Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP)

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.     

Deforestation and Reforestation of Latin America and the Caribbean (2001–2010)

Forest cover change directly affects biodiversity, the global carbon budget, and ecosystem function. Within Latin American and the Caribbean region (LAC), many studies have documented extensive deforestation, but there are also many local studies reporting forest recovery. These contrasting dynamics have been largely attributed to demographic and socio‐economic change. For example, local population change due to migration can stimulate forest recovery, while the increasing global demand for food can drive agriculture expansion. However, as no analysis has simultaneously evaluated deforestation and reforestation from the municipal to continental scale, we lack a comprehensive assessment of the spatial distribution of these processes. We overcame this limitation by producing wall‐to‐wall, annual maps of change in woody vegetation and other land‐cover classes between 2001 and 2010 for each of the 16,050 municipalities in LAC, and we used nonparametric Random Forest regression analyses to determine which environmental or population variables best explained the variation in woody vegetation change. Woody vegetation change was dominated by deforestation (?541,835 km²), particularly in the moist forest, dry forest, and savannas/shrublands biomes in South America. Extensive areas also recovered woody vegetation (+362,430 km²), particularly in regions too dry or too steep for modern agriculture. Deforestation in moist forests tended to occur in lowland areas with low population density, but woody cover change was not related to municipality‐scale population change. These results emphasize the importance of quantitating deforestation and reforestation at multiple spatial scales and linking these changes with global drivers such as the global demand for food.     

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C–mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca²⁺–CaM, Ca²⁺–CaM–PDE1A, and Ca²⁺–CaM–MLCK complexes, CPZ apparent dissociation constants (K_ds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC K_ds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca²⁺–hCaM M124C–mBBr quenched the fluorescence (K_d = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca²⁺–hCaM M124C–mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca²⁺–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca²⁺–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd.     

Richness and composition of Calliphoridae in an Atlantic Forest fragment: implication for the use of dipteran species as bioindicators

Studies measuring the damage in degraded environments have increased, and it is necessary to obtain reliable biological and ecological indicators for the recovery of such degraded environments and subsequent preservation. This study aimed to conduct a survey of local insects and evaluate their utility in monitoring forest restoration in a degraded area in the Atlantic Forest biome in Ribeirao Grande, State of São Paulo, Brazil. Collections with Shuey traps were conducted from 2006 to 2011 in four distinct forest fragments characterized by environmental impacts, human actions, and phytophysiognomic profiles: Forest (FO), a preserved area with native plant species; “Capoeira” (CA), an area in the natural regeneration process; Planting (PL), an area reforested with native plants; and Pasture (PA), an area with only shrubs. A total of 2,456 specimens of Calliphoridae (Diptera) were collected. Mesembrinella bellardiana was the most abundant (n = 1,884) and dominant species in all environments sampled. The relative abundance of M. bellardiana in the most preserved environment (60.2 % FO, 24.4 % CA, 8.6 % PL, and 6.7 % PA) and other ecological parameters showed that it could be a bioindicator species, i.e., data on its presence or absence directly reflected the status of local preservation. Information on Neotropical dipterans associated with the forested environment is very scarce in the literature. To our knowledge, this is the first study describing the occurrence of Calliphora lopesi (Calliphoridae) in the State of São Paulo.     

Diurnal Variation in Oral Temperature,Sleepiness, and Performance of High School Girls

Evaluation of rhythmic fluctuations of physical and mental variables should be of special significance for the understanding of students' performance and setting the schedules of school activities. The present study investigated the pattern of diurnal variation in oral temperature, sleepiness and performance of a group of adolescents undergoing a daytime school schedule. Eighteen girls (mean age 16 years-old), who attended the same class from 0715h to 1645h, were tested on seven days. They measured their oral temperature, quantified their sleepiness level by means of a visual analogue scale, and completed the following tests: letter cancellation test, addition test, and a simple motor task. One-way ANOVA statistics for repeated measures was used in order to verify the effect of test time on oral temperature, sleepiness and performance. Possible correlations between the level of sleepiness and performance were investigated by means of Spearman rank correlation. The results revealed significant time of day effect on all variables, except for the number of addition errors. Oral temperature values showed an increase from morning to afternoon. Letter cancellation, motor task and addition scores increased from early morning to late afternoon, showing rapid fluctuations throughout the day. Sleepiness level was negatively correlated with letter cancellation scores during the first three tests of the day. In agreement with other work, the diurnal variation of oral temperature, letter cancellation and addition test showed an improvement as the day progressed. Sleepiness, on the other hand, decreased throughout the day, with the highest level associated with the first test of the day, suggesting a circadian pattern of variation rather than a cumulative effect due to school activities.     

Simulation of the inhibition of microbial sulfate reduction in a two-compartment upflow bioreactor subjected to molybdate injection

Souring of oil fields during secondary oil recovery by water injection occurs mainly due to the action of sulfate-reducing bacteria (SRB) adhered to the rock surface in the vicinity of injection wells. Upflow packed-bed bioreactors have been used in petroleum microbiology because of its similarity to the oil field near the injection wells or production. However, these reactors do not realistically describe the regions near the injection wells, which are characterized by the presence of a saturated zone and a void region close to the well. In this study, the hydrodynamics of the two-compartment packing-free/packed-bed pilot bioreactor that mimics an oil reservoir was studied. The packed-free compartment was modeled using a continuous stirred tank model with mass exchange between active and stagnant zones, whereas the packed-bed compartment was modeled using a piston-dispersion-exchange model. The proposed model adequately represents the hydrodynamic of the packed-free/packed-bed bioreactor while the simulations provide important information about the characteristics of the residence time distribution (RTD) curves for different sets of model parameters. Simulations were performed to represent the control of the sulfate-reducing bacteria activity in the bioreactor with the use of molybdate in different scenarios. The simulations show that increased amounts of molybdate cause an effective inhibition of the souring sulfate-reducing bacteria activity.     

OpenZika: An IBM World Community Grid Project to Accelerate Zika Virus Drug Discovery

The Zika virus outbreak in the Americas has caused global concern. To help accelerate this fight against Zika, we launched the OpenZika project. OpenZika is an IBM World Community Grid Project that uses distributed computing on millions of computers and Android devices to run docking experiments, in order to dock tens of millions of drug-like compounds against crystal structures and homology models of Zika proteins (and other related flavivirus targets). This will enable the identification of new candidates that can then be tested in vitro, to advance the discovery and development of new antiviral drugs against the Zika virus. The docking data is being made openly accessible so that all members of the global research community can use it to further advance drug discovery studies against Zika and other related flaviviruses.The Zika virus (ZIKV) has emerged as a major public health threat to the Americas as of 2015 [1]. We have previously suggested that it represents an opportunity for scientific collaboration and open scientific exchange [2]. The health of future generations may very well depend on the decisions we make, our willingness to share our findings quickly, and open collaboration to rapidly find a cure for this disease. Since February 1, 2016, when the World Health Organization deemed the cluster of microcephaly cases, Guillain-Barré, and other neurological disorders associated with ZIKV in Latin America and the Caribbean as constituting a Public Health Emergency of International Concern [3] (PHEIC), we have seen a rapid increase in publications (S1 References and main references). We [2] and others [4,5] described steps that could be taken to initiate a drug discovery program on ZIKV. For example, computational approaches, such as virtual screening of chemical libraries or focused screening to repurpose FDA and/or EU-approved drugs, can be used to help accelerate the discovery of an anti-ZIKV drug. An antiviral drug discovery program can be initiated using structure-based design, based on homology models of the key ZIKV proteins. With the lack of structural information regarding the proteins of ZIKV, we built homology models for all the ZIKV proteins, based on close homologs such as dengue virus, using freely available software [6] (S1 Table). These were made available online on March 3, 2016. We also predicted the site of glycosylation of glycoprotein E as Asn154, which was recently experimentally verified [7].Since the end of March 2016, we have now seen two cryo-EM structures and 16 crystal structures of five target classes (S1 Table). These structures, alongside the homology models, represent potential starting points for docking-based virtual screening campaigns to help find molecules that are predicted to have high affinity with ZIKV proteins. These predictions can then be tested against the virus in cell-based assays and/or using individual protein-based assays. There are millions of molecules available that can be assayed, but which ones are likely to work, and how should we prioritize them?In March, we initiated a new open collaborative project called OpenZika (Fig 1), with IBM’s World Community Grid (WCG, worldcommunitygrid.org), which has been used previously for distributed computing projects (S2 Table). On May 18, 2016, the OpenZika project began the virtual screening of ~6 million compounds that are in the ZINC database (Fig 1), as well as the FDA-approved drugs and the NIH clinical collection, using AutoDock Vina and the homology models and crystal structures (S1 Table, S1 Text, S1 References), to discover novel candidate compounds that can potentially be developed into new drugs for treating ZIKV. These will be followed by additional virtual screens with a new ZINC library of ~38 million compounds, and the PubChem database (at most ~90 million compounds), after their structures are prepared for docking.Open in a separate windowFig 1Workflow for the OpenZika project.A. Docking input files of the targets and ligands are prepared, and positive control docking studies are performed. The crystallographic binding mode of a known inhibitor is shown as sticks with dark purple carbon atoms, while the docked binding mode against the NS5 target from HCV has cyan carbons. Our pdbqt files of the libraries of compounds we screen are also openly accessible (http://zinc.docking.org/pdbqt/). B. We have already prepared the docking input files for ~6 million compounds from ZINC (i.e., the libraries that ALP previously used in the GO Fight Against Malaria project on World Community Grid), which are currently being used in the initial set of virtual screens on OpenZika. C. IBM’s World Community Grid is an internet-distributed network of millions of computers (Mac, Windows, and Linux) and Android-based tablets or smartphones in over 80 countries. Over 715,000 volunteers donate their dormant computer time (that would otherwise be wasted) towards different projects that are both (a) run by an academic or nonprofit research institute, and (b) are devoted to benefiting humanity. D. OpenZika is harnessing World Community Grid to dock millions of commercially available compounds against multiple ZIKV homology models and crystal structures (and targets from related viruses) using AutoDock Vina (AD Vina). This ultimately produces candidates (virtual hits that produced the best docking scores and displayed the best interactions with the target during visual inspection) against individual proteins, which can then be prioritized for in vitro testing by collaborators. After it is inspected, all computational data against ZIKV targets will be made open to the public on our website (http://openzika.ufg.br/experiments/#tab-id-7), and OpenZika results are also available upon request. The computational and experimental data produced will be published as quickly as possible.Initially, compounds are being screened against the ZIKV homologs of drug targets that have been well-validated in research against dengue and hepatitis C viruses, such as NS5 and Glycoprotein E (S1 Table, S1 Text, S1 References). These may allow us to identify broad-spectrum antivirals against multiple flaviviruses, such as dengue virus, West Nile virus, and yellow fever virus. In addition, docking against the crystal structure of a related protein from a different pathogen can sometimes discover novel hits against the pathogen of interest [8].As well as applying docking-based filters, the compounds virtually screened on OpenZika will also be filtered using machine learning models (S1 Text, S1 References). These should be useful selection criteria for subsequent tests by our collaborators in whole-cell ZIKV assays, to verify their antiviral activity for blocking ZIKV infection or replication. Since all OpenZika docking data will be in the public domain soon after they are completed and verified, we and other labs can then advance the development of some of these new virtual candidates into experimentally validated hits, leads, and drugs through collaborations with wet labs.This exemplifies open science, which should help scientists around the world as they address the long and arduous process of discovering and developing new drugs. Screening millions of compounds against many different protein models in this way would take far more resources and time than any academic researcher could generally obtain or spend. As of August 16, 2016, we have submitted 894 million docking jobs. Over 6,934 CPU years have been donated to us, enabling over 439 million different docking jobs. We recently selected an initial batch of candidates for NS3 helicase (data openly available at http://openzika.ufg.br/experiments/#tab-id-7), for in vitro testing. Without the unique community of volunteers and tremendous resources provided by World Community Grid, this project would have been very difficult to initiate in a reasonable time frame at this scale.The OpenZika project will ultimately generate several billion docking results, which could make it the largest computational drug discovery project ever performed in academia. The potential challenges we foresee will be finding laboratories with sufficient funding to pursue compounds, synthesize analogs, and develop target-based assays to validate our predictions and generate SAR (Structure-Activity Relationship) data to guide the process of developing the new hits into leads and then drugs. Due to the difficult nature of drug discovery and the eventual evolution of drug resistance, funding of ZIKV research once initiated will likely need to be sustained for several years, if not longer (e.g., HIV research has been funded for decades). As with other WCG projects, once scientists identify experimentally validated leads, finding a company to license them and pursue them in clinical trials and beyond will need incentives such as the FDA Tropical Disease Priority voucher, [9] which has a financial value on the open market [10].By working together and opening our research to the scientific community, many other labs will also be able to take promising molecular candidates forward to accelerate progress towards defeating the ZIKV outbreak. We invite any interested researcher to join us (send us your models or volunteer to assay the candidates we identify through this effort against any of the flaviviruses), and we hope new volunteers in the general public will donate their dormant, spare computing cycles to this cause. We will ultimately report the full computational and experimental results of this collaboration.

Advantages and Disadvantages of OpenZika

Advantages

• Open Science could accelerate the discovery of new antivirals using docking and virtual screening
• Docking narrows down compounds to test, which saves time and money
• Free to use distributed computing on World Community Grid, and the workflow is simpler than using conventional supercomputers

Disadvantages

• Concern around intellectual property ownership and whether companies will develop drugs coming from effort
• Need for experimental assays will always be a factor
• Testing in vitro and in vivo is not free, nor are the samples of the compounds

     

Glucose Metabolism Disorder Is Associated with Pulmonary Tuberculosis in Individuals with Respiratory Symptoms from Brazil

BackgroundDiabetes mellitus (DM) has been associated with increased risk for pulmonary tuberculosis (PTB) in endemic settings but it is unknown whether PTB risk is also increased by pre-DM. Here, we prospectively examined the association between glucose metabolism disorder (GMD) and PTB in patients with respiratory symptoms at a tuberculosis primary care reference center in Brazil.MethodsOral glucose tolerance test was performed and levels of fasting plasma glucose and glycohemoglobin (HbA1c) were measured in a cohort of 892 individuals presenting with respiratory symptoms of more than two weeks duration. Patients were also tested for PTB with sputum cultures. Prevalence of pre-DM and DM (based on HbA1c) was estimated and tested for association with incident PTB. Other TB risk factors including smoking history were analyzed.ResultsThe majority of the study population (63.1%) exhibited GMD based on HbA1c ≥5.7%. Patients with GMD had higher prevalence of PTB compared to normoglycemic patients. Individuals with DM exhibited increased frequency of TB-related symptoms and detection of acid-fast bacilli in sputum smears. Among patients with previous DM diagnosis, sustained hyperglycemia (HbA1c ≥7.0%) was associated with increased TB prevalence. Smoking history alone was not significantly associated with TB in our study population but the combination of smoking and HbA1c ≥7.0% was associated with 6 times higher odds for PTB.ConclusionsSustained hyperglycemia and pre-DM are independently associated with active PTB. This evidence raises the question whether improving glycemic control in diabetic TB patients would reduce the risk of TB transmission and simultaneously reduce the clinical burden of disease. A better understanding of mechanisms underlying these associations, especially those suggesting that pre-DM may be a factor driving susceptibility to TB is warranted.