Inactivation Mechanism of the Fatty Acid Amide Hydrolase Inhibitor BIA 10-2474.

BIA 10-2474 is a time-dependent inhibitor of fatty acid amide hydrolase (FAAH) that was under clinical development for the treatment of neurological conditions when the program was terminated after one subject died and four were hospitalized with neurological symptoms during a first-in-human clinical study. The present work describes the mechanism of FAAH inhibition by BIA 10-2474 as a target-specific covalent inhibition, supported by quantum mechanics and molecular modelling studies. The inhibitor incorporates a weakly reactive electrophile which, upon specific binding to the enzyme's active site, is positioned to react readily with the catalytic residues. The reactivity is enhanced on-site by the increased molarity at the reaction site and by specific inductive interactions with FAAH. In the second stage, the inhibitor reacts with the enzyme's catalytic nucleophile to form a covalent enzyme-inhibitor adduct. The hydrolysis of this adduct is shown to be unlikely under physiological conditions, therefore leading to irreversible inactivation of FAAH. The results also reveal the important role played by FAAH Thr236 in the reaction with BIA 10-2474, which is specific to FAAH and is not present in other serine hydrolases. It forms a hydrogen bond with the imidazole nitrogen of the inhibitor and helps lowering the activation free energy of the first step of the reaction, by pre-orienting and stabilizing the inhibitor in a near-reactive configuration. In the second step, Thr236 can also serve as a mechanistic alternative to protonate the leaving group.

Epstein-Barr virus strains and variations: Geographic or disease-specific variants?

The Epstein-Barr Virus (EBV) is associated with the development of several diseases, including infectious mononucleosis (IM), Burkitt's Lymphoma (BL), Nasopharyngeal Carcinoma, and other neoplasias. The publication of EBV genome 1984 led to several studies regarding the identification of different viral strains. Currently, EBV is divided into EBV type 1 (B95-8 strain) and EBV type 2 (AG876 strain), also known as type A and type B, which have been distinguished based upon genetic differences in the Epstein-Barr nuclear antigens (EBNAs) sequence. Several other EBV strains have been described in the past 10 years considering variations on EBV genome, and many have attempted to clarify if these variations are ethnic or geographically correlated, or if they are disease related. Indeed, there is an increasing interest to describe possible specific disease associations, with emphasis on different malignancies. These studies aim to clarify if these variations are ethnic or geographically correlated, or if they are disease related, thus being important to characterize the epidemiologic genetic distribution of EBV strains on our population. Here, we review the current knowledge on the different EBV strains and variants and its association with different diseases. J. Med. Virol. 89:373-387, 2017. © 2016 Wiley Periodicals, Inc.

Low Sensitivity of the Mini-Mental State Examination for Cognitive Assessment of Brazilian Patients With Parkinson Disease.

INTRODUCTION: Recent publications have highlighted the low sensitivity of the Mini-Mental State Examination (MMSE) for the cognitive assessment of patients with Parkinson disease (PD). The Montreal Cognitive Assessment (MoCA), otherwise, has shown greater sensitivity when compared to the MMSE. Based on this, we have searched for the cognitive impairment measurable by the MoCA and the functional performance on activities of daily living in a sample of Brazilian patients with PD and normal MMSE. We hypothesized that the low sensitivity of the MMSE, already shown by other authors, could be replicated in a low-income country. OBJECTIVE: To describe the performance on the MoCA and the dependence on third parties for activities of daily living in a sample of Brazilian patients with PD and normal MMSE. METHODS: We evaluated 43 volunteers with PD and normal MMSE considering the Brazilian cutoffs. Cognitive performance was assessed through the MoCA and functional performance through a modified version of the Disability Assessment for Dementia Scale. RESULTS: Despite normal score on the MMSE, considering the Brazilian cutoffs, 62.7% of the volunteers performed below the literature cutoff for the MoCA (26 points). Furthermore, 30.2% had dependence on third party for activities of daily living. By using a strict cutoff for the MMSE (26 points), 56.7% performed below the MoCA cutoff and 24.3% had dependence for activities of daily living. CONCLUSIONS: Our findings confirm the limitations of the MMSE for the cognitive screening of patients with PD in a low-income country.

Acute effects of neural mobilization and infrared on the mechanics of the median nerve.

[Purpose] This study analyzed the acute effects of infrared and neural mobilization on the median nerve on the range of elbow extension of the dominant limb. [Subjects and Methods] Forty participants from university, neurologically asymptomatic, 12 males and 28 females (22.8 ± 1.9 years), were randomly divided into four groups: Group 1 (control) rested for 25 minutes in the supine position; Group 2 received the specific neural mobilization for the median nerve; Group 3 received an application of infrared for 15 minutes on the forearm; Group 4 received the same application of infrared followed by neural mobilization. The goniometric parameters of elbow extension were evaluated after the intervention. [Results] Significant differences of extension value were observed between Group 1 and Group 3 (15.75 degrees), and between Group 1 and Group 4 (14.60 degrees), and the average higher in Group 3 (26.35 degrees). [Conclusion] This research provides new experimental evidence that NM in relation to superficial heat produces an immediate effect on elbow range of motion versus NM isolated.

Characterization of Epstein-Barr virus strains and LMP1-deletion variants in Portugal.

Variations in the genome sequence of Epstein-Barr Virus (EBV) are thought to lead to differential interaction with host cells, immune evasion, and transformation. The discussion regarding EBV strains as having a geographic or disease-association has been increasing and the majority of studies are performed in Asiatic populations. We developed a case-control study with 139 individuals, including 96 subjects with different malignancies and 43 healthy individuals, from the North region of Portugal. We have used PCR protocols for the characterization of EBV strains (type A or B) based on EBNA3C genome variation and for the LMP1 30bp-deletion variants (wt-LMP1 or del-LMP1). Our study showed that type A is the most prevalent in our population (100% of healthy controls, 96.9% of aHSCT patients, 90.8% of HNSCC patients, and 94.9% of NPC patients) and that type B was significantly associated with NPC (P = 0.019; RR = 8.90). Regarding the LMP1 30bp-deletion, we found a similar distribution of both wt- and del-LMP1 variants in controls and dispare results in cases: del-LMP1 was more frequent in aHSCT and HNSCC patients (64.7% and 63.2%, respectively) and wt-LMP1 in NPC patients (100%). In fact, the study reveals that wt-LPM1 was significantly associated with NPC (P < 0.001; RR = 18.4). Hence, our study showed that EBV type B and wt-LMP1 variant seem to be associated with NPC in our population, with a clear disease-association for wt-LMP1. These results contribute for the knowledge of EBV genetic diversity among Caucasian populations.

Implementation of the Fast-track Protocol for Total Hip Arthroplasty in a Public Hospital in the State of São Paulo - Brazil.

Objective Evaluate the results of the implementation of the Fast Track Protocol (FTP), a medical practice based on scientific evidence, for elective total hip arthroplasty surgery, mainly comparing the National Average Hospital Admission Rate of 7.1 days. Methods 98 patients who underwent elective total hip arthroplasty surgery via the direct anterior approach, anterolateral approach and posterior approach were included in the FTP from December 2018 to March 2020, being followed up preoperatively, intraoperatively and immediately postoperatively. Results The average length of hospital stay was 2.8 days, being 2.1 days for the direct anterior approach, 3.0 days for the anterolateral access approach and 4.1 days for the posterior access approach. The average surgery time was 90 minutes, 19 (19.39%) of the patients were referred to the ICU in the postoperative period, however, none of them underwent surgery using the direct anterior approach. We had no cases of deep vein thrombosis (DVT), pulmonary embolism (PTE) or neurological injury, 19 (19.39%) patients had postoperative bleeding requiring dressing change, 4 (4.08%) needed blood transfusion, 2 (2.04%) patients had implant instability, 1 (1.02%) patient had a fracture during surgery and 1 (1.02%) patient died of cardiac complications. Conclusion FTP may be a viable alternative to reduce the length of stay and immediate postoperative complications for elective total hip arthroplasty surgery decreasing the length of stay of patients by 2 to 3 times when compared to the national average of 7.1 days.

Tumor-Infiltrating T Cells in Skin Basal Cell Carcinomas and Squamous Cell Carcinomas: Global Th1 Preponderance with Th17 Enrichment-A Cross-Sectional Study.

Basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) are high-incidence, non-melanoma skin cancers (NMSCs). The success of immune-targeted therapies in advanced NMSCs led us to anticipate that NMSCs harbored significant populations of tumor-infiltrating lymphocytes with potential anti-tumor activity. The main aim of this study was to characterize T cells infiltrating NMSCs. Flow cytometry and immunohistochemistry were used to assess, respectively, the proportions and densities of T cell subpopulations in BCCs (n = 118), SCCs (n = 33), and normal skin (NS, n = 30). CD8+ T cells, CD4+ T cell subsets, namely, Th1, Th2, Th17, Th9, and regulatory T cells (Tregs), CD8+ and CD4+ memory T cells, and γδ T cells were compared between NMSCs and NS samples. Remarkably, both BCCs and SCCs featured a significantly higher Th1/Th2 ratio (~four-fold) and an enrichment for Th17 cells. NMSCs also showed a significant enrichment for IFN-γ-producing CD8+T cells, and a depletion of γδ T cells. Using immunohistochemistry, NMSCs featured denser T cell infiltrates (CD4+, CD8+, and Tregs) than NS. Overall, these data favor a Th1-predominant response in BCCs and SCCs, providing support for immune-based treatments in NMSCs. Th17-mediated inflammation may play a role in the progression of NMSCs and thus become a potential therapeutic target in NMSCs.

Anticancer steroids: linking natural and semi-synthetic compounds.

Steroids, a widespread class of natural organic compounds occurring in animals, plants and fungi, have shown great therapeutic value for a broad array of pathologies. The present overview is focused on the anticancer activity of steroids, which is very representative of a rich structural molecular diversity and ability to interact with various biological targets and pathways. This review encompasses the most relevant discoveries on steroid anticancer drugs and leads through the last decade and comprises 668 references.

Identification of a new class of FtsZ inhibitors by structure-based design and in vitro screening.

The Filamenting temperature-sensitive mutant Z (FtsZ), an essential GTPase in bacterial cell division, is highly conserved among Gram-positive and Gram-negative bacteria and thus considered an attractive target to treat antibiotic-resistant bacterial infections. In this study, a new class of FtsZ inhibitors bearing the pyrimidine-quinuclidine scaffold was identified from structure-based virtual screening of natural product libraries. Iterative rounds of in silico studies and biological evaluation established the preliminary structure-activity relationships of the new compounds. Potent FtsZ inhibitors with low micromolar IC50 and antibacterial activity against S. aureus and E. coli were found. These findings support the use of virtual screening and structure-based design for the rational development of new antibacterial agents with innovative mechanisms of action.

What is better in TRAM flap survival: LLLT single or multi-irradiation?

Low-level laser therapy (LLLT) has been used with the aim of improving vascular perfusion of the skin and musculocutaneous flaps. This study evaluated the effect of LLLT on transverse rectus abdominis musculocutaneous flap (TRAM) viability, vascular angiogenesis, and VEGF release. Eighty-four Wistar rats were randomly divided into seven groups with 12 rats in each group. Group 1 received sham laser treatment; group 2, 3 J/cm(2) at 1 point; group 3, 3 J/cm(2) at 24 points; group 4, 72 J/cm(2) at 1 point; group 5, 6 J/cm(2) at 1 point; group 6, 6 J/cm(2) at 24 points; and group 7, 144 J/cm(2) at 1 point. All experimental groups underwent LLLT immediately after the TRAM operation and on the following 2 days; thus, animals underwent 3 days of treatment. The percentage of skin flap necrosis area was calculated on the fourth postoperative day using the paper template method, and two skin samples were collected using a 1-cm(2) punch to evaluate alpha smooth muscle actin (1A4) and VEGF levels in blood vessels. Significant differences were found in necrosis percentage, and higher values were seen in group 1 than in the other groups. Statistically significant differences were not found among groups 3 to 7 (p<0.292). Groups 5 and 7 showed significantly higher VEGF levels compared to other groups. Groups 3 and 5 had an increase in levels of blood vessels compared to other groups. LLLT at energy densities of 6 to 144 J/cm(2) was efficient to increase angiogenesis and VEGF levels and promote viability in TRAM flaps in rats.

Smoking induces increased apoptosis in osteoblasts: changes in bone matrix organic components.

Clinical studies demonstrate the impact of smoking on bone tissue fragility and higher incidence of fractures. However, it is not totally understood which physiological mechanisms could be involved in these events. Previously, we showed important changes in bone tissue components in experimental model of cigarette smoke (CS) exposure. CS exposure induces worsening in bone mineralization and a decrease in collagen type I deposition, leading to bone fragility. Considering that the majority of clinical studies described bone structural changes by radiographic images, in this study we performed analyses "in situ" using tissue samples from smokers, former smokers and non-smokers to better understand how the increase in inflammatory mediators induced by smoking exposure could interfere in bone cells activity leading bone structural changes. We observed increased levels of IL-1ß, IL-6 and TNF-α in bone tissue homogenates with a concomitant increase in osteoblast apoptosis in smokers and former smokers compared with non-smokers. Histological changes in both smokers and former smokers were characterized by reduction in collagen type I. Only in smokers, it was observed decrease in trabecular area, suggesting increased bone resorption and increase in collagen type V. These results showed that osteoblasts apoptosis in association with increased bone resorption leads bone structural changes in smokers.

Validation of the AmpC ß-lactamase binding site and identification of inhibitors with novel scaffolds.

AmpC ß-lactamase confers resistance to ß-lactam antibiotics in multiple Gram-negative bacteria. Therefore, identification of non-ß-lactam compounds that inhibit the enzyme is considered crucial to the development of novel antibacterial therapies. Given the highly solvent-exposed active site, it is important to study the induced-fit movements and water-mediated interactions to improve docking accuracy and virtual screening enrichments in structure-based design of new AmpC inhibitors. Here, we tested multiple models of the AmpC binding site to investigate the importance of conserved water molecules and binding site plasticity on molecular docking. The results indicate that at least one conserved water molecule greatly improves the binding pose predictions and virtual screening enrichments of known noncovalent AmpC inhibitors. The best model was tested prospectively in the virtual screening of about 6 million commercially available compounds. Sixty-one chemically diverse top-scoring compounds were experimentally tested, which led to the identification of seven previously unknown inhibitors. These findings validate the essential features of the AmpC binding site for molecular recognition and are useful for further optimization of identified inhibitors.

Docking and scoring with ICM: the benchmarking results and strategies for improvement.

Flexible docking and scoring using the internal coordinate mechanics software (ICM) was benchmarked for ligand binding mode prediction against the 85 co-crystal structures in the modified Astex data set. The ICM virtual ligand screening was tested against the 40 DUD target benchmarks and 11-target WOMBAT sets. The self-docking accuracy was evaluated for the top 1 and top 3 scoring poses at each ligand binding site with near native conformations below 2 Å RMSD found in 91 and 95% of the predictions, respectively. The virtual ligand screening using single rigid pocket conformations provided the median area under the ROC curves equal to 69.4 with 22.0% true positives recovered at 2% false positive rate. Significant improvements up to ROC AUC = 82.2 and ROC((2%)) = 45.2 were achieved following our best practices for flexible pocket refinement and out-of-pocket binding rescore. The virtual screening can be further improved by considering multiple conformations of the target.

Cancer mortality in the State of Mato Grosso from 2000 to 2015: temporal trend and regional differences.

OBJECTIVE: To analyze the trend of standardized cancer mortality rate in the state of Mato Grosso according to health regions, from 2000 to 2015. METHODS: Ecological time series study with data on deaths by cancer from the Mortality Information System. The rates were standardized using direct method and calculated by year and health regions. The annual percentage changes (APC) and respective confidence interval (95%CI) were obtained through simple linear regression. Thematic maps were built to show the spatial distribution of rates. RESULTS: There were 28,525 deaths by cancer registered in Mato Grosso, with the main types being lung, prostate, stomach, breast and liver cancer. The highest mortality rates were found in regions Médio Norte, Baixada Cuiabana and Sul Mato-Grossense. From 2000 to 2015, an upward trend was seen in the mortality rate by cancer in Mato Grosso (APC=0.81%; 95%CI 0.38-1.26), and in four health regions, Garças Araguaia (APC=2.27%; 95%CI 1.46-3.08), Sul Mato-Grossense (APC=1.12%; 95%CI 0.28-1.97), Teles Pires (APC=1.93%; 95%CI 0,11-3,74) and Vale dos Arinos (APC=2.61%; 95%CI 1.10-4.70), while the other regions remained stable. CONCLUSION: In the state of Mato Grosso and in the four health regions, cancer mortality rate showed a growing trend. The results point to the need to consider regional differences when thinking about actions for cancer prevention, control and assistance.

Incidence, mortality and survival of prostate cancer in two municipalities with a high human development index in Mato Grosso, Brazil.

OBJECTIVE: To analyze the incidence, mortality and survival of prostate cancer in Cuiabá and Várzea Grande, Brazil from 2000 to 2016. METHODS: Data from the Population-based Cancer Registry and the Mortality Information System were used. Mortality and incidence trends were analyzed using joinpoint regression models by age group. Survival analyses were performed using the Kaplan-Meier method, and hazard ratio was estimated by age group. RESULTS: From 2000 to 2016, 3,671 new cases and 892 deaths for prostate cancer were recorded. The average incidence and mortality rates were 87.96 and 20.22 per 100,000, respectively. Decreasing incidence trend was noted for all age groups from 2006 to 2016 (APC=-3.2%) and for men with 80+ years of age from 2000 to 2016 (APC=-3.0%), and increasing mortality trend for men 60-69 years of age from 2000 to 2009 (APC=3.2%). The specific five-year survival rate for prostate cancer was 79.6% (95%CI 77.2-81.9), and the rate decreased with advanced age (HR=2.43, 95%CI 1.5-3.9, for those 70 to 79 years old and HR=7.20, 95%CI 4.5-11.5, for those 80 or older). CONCLUSION: The incidence rate of prostate cancer showed a decreasing trend from 2006 for all age groups; the mortality rate was stable in that period, and worse prognosis was observed in men 70 years or older.

Cancer surveillance in Mato Grosso, Brazil: methodological and operational aspects of a university extension/research project.

OBJECTIVE: To describe the methodological and operational aspects of the "Project for surveillance of cancer and its associated factors: population-based and hospital-based registry" (VIGICAN), in the state of Mato Grosso (MT), Brazil. METHODS: VIGICAN was divided into two projects: a university extension one, which updated the data from the Population-based Cancer Registry (PBCR) of MT in the 2008-2016 period; and a research project, which collected primary data, through individual interviews and analysis of medical records of people with a diagnosis of cancer, aged 18 years or older, treated at reference hospitals for oncology. To analyze the factors associated with cancer, the following variables were collected: socioeconomic and demographic, social support, health status and behavior, and environmental exposure. RESULTS: In the 2008-2016 period, approximately one hundred thousand cases of cancer (incident and prevalent) were reported in the PBCR Cuiabá and PBCR Interior. After validation procedures, 50 thousand incident cases were elected. The survey interviewed 1,012 patients, 38.2% living in the municipalities of Cuiabá and Várzea Grande, 60.4% in small cities of the state, and 1.4% in other states. Preliminary data showed that the majority were women (55.0%) and younger than 60 years of age (54.3%). Among the interviewees, 7.2% reported smoking tobacco, 15.5% consumed alcoholic beverages (15.5%), and 32.7% lived nearby crops. CONCLUSION: The development of these projects allowed the integration of education with health services and will enable the recognition of specificities and different exposure scenarios and factors associated with cancer in the Mato Grosso territory.

Non-peptidic thrombospondin-1 mimics as fibroblast growth factor-2 inhibitors: an integrated strategy for the development of new antiangiogenic compounds.

Endogenous inhibitors of angiogenesis, such as thrombospondin-1 (TSP-1), are promising sources of therapeutic agents to treat angiogenesis-driven diseases, including cancer. TSP-1 regulates angiogenesis through different mechanisms, including binding and sequestration of the angiogenic factor fibroblast growth factor-2 (FGF-2), through a site located in the calcium binding type III repeats. We hypothesized that the FGF-2 binding sequence of TSP-1 might serve as a template for the development of inhibitors of angiogenesis. Using a peptide array approach followed by binding assays with synthetic peptides and recombinant proteins, we identified a FGF-2 binding sequence of TSP-1 in the 15-mer sequence DDDDDNDKIPDDRDN. Molecular dynamics simulations, taking the full flexibility of the ligand and receptor into account, and nuclear magnetic resonance identified the relevant residues and conformational determinants for the peptide-FGF interaction. This information was translated into a pharmacophore model used to screen the NCI2003 small molecule databases, leading to the identification of three small molecules that bound FGF-2 with affinity in the submicromolar range. The lead compounds inhibited FGF-2-induced endothelial cell proliferation in vitro and affected angiogenesis induced by FGF-2 in the chicken chorioallantoic membrane assay. These small molecules, therefore, represent promising leads for the development of antiangiogenic agents. Altogether, this study demonstrates that new biological insights obtained by integrated multidisciplinary approaches can be used to develop small molecule mimics of endogenous proteins as therapeutic agents.

EDU (5-Ethynyl-2'-Deoxyuridine)-Coupled Fluorescence-Intensity Analysis: Determining Absolute Parameters of the Cell Cycle.

The principles and practice of a methodology of cell cycle analysis that allows the estimation of the absolute length (in units of time) of all cell cycle stages (G1, S, and G2) are detailed herein. This methodology utilizes flow cytometry to take full advantage of the excellent stoichiometric properties of click chemistry. This allows detection, via azide-fluorochrome coupling, of the modified deoxynucleoside 5-ethynyl-2'-deoxyuridine (EDU) incorporated into replicated DNA through incremental pulsing times. This methodology, which we designated as EdU-Coupled Fluorescence Intensity (E-CFI) analysis, can be applied to cell types with very distinct cell cycle features, and has shown excellent agreement with established techniques of cell cycle analysis. Useful modifications to the original protocol (Pereira et al., Oncotarget, 8:40514-40,532, 2017) have been introduced to increase flexibility in data collection and facilitate data analysis.

Lead-DBS: an additional tool for stereotactic surgery.

OBJECTIVE: Use Lead-DBS software to analyze stereotactical surgical outcome of an operated population and demonstrate that small target deviations do not compromise the stimulation of desired structures, even with small amperages. METHODS: Image exams of patients submitted to deep brain stimulation for movement disorders treatment were processed in Lead-DBS software. Electrode stereotactic coordinates were subtracted from the planned target and those deviations, compared among different anatomical targets and sides operated firstly and secondly. We also quantified the frequency of relation between the activated tissue volume and the planned target through computer simulations. RESULTS: None of the 16 electrodes were exactly implanted at the planned coordinates. A stimulation of 3 mA reached 62.5% of the times the planned coordinates, rising to 68.75% with a 3,5 mA. No statistical significance was demonstrated in any comparison of laterality and anatomical sites. CONCLUSIONS: The simulation of small amperage fields could reach the intended target even when electrode placement is suboptimal. Furthermore, such a goal can be achieved without overlapping the volume of activated tissue with undesired structures. Software Lead-DBS proved to be a valuable complementary asset for surgical stereotactical result assessment.

A new microtubule-stabilizing agent shows potent antiviral effects against African swine fever virus with no cytotoxicity.

African swine fever virus (ASFV) is the causal agent of a fatal disease of domestic swine for which no effective antiviral drugs are available. Recently, it has been shown that microtubule-targeting agents hamper the infection cycle of different viruses. In this study, we conducted in silico screening against the colchicine binding site (CBS) of tubulin and found three new compounds with anti-ASFV activity. The most promising antiviral compound (6b) reduced ASFV replication in a dose-dependent manner (IC50 = 19.5 µM) with no cellular (CC50 > 500 µM) and animal toxicity (up to 100 mg/kg). Results also revealed that compound 6b interfered with ASFV attachment, internalization and egress, with time-of-addition assays, showing that compound 6b has higher antiviral effects when added within 2-8 h post-infection. This compound significantly inhibited viral DNA replication and disrupted viral protein synthesis. Experiments with ASFV-infected porcine macrophages disclosed that antiviral effects of the compound 6b were similar to its effects in Vero cells. Tubulin polymerization assay and confocal microscopy demonstrated that compound 6b promoted tubulin polymerization, acting as a microtubule-stabilizing, rather than a destabilizing agent in cells. In conclusion, this work emphasizes the idea that microtubules can be targets for drug development against ASFV.