell epitopes (positions 178–186, 241–249, 335–343, and 402–410) were predicted. These results established a theoretical basis for further studies and eventual epitope-based vaccine design against Der f 33.

Resumo em Inglês:

The aim of this study was to find related pathogenic genes in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy in (CADASIL)-like patients. The direct sequencing and high-throughput multiplex polymerase chain reaction (PCR) was performed to screen for related genes. The clinical and imaging data of a CADASIL-like patient (the pro-band) and his family members were collected. At first, the known hereditary cerebral vascular genes of the pro-band were screened with direct sequencing to find candidate gene mutations. High-throughput multiplex PCR was then used to analyze the single nucleotide polymorphism of the candidate gene in the family members. The results showed that there was missense mutation of the high temperature requirement protease A1 (HTRA1) gene in the pro-band, which may be a pathogenic factor according to the biological software analysis. The following SNP results revealed that the other family members also had the HTRA1 gene mutation. Thus, the CADASIL-like family disease may be caused by heterozygous HTRA1 gene mutation, which leads to autosomal dominant hereditary cerebral small vessel disease.

Resumo em Inglês:

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

Resumo em Inglês:

This study aimed to investigate the protective effect of salvinorin A on the cerebral pial artery after forebrain ischemia and explore related mechanisms. Thirty Sprague-Dawley rats received forebrain ischemia for 10 min. The dilation responses of the cerebral pial artery to hypercapnia and hypotension were assessed in rats before and 1 h after ischemia. The ischemia reperfusion (IR) control group received DMSO (1 µL/kg) immediately after ischemia. Two different doses of salvinorin A (10 and 20 µg/kg) were administered following the onset of reperfusion. The 5th, 6th, and 7th groups received salvinorin A (20 µg/kg) and LY294002 (10 µM), L-NAME (10 μM), or norbinaltorphimine (norBIN, 1 μM) after ischemia. The levels of cGMP in the cerebrospinal fluid (CSF) were also measured. The phosphorylation of AKT (p-AKT) was measured in the cerebral cortex by western blot at 24 h post-ischemia. Cell necrosis and apoptosis were examined by hematoxylin-eosin staining (HE) and TUNEL staining, respectively. The motor function of the rats was evaluated at 1, 2, and 5 days post-ischemia. The dilation responses of the cerebral pial artery were significantly impaired after ischemia and were preserved by salvinorin A treatment. In addition, salvinorin A significantly increased the levels of cGMP and p-AKT, suppressed cell necrosis and apoptosis of the cerebral cortex and improved the motor function of the rats. These effects were abolished by LY294002, L-NAME, and norBIN. Salvinorin A preserved cerebral pial artery autoregulation in response to hypercapnia and hypotension via the PI3K/AKT/cGMP pathway.

Resumo em Inglês:

2-Methyl-2-butanol (MBT) is a chemical compound from the group of alcohols more specifically pentanols, which has shown an excellent anti-cancer activity in our previous study. However, its mechanism of action remains unclear. The present study was designed to investigate the anti-cancer effect of MBT on human retinoblastoma cells. The results showed that the use of MBT leads to HXO-RB44 cell death but is cytotoxic to normal cells at higher concentrations. It showed a dose- as well as a time-dependent inhibition of HXO-RB44 cells. P27 is a cell cycle inhibitory protein, which plays an important role in cell cycle regulation whereas cyclin-B1 is a regulatory protein involved in mitosis. MBT increased the cell cycle arrest in a dose-dependent manner by augmenting p27 and reducing cyclin B1 expression. Moreover, it also accelerated apoptosis, increased light chain-3 (LC-3) conversion in a dose-dependent manner, and helped to debulk cancerous cells. LC3 is a soluble protein, which helps to engulf cytoplasmic components, including cytosolic proteins and organelles during autophagy from autophagosomes. In order to verify the effect of MBT, bafilomycin A1, an autophagy inhibitor, was used to block the MTB-induced apoptosis and necrosis. Additionally, a specific Akt agonist, SC-79, reversed the MBT-induced cell cycle arrest and autophagy. Thus, from the present study, it was concluded that MBT induced cell cycle arrest, apoptosis and autophagy through the PI3K/Akt pathway in HXO-RB44 cells.

Resumo em Inglês:

We aimed to outline the profile of medical professionals in Brazil who have violated the deontological norms set forth in the ethics code of the profession, and whose cases were judged by the higher tribunal for medical ethics between 2010 and 2016. This survey was conducted using a database formed from professional ethics cases extracted from the plenary of the medical ethics tribunal of the Federal Council of Medicine. These were disciplinary ethics cases that were judged at appeal level between 2010 and 2016. Most of these professionals were male (88.5%) and their mean age was 59.9 years (SD=11.62) on the date of judgment of their appeals, ranging from 28 to 95 years. Most of them were based in the southeastern region of Brazil (50.89%). Articles 1 and 18 of the medical ethics code were the rules most frequently violated. The sentence given most often was the cancellation of their professional license (37.6%) and the acts most often sentenced involved malpractice, imprudence, and negligence (18.49%). It is acknowledged that concern for the principles of bioethics was present in the appeal decisions made by the plenary of the medical ethics tribunal of the Federal Council of Medicine.

Resumo em Inglês:

Gastroschisis (GS) is an abdominal wall defect that results in histological and morphological changes leading to intestinal motility perturbation and impaired absorption of nutrients. Due to its anti-inflammatory, antioxidant, and neuroprotective effects, cannabidiol (CBD) has been used as a therapeutic agent in many diseases. Our aim was to test the effect of maternal CBD in the intestine of an experimental model of GS. Pregnant rats were treated over 3 days with CBD (30 mg/kg) after the surgical induction of GS (day 18.5 of gestation) and compared to controls. Fetuses were divided into 4 groups: 1) control (C); 2) C+CBD (CCBD); 3) gastroschisis (G), and 4) G+CBD (GCBD). On day 21.5 of gestation, the fetuses were harvested and evaluated for: a) body weight (BW), intestinal weight (IW), and IW/BW ratio; b) histometric analysis of the intestinal wall; c) immunohistochemically analysis of inflammation (iNOS) and nitrite/nitrate level. BW: GCBD was lower than CCBD (P<0.005), IW and IW/BW ratio: GCBD was smaller than G (P<0.005), GCBD presented lower thickness in all parameters compared to G (P<0.005), iNOS and nitrite/nitrate were lower concentration in GCBD than to G (P<0.005). Maternal use of CBD had a beneficial effect on the intestinal loops of GS with decreased nitrite/nitrate and iNOS expression.

Resumo em Inglês:

Human pluripotent stem cells (hPSCs)/OP9 coculture system is a widely used hematopoietic differentiation approach. The limited understanding of this process leads to its low efficiency. Thus, we used single-cell qPCR to reveal the gene expression profiles of individual CD34+ cells from different stages of differentiation. According to the dynamic gene expression of hematopoietic transcription factors, we overexpressed specific hematopoietic transcription factors (Gata2, Lmo2, Etv2, ERG, and SCL) at an early stage of hematopoietic differentiation. After overexpression, we generated more CD34+ cells with normal expression level of CD43 and CD31, which are used to define various hematopoietic progenitors. Furthermore, these CD34+ cells possessed normal differentiation potency in colony-forming unit assays and normal gene expression profiles. In this study, we demonstrated that single-cell qPCR can provide guidance for optimization of hematopoietic differentiation and transient overexpression of selected hematopoietic transcription factors can enhance hematopoietic differentiation.

Resumo em Inglês:

Data on the association between subclinical thyroid dysfunction and coronary artery disease (CAD) is scarce. We aimed to analyze the association between thyroid function and CAD using baseline data from the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). We included subjects with normal thyroid function (0.4-4.0 mIU/L, and normal free thyroxine, FT4, or 0.8 to 1.9 ng/dL), subclinical hypothyroidism (SCHypo; TSH>4.0 mIU/L and normal FT4), and subclinical hyperthyroidism (SCHyper; TSH<0.4 mIU/L and normal FT4) evaluated by coronary computed tomography angiography. We excluded individuals using medications that interfere in thyroid function or with past medical history of cardiovascular disease. Logistic regression models evaluated the presence of CAD, segment involvement score (SIS) >4, and segment severity score (SSS) >4 of coronary arteries as the dependent variables, and quintiles of TSH and FT4 as the independent variables, adjusted for demographical data and cardiovascular risk factors. We included 767 subjects, median age 58 years (IQR=55-63), 378 (49.3%) women, 697 euthyroid (90.9%), 57 (7.4%) with SCHypo, and 13 (1.7%) with SCHyper. No association between TSH and FT4 quintiles and CAD prevalence was noted. Similarly, no association between TSH levels and the extent or severity of CAD, represented by SIS>4 and SSS>4 were seen. Restricting analysis to euthyroid subjects did not alter the results. TSH levels were not significantly associated with the presence, extent, or severity of CAD in a middle-aged healthy population.

Resumo em Inglês:

MicroRNAs play a crucial role in the progression of spinal cord ischemia/reperfusion injury (SCII). The role of miR-448 and SIRT1 in SCII was investigated in this study, to provide further insights into prevention and improvement of this disorder. In this study, expressions of miR-448 and SIRT1 protein were determined by qRT-PCR and western blot, respectively. Flow cytometry was used to analyze cell apoptosis. The endogenous expression of genes was modulated by recombinant plasmids and cell transfection. Dual-luciferase reporter assay was performed to determine the interaction between miR-448 and SIRT1. The Basso, Beattie, and Bresnahan score was used to measure the hind-limb function of rat. The spinal cord ischemia reperfusion injury model of adult rats was developed by abdominal aorta clamping, and the nerve function evaluation was completed by motor deficit index score. In SCII tissues and cells treated with hypoxia, miR-448 was up-regulated while SIRT1 was down-regulated. Hypoxia treatment reduced the expression of SIRT1 through up-regulating miR-448 in nerve cells. Up-regulation of miR-448 induced by hypoxia promoted apoptosis of nerve cells through down-regulating SIRT1. Down-regulated miR-448 improved neurological function and hind-limb motor function of rats with SCII by up-regulating SIRT1. Down-regulated miR-448 inhibited apoptosis of nerve cells and improved neurological function by up-regulating SIRT1, which contributes to relieving SCII.

Resumo em Inglês:

Reactive oxygen species and lipid peroxidation are important factors that contribute to the development of age-related cataract. The study included 130 patients with age-related cataract, 69 of whom were diagnosed with hypertension (HT), 20 with hypertension and type 2 diabetes mellitus (DM), and 41 had no accompanying condition. The following parameters were measured in the serum of the examinees: products of lipid peroxidation malondialdehyde (MDA) and lipofuscin-like fluorophores (LLF), activity of prooxidative enzymes xanthine oxidase (XO) and myeloperoxidase (MPO), antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), the concentration of thiol groups, and the ferric reducing activity of plasma. The activity of prooxidative enzymes XO and MPO was higher in the plasma of patients with HT (XO=9.0±1.2 U/L; MPO=77.3±8.4 U/L) and with HT and DM (XO=11.9±0.9 U/L; MPO=89.5±5.0 U/L) compared to patients with age-related cataract (XO=6.2±0.9 U/L; MPO=52.4±6.3 U/L; P<0.01). Our research has shown that patients with age-related cataract and hypertension were exposed to increased oxidative damage of biomolecules, based on the increased plasma LLF and MDA content and decreased levels of thiol groups. Oxidative changes of biomolecules in these patients were associated with increased activity of the XO, MPO, and GPx enzymes and a lower extracellular SOD activity and total ferric reductive ability of plasma.

Resumo em Inglês:

Spirometry has been used as the main strategy for assessing ventilatory changes related to occupational exposure to particulate matter (OEPM). However, in some cases, as one of its limitations, it may not be sensitive enough to show abnormalities before extensive damage, as seen in restrictive lung diseases. Therefore, we hypothesized that cardiopulmonary exercise testing (CPET) may be better than spirometry to detect early ventilatory impairment caused by OEPM. We selected 135 male workers with at least one year of exposure. After collection of self-reported socioeconomic status, educational level, and cardiovascular risk data, participants underwent spirometry, CPET, body composition assessment (bioelectrical impedance), and triaxial accelerometry (for level of physical activity in daily life). CPET was performed using a ramp protocol on a treadmill. Metabolic, cardiovascular, ventilatory, and submaximal relationships were measured. We compared 52 exposed to 83 non-exposed workers. Multiple linear regressions were developed using spirometry and CPET variables as outcomes and OEPM as the main predictor, and adjusted by the main covariates. Our results showed that OEPM was associated with significant reductions in peak minute ventilation, peak tidal volume, and breathing reserve index. Exposed participants presented shallower slope of ΔVT/ΔlnV̇E (breathing pattern), i.e., increased tachypneic breathing pattern. The OEPM explained 7.4% of the ΔVT/ΔlnV̇E variability. We found no significant influence of spirometric indices after multiple linear regressions. We conclude that CPET might be a more sensitive feature of assessing early pulmonary impairment related to OEPM. Our cross-sectional results suggested that CPET is a promising tool for the screening of asymptomatic male workers.

Resumo em Inglês:

The purpose of this study was to look at the determinants of the unsteady walking (UW) symptom in patients with type 2 diabetes mellitus (T2DM) by defining if UW and/or the Diabetic Neuropathy Symptoms Score (DNSS) are associated with positive scores in Beck's Depression Inventory (BDI) and with a positive Michigan Neuropathy Screening Instrument score (MNSI). We evaluated 203 T2DM patients without visible gait disturbances. They were divided into UW (+) and UW (−) or DNSS (+) and DNSS (−) according to symptoms. We found a prevalence of 48.3% for UW (+) and of 63% for DNSS (+) in our sample. In univariate analysis, the presence of UW was significantly associated with waist circumference (P=0.024), number of comorbidities (P=0.012), not practicing physical exercise (P=0.011), positive BDI score (P=0.003), presence of neuropathic symptoms by the MNSI questionnaire (P<0.001), and positive diabetic neuropathy screening by MNSI (P=0.021). In multivariate analysis, UW (used as a dependent variable) was independently associated with a positive BDI score (P<0.001; 95%CI=1.01-1.03), T2DM duration (P=0.023; 95%CI=1.00–1.03), number of co-morbidities (P=0.032; 95%CI=1.01–1.37), and a sedentary lifestyle (P=0.025; 95%CI=1.06–2.5). The UW symptom and a positive DNSS are more closely related to a positive score for depression than to presence of neuropathy in T2DM.

Resumo em Inglês:

Trypanosoma cruzi triggers a progressive inflammatory response affecting cardiovascular functions in humans and experimental models. Angiotensin II, a key effector of the renin-angiotensin system, plays roles in mediating hypertension, heart failure, and inflammatory responses. T. cruzi and AngII can induce inflammatory responses by releasing inflammatory mediators. The aim of this study was to evaluate systemic AngII, tumor necrosis factor (TNF), and CX3CL1 mediators in a two-kidney one-clip (2K1C) renovascular hypertension model using Wistar rats infected with T. cruzi. Our data showed an increase in serum AngII in uninfected and T. cruzi-infected rats 1 week after 2K1C surgery compared to non-2K1C (Sham) animals. The baseline systolic blood pressure was higher in both uninfected and infected 2K1C rats. Despite no difference in circulating parasites in the acute phase of infection, elevated serum TNF and CX3CL1 were observed at 8 weeks post-infection in 2K1C rats in association with higher cardiac inflammatory infiltration. In summary, AngII-induced hypertension associated with T. cruzi infection may act synergistically to increase TNF and CX3CL1 in the 2K1C rat model, thereby intensifying cardiac inflammatory infiltration and worsening the underlying inflammation triggered by this protozoan.

Resumo em Inglês:

Cell adhesion in three-dimensional scaffolds plays a key role in tissue development. However, stem cell behavior in electrospun scaffolds under perfusion is not fully understood. Thus, an investigation was made on the effect of flow rate and shear stress, adhesion time, and seeding density under direct perfusion in polycaprolactone electrospun scaffolds on human dental pulp stem cell detachment. Polycaprolactone scaffolds were electrospun using a solvent mixture of chloroform and methanol. The viable cell number was determined at each tested condition. Cell morphology was analyzed by confocal microscopy after various incubation times for static cell adhesion with a high seeding density. Scanning electron microscopy images were obtained before and after perfusion for the highest flow rate tested. The wall pore shear stress was calculated for all tested flow rates (0.005–3 mL/min). An inversely proportional relationship between adhesion time with cell detachment under perfusion was observed. Lower flow rates and lower seeding densities reduced the drag of cells by shear stress. However, there was an operational limit for the lowest flow rate that can be used without compromising cell viability, indicating that a flow rate of 0.05 mL/min might be more suitable for the tested cell culture in electrospun scaffolds under direct perfusion.

Resumo em Inglês:

Bacterial infections occur worldwide and are a major public health problem. Among pathogens, Staphylococcus aureus is the main causative agent of bacterial diseases in the world. This study aimed to evaluate which components of the immune system could act protectively against a S. aureus infection in intradermally immunized mice. C57BL/6 and A/j mice were immunized intradermally with S. aureus inactivated by heat and then challenged with viable strains in an air pouch model. At 6, 12, and 24 h after the challenge, euthanasia was performed, and the cellular profile of the inflammatory infiltrate, cytokines, and the bacterial load were evaluated in the air pouch lavages. Immunized mice demonstrated that the intradermal immunization with S. aureus promoted protection in C57BL/6 mice by reducing the bacterial, which was correlated with increased serum concentration of IgG antibodies (IgG1 and IgG2a) against S. aureus. The increase in IgG2a antibody levels was correlated with a decrease of bacterial load in intradermally immunized C57BL/6 mice, along with production of IL-17A at the inflammation site, as well as IgG1consumption. Similar results were not found in the A/j lineage. In conclusion, a vaccine against S. aureus should focus more on the individual characteristics of the host because it is a determinant factor for the success of the immunization.

Resumo em Inglês:

The aim of this study was to test the hypothesis that reduced pre-exercise carbohydrate (CHO) availability potentiates fat oxidation after an exhaustive high-intensity exercise bout. Eight physically active men underwent a high-intensity exercise (∼95% V̇O2max) until exhaustion under low or high pre-exercise CHO availability. The protocol to manipulate pre-exercise CHO availability consisted of a 90-min cycling bout at ∼70% V̇O2max + 6 × 1-min at 125% V̇O2max with 1-min rest, followed by 48 h under a low- (10% CHO, low-CHO availability) or high-CHO diet (80% CHO, high-CHO availability). Time to exhaustion was shorter and energy expenditure (EE) lower during the high-intensity exercise in low- compared to high-CHO availability (8.6±0.8 and 11.4±1.6 min, and 499±209 and 677±343 kJ, respectively, P<0.05). Post-exercise EE was similar between low- and high-CHO availability (425±147 and 348±54 kJ, respectively, P>0.05), but post-exercise fat oxidation was significantly higher (P<0.05) in low- (7,830±1,864 mg) than in high-CHO availability (6,264±1,763 mg). The total EE (i.e., exercise EE plus post-exercise EE) was similar between low- and high-CHO availability (924±264 and 1,026±340 kJ, respectively, P>0.05). These results suggest that a single bout of high-intensity exercise performed under low-CHO availability increased post-exercise fat oxidation, and even with shorter exercise duration, both post-exercise EE and total EE were not impaired.

Resumo em Inglês:

The aim of this study was to investigate the effect of lactatemia elevation and glycemia reduction on strenuous swimming performance in fasted rats. Three rats were placed in a swimming tank at the same time. The first rat was removed immediately (control group) and the remaining ones were submitted to a strenuous swimming session. After the second rat was exhausted (Exh group), the third one was immediately removed from the water (Exe group). According to the period of time required for exhaustion, the rats were divided into four groups: low performance (3–7 min), low-intermediary performance (8–12 min), high-intermediary performance (13–17 min), and high performance (18–22 min). All rats were removed from the swimming tanks and immediately killed by decapitation for blood collection or anesthetized for liver perfusion experiments. Blood glucose, lactate, and pyruvate concentrations, blood lactate/pyruvate ratio, and liver lactate uptake and its conversion to glucose were evaluated. Exhaustion in low and low-intermediary performance were better associated with higher lactate/pyruvate ratio. On the other hand, exhaustion in high-intermediary and high performance was better associated with hypoglycemia. Lactate uptake and glucose production from lactate in livers from the Exe and Exh groups were maintained. We concluded that there is a time sequence in the participation of lactate/pyruvate ratio and hypoglycemia in performance during an acute strenuous swimming section in fasted rats. The liver had an important participation in preventing hyperlactatemia and hypoglycemia during swimming through lactate uptake and its conversion to glucose.

Resumo em Inglês:

Persistent human papillomavirus (HPV) infection is an essential factor of cervical cancer. This study evaluated the analytical performance of restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) assay compared to PapilloCheck® microarray to identify human papilloma virus (HPV) in cervical cells. Three hundred and twenty-five women were analyzed. One sample was used for conventional cytology and another sample was collected using BD SurePath™ kit for HPV tests. Eighty samples (24.6%) were positive for HPV gene by PCR-Multiplex and were then submitted to PCR-RFLP and PapilloCheck® microarray. There was a genotyping agreement in 71.25% (57/80) on at least one HPV type between PCR-RFLP and PapilloCheck® microarray. In 22 samples (27.5%), the results were discordant and those samples were additionally analyzed by DNA sequencing. HPV 16 was the most prevalent HPV type found in both methods, followed by HPVs 53, 68, 18, 39, and 66 using PCR-RFLP analysis, and HPVs 39, 53, 68, 56, 31, and 66 using PapilloCheck® microarray. In the present study, a perfect agreement using Cohen's kappa (κ) was found in HPV 33 and 58 (κ=1), very good for HPV 51, and good for types 16, 18, 53, 59, 66, 68, 70, and 73. PCR-RFLP analysis identified only 25% (20/80) HPV coinfection, and PapilloCheck® microarray found 62.5% (50/80). Our Cohen's kappa results indicate that our in-house HPV genotyping testing (PCR-RFLP analysis) could be applied as a primary HPV test screening, especially in low income countries. If multiple HPV types are found in this primary test, a more descriptive test, such as PapilloCheck® microarray, could be performed.