SMAD3 rs17228212 Gene Polymorphism Is Associated with Reduced Risk to Cerebrovascular Accidents and Subclinical Atherosclerosis in Anti-CCP Negative Spanish Rheumatoid Arthritis Patients

Rheumatoid arthritis (RA) is a complex polygenic inflammatory disease associated with accelerated atherosclerosis and increased risk of cardiovascular (CV) disease. Previous genome-wide association studies have described SMAD3 rs17228212 polymorphism as an important signal associated with CV events. The aim of the present study was to evaluate for the first time the relationship between this gene polymorphism and the susceptibility to CV manifestations and its potential association with the presence of subclinical atherosclerosis assessed by the evaluation of carotid intima-media thickness (cIMT) in patients with RA.

Methods

One thousand eight hundred and ninety-seven patients fulfilling classification criteria for RA were genotyped for SMAD3 rs17228212 gene polymorphism through TaqMan genotyping assay. Also, subclinical atherosclerosis determined by the assessment of cIMT was analyzed in a subgroup of these patients by carotid ultrasonography.

Results

No statistically significant differences were observed when allele frequencies of RA patients with or without CV events were compared. Nevertheless, when RA patients were stratified according to anti-cyclic citrullinated peptide (anti-CCP) status, we found that in RA patients who were negative for anti-CCP antibodies, the presence of C allele of SMAD3 rs17228212 polymorphism conferred a protective effect against the risk of cerebrovascular accident (CVA) after adjustment for demographic and classic CV risk factors (HR [95%CI]=0.36 [0.14–0.94], p=0.038) in a Cox regression model. Additionally, correlation between the presence of C allele of SMAD3 rs17228212 polymorphism and lower values of cIMT was found after adjustment for demographic and classic CV risk factors (p-value=0.0094) in the anti-CCP negative RA patients.

Conclusions

Our results revealed that SMAD3 rs17228212 gene variant is associated with lower risk of CVA and less severe subclinical atherosclerosis in RA patients negative for anti-CCP antibodies. These findings may have importance to establish predictive models of CV disease in RA patients according to anti-CCP status.     

Expansion in CD39+ CD4+ Immunoregulatory T Cells and Rarity of Th17 Cells in HTLV-1 Infected Patients Is Associated with Neurological Complications

HTLV-1 infection is associated with several inflammatory disorders, including the neurodegenerative condition HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is unclear why a minority of infected subjects develops HAM/TSP. CD4⁺ T cells are the main target of infection and play a pivotal role in regulating immunity to HTLV and are hypothesized to participate in the pathogenesis of HAM/TSP. The CD39 ectonucleotidase receptor is expressed on CD4⁺ T cells and based on co-expression with CD25, marks T cells with distinct regulatory (CD39⁺CD25⁺) and effector (CD39⁺CD25⁻) function. Here, we investigated the expression of CD39 on CD4⁺ T cells from a cohort of HAM/TSP patients, HTLV-1 asymptomatic carriers (AC), and matched uninfected controls. The frequency of CD39⁺ CD4⁺ T cells was increased in HTLV-1 infected patients, regardless of clinical status. More importantly, the proportion of the immunostimulatory CD39⁺CD25⁻ CD4⁺ T-cell subset was significantly elevated in HAM/TSP patients as compared to AC and phenotypically had lower levels of the immunoinhibitory receptor, PD-1. We saw no difference in the frequency of CD39⁺CD25⁺ regulatory (Treg) cells between AC and HAM/TSP patients. However, these cells transition from being anergic to displaying a polyfunctional cytokine response following HTLV-1 infection. CD39⁻CD25⁺ T cell subsets predominantly secreted the inflammatory cytokine IL-17. We found that HAM/TSP patients had significantly fewer numbers of IL-17 secreting CD4⁺ T cells compared to uninfected controls. Taken together, we show that the expression of CD39 is upregulated on CD4⁺ T cells HAM/TSP patients. This upregulation may play a role in the development of the proinflammatory milieu through pathways both distinct and separate among the different CD39 T cell subsets. CD39 upregulation may therefore serve as a surrogate diagnostic marker of progression and could potentially be a target for interventions to reduce the development of HAM/TSP.     

High‐throughput cloning and expression library creation for functional proteomics

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single‐gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high‐throughput proteomics platforms, such as protein microarrays and cell‐based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high‐throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator^TM DNA Cloning System) and compare them side‐by‐side. We also report an example of high‐throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12).     

Antioxidant therapy: Still in search of the ‘magic bullet’

The therapeutic potential of natural phenolic antioxidants in human diseases associated with oxidative damage has received great attention to date. Appraisal of literature evidences that, in general, antioxidant therapy has enjoyed relative successes in preclinical studies but little benefits in human intervention studies or clinical trials. In fact, despite the huge, largely untapped potential therapeutic benefit of natural phenolic antioxidants, such as vitamins, non-flavonoid and flavonoid compounds, they appear not to be suitable drug candidates. The problem may be related, among others, to their non-drug-likeness properties. Though controversial the results obtained so far confirm the importance of exploring phenolic natural systems as safe templates for the design of new antioxidants. To support the assumption an outlook of the lead structural optimization process to improve ADME properties was given by means of natural hydroxycinnamic acids as a case study. The optimization of drug physicochemical properties and the development of appropriate delivery antioxidant systems can provide in the next future a way out to attain effective therapeutic antioxidant agents.     

Isolation, Characterization and Antifungal Activity of Proteinase Inhibitors from Capsicum chinense Jacq. Seeds

Capsicum species belong to the Solanaceae family and have great social, economic and agronomical significance. The present research presents data on the isolation and characterization of Capsicum chinense Jacq. peptides which were scrutinized in relation to their toxicity towards a diverse set of yeast species. The protein extract was separated with C18 reverse-phase chromatography in high performance liquid chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Tricine-SDS-PAGE of the PEF2 revealed peptides with molecular masses of approximately 5.0 and 8.5 kDa. These PEFs also exhibited strong antifungal activity against different yeasts. In the presence of the PEF2, Candida tropicalis exhibited morphological changes, including cellular agglomeration and formation of pseudohyphae. Determined N-terminal sequences of PEF2 and PEF3 were proven to be highly homologous to serine proteinase inhibitors, when analysed by comparative database sequence tools. For this reason were performed protease inhibitory activity assay. The PEFs displayed high inhibitory activity against trypsin and low inhibitory activity against chymotrypsin. PEF2 and PEF3 were considerably unsusceptible to a broad interval of pH and temperatures. Due to the myriad of application of Proteinase inhibitors (PIs) in fields ranging from plant protection against pathogens and pests to medicine such as in cancer and virus replication inhibition, the discovery of new PIs with new properties are of great interest.     

Sustained Reduction of the Dengue Vector Population Resulting from an Integrated Control Strategy Applied in Two Brazilian Cities

Aedes aegypti has developed evolution-driven adaptations for surviving in the domestic human habitat. Several trap models have been designed considering these strategies and tested for monitoring this efficient vector of Dengue. Here, we report a real-scale evaluation of a system for monitoring and controlling mosquito populations based on egg sampling coupled with geographic information systems technology. The SMCP-Aedes, a system based on open technology and open data standards, was set up from March/2008 to October/2011 as a pilot trial in two sites of Pernambuco -Brazil: Ipojuca (10,000 residents) and Santa Cruz (83,000), in a joint effort of health authorities and staff, and a network of scientists providing scientific support. A widespread infestation by Aedes was found in both sites in 2008–2009, with 96.8%–100% trap positivity. Egg densities were markedly higher in SCC than in Ipojuca. A 90% decrease in egg density was recorded in SCC after two years of sustained control pressure imposed by suppression of >7,500,000 eggs and >3,200 adults, plus larval control by adding fishes to cisterns. In Ipojuca, 1.1 million mosquito eggs were suppressed and a 77% reduction in egg density was achieved. This study aimed at assessing the applicability of a system using GIS and spatial statistic analysis tools for quantitative assessment of mosquito populations. It also provided useful information on the requirements for reducing well-established mosquito populations. Results from two cities led us to conclude that the success in markedly reducing an Aedes population required the appropriate choice of control measures for sustained mass elimination guided by a user-friendly mosquito surveillance system. The system was able to support interventional decisions and to assess the program’s success. Additionally, it created a stimulating environment for health staff and residents, which had a positive impact on their commitment to the dengue control program.     

Isolation and characterization of microsatellite loci in Pimelodus microstoma (Siluriformes: Pimelodidae)

Ten microsatellite loci were developed and described for Pimelodus microstoma, identified in eggs collected from the lower Das Cinzas river. Nine loci were polymorphic, with 02–19 alleles per locus. The observed heterozygosity values were up to 0.898. Five species of the Pimelodidae family were also tested for amplification of loci, among which Pimelodus maculatus was the most successful in the transferability of primers. These loci have proven to be useful for studying the variability and structuring of populations of Pimelodus microstoma.     

Fish biodiversity of Saint Peter and Saint Paulʼs Archipelago,Mid-Atlantic Ridge,Brazil: new records and a species database

Saint Peter and Saint Paul's Archipelago (SPSPA), one of the smallest and most isolated island groups in the world, is situated on the Mid-Atlantic Ridge, between Brazil and the African continent. SPSPA has low species richness and high endemism; nonetheless, the diversity of fishes from deep habitats (>30 m depth) had not been previously studied in detail. Several expeditions conducted between 2009 and 2018 explored the shallow and deep reefs of SPSPA using scuba, closed-circuit rebreathers, manned submersibles, baited remote underwater stereo-videos (stereo-BRUV) and fishing between 0 and 1050 m depth. These expeditions yielded 41 new records of fishes for SPSPA: 9 in open waters, 9 in shallow waters (0–30 m), 8 in mesophotic ecosystems (30–150 m) and 15 in deeper reefs (>150 m). Combined with literature records of adult pelagic, shallow and deep-reef species, as well as larvae, the database of the fish biodiversity for SPSPA currently comprises 225 species (169 recorded as adult fishes and 79 as larvae, with 23 species found in both stages). Most of them (112) are pelagic, 86 are reef-associated species and 27 are deep-water specialists. Species accumulation curves show that the number of fish species has not yet reached an asymptote. Whereas the number of species recorded in SPSPA is similar to that in other oceanic islands in the Atlantic Ocean, the proportion of shorefishes is relatively lower, and the endemism level is the third highest in the Atlantic. Twenty-nine species are listed as threatened with extinction. Observations confirm the paucity of top predators on shallow rocky reefs of the island, despite the presence of several pelagic shark species around SPSPA. Because all of the endemic species are reef associated, it is argued that the new marine-protected areas created by the Brazilian government do not ensure the protection and recovery of SPSPA's biodiversity because they allow exploitation of the most vulnerable species around the archipelago itself. This study suggests a ban on reef fish exploitation inside an area delimited by the 1000 m isobath around the islands (where all known endemics are concentrated) as the main conservation strategy to be included in the SPSPA management plan being prepared by the Brazilian government.     

Improvement in cyst recovery and molecular detection of Giardia duodenalis from stool samples

Molecular Biology Reports - Molecular detection of Giardia duodenalis by polymerase chain reaction (PCR) is difficult in faecal samples due to inhibitors that contaminate DNA preparations, or due...