Molecular analysis on resistant TB isolates in Portugal

Abstrat publicado em: http://www.pasteur.fr/infosci/conf/sb/tuberculosis2012/images/TB2012-Program-Abstract-book-LD.pdfPortugal has one of the highest tuberculosis (TB) incidence (20/100 000 inhabitants) in Europe. The emergence of multidrug resistant (MDR) TB and extensive drug-resistant (XDR) TB infection is the biggest threat to TB control. Most strains of MDR-TB circulating in the Lisbon area belong to a particular family of genetically related strains, the Lisboa family, detected in the 90’s. The prevalence of this family of strains has increased over the years, and represented more than 85% of the MDR-TB strains in the year of 2008. XDR-TB has been recently derived from MDR-TB strains and account for about 50% of these, the majority belonging to Lisboa family. Lisboa family represents a serious problem regarding TB control in Portugal and its prevalance in recent years suggests that these strains may have selective advantages over others. In order to establish a link between the most prevalent mutations in drug-resistance associated genes and spread of Lisboa strains in the Portuguese setting, 54 resistant-TB clinical isolates in Portugal were study. The isolates were characterized by 24 loci MIRU-VNTR and analyzed for inhA, katG, rpoB, rpsL, rrs, embB and pncA genes, for resistance to first-line drugs. The MDR isolates (n=35) were further analyzed for mutations in gyrA, tlyA, eis and rrs for resistance to fluoroquinolones and second-line injectables. MIRU-VNTR analysis showed that Lisboa family strains and Q1 cluster were the most prevalent, with 26 (48%) and 6 (11%) isolates respectively, including the majority of the MDR-TB and XDR- TB isolates. No mutations in first-line drug resistance associated genes specifically related with MDR were found. However, mutation analysis to second-line drug resistance in 17 MDR-TB isolates shown that specific mutations arepresent in particular families. Therefore, XDR-TB from Lisboa family exhibits gyrA D94G/S91P, tlyA Ins755GT and eis G-10A mutations, and XDR-TB from Q1 presents gyrA D94A and rrs A1401G mutations. The remaining isolates are still under study, and further analyses are ongoing. We conclude that XDR-TB isolates from Lisboa family and Q1 cluster have shown a marked difference between them, regarding second-line mutations. Such analysis may be useful to define mutation profiles that distinguish Lisboa family from Q1 isolates

An evaluation of 2D SLAM techniques available in Robot Operating System

n this work, a study of several laser-based 2D Simultaneous Localization and Mapping (SLAM) techniques available in Robot Operating System (ROS) is conducted. All the approaches have been evaluated and compared in 2D simulations and real world experiments. In order to draw conclusions on the performance of the tested techniques, the experimental results were collected under the same conditions and a generalized performance metric based on the k-nearest neighbours concept was applied. Moreover, the CPU load of each technique is examined. This work provides insight on the weaknesses and strengths of each solution. Such analysis is fundamental to decide which solution to adopt according to the properties of the intended final application

Fusing sonars and LRF data to perform SLAM in reduced visibility scenarios

Simultaneous Localization and Mapping (SLAM) approaches have evolved considerably in recent years. However, there are many situations which are not easily handled, such as the case of smoky, dusty, or foggy environments where commonly used range sensors for SLAM are highly disturbed by noise induced in the measurement process by particles of smoke, dust or steam. This work presents a sensor fusion method for range sensing in Simultaneous Localization and Mapping (SLAM) under reduced visibility conditions. The proposed method uses the complementary characteristics between a Laser Range Finder (LRF) and an array of sonars in order to ultimately map smoky environments. The method was validated through experiments in a smoky indoor scenario, and results showed that it is able to adequately cope with induced disturbances, thus decreasing the impact of smoke particles in the mapping task

Efflux Activity Differentially Modulates the Levels of Isoniazid and Rifampicin Resistance among Multidrug Resistant and Monoresistant Mycobacterium tuberculosis Strains

With the growing body of knowledge on the contribution of efflux activity to Mycobacterium tuberculosis drug resistance, increased attention has been given to the use of efflux inhibitors as adjuvants of tuberculosis therapy. Here, we investigated how efflux activity modulates the levels of efflux between monoresistant and multi- and extensively drug resistant (M/XDR) M. tuberculosis clinical isolates. The strains were characterized by antibiotic susceptibility testing in the presence/absence of efflux inhibitors, molecular typing, and genetic analysis of drug-resistance-associated genes. Efflux activity was quantified by real-time fluorometry. The results demonstrated that all the M. tuberculosis clinical strains, susceptible or resistant, presented a faster, rapid, and non-specific efflux-mediated short-term response to drugs. The synergism assays demonstrated that the efflux inhibitors were more effective in reducing the resistance levels in the M/XDR strains than in the monoresistant strains. This indicated that M/XDR strains presented a more prolonged response to drugs mediated by efflux compared to the monoresistant strains, but both maintain it as a long-term stress response. This work shows that efflux activity modulates the levels of drug resistance between monoresistant and M/XDR M. tuberculosis clinical strains, allowing the bacteria to survive in the presence of noxious compounds.publishersversionpublishe

Dynamics and Development of Extensively Drug-resistant Tuberculosis, Portugal

Abstrat publicado em: http://www.pasteur.fr/infosci/conf/sb/tuberculosis2012/images/TB2012-Program-Abstract-book-LD.pdfThe development of multidrug-resistant (MDR) and extensive drug-resistant (XDR) tuberculosis(TB) combined with subsequent transmission constitutes a serious threat to the effective control of tuberculosis in several countries. Lisbon Health Region, despite great progresses in TB management still presents a high number of MDR/XDR-TB cases. The development of this type of resistance is the result of adaptative selection of Mycobacterium tuberculosis strains that acquire and accumulate specific mutations at specific genes. The presently known mechanisms of drug resistance include the modification or overexpression of drug targets, inactivation of drug- activator enzymes and overexpression of drug-modifying enzymes. Although the molecular basis of resistance of MDR/XDR-TB strains circulating in Lisbon has already been addressed in different studies, the dynamics or mode of resistance acquisition that have lead to the different circulating strains is still partially unclear. In the present study we have genotyped and screened a set of 44 MDR/XDR-TB isolates for mutations in tlyA, gyrA, rrs and eis genes. We have determined the most prevalent mutations found in each gene to be Ins755GT in tlyA, A1401G in rrs, G-10A in eis and S91P in gyrA. Two genetic clusters previously known to be associated with XDR-TB were detected, Lisboa3 and Q1, containing 27 and 17 isolates, respectively. Lisboa3 strains isolated in the 90’s with the same mutational profile of recent XDR-TB Lisboa3 strains were found, emphasizing the ancient XDR-TB problem in the region. Also investigated was the resistance level conferred by eis G-10A mutations, revealing that eis G-10A mutations may result in an undetectable AMK resistance. We concluded by analyzing the mutational distribution found by genetic cluster that in Q1 cluster two mutations, gyrA D94A and rrs A1401G, were enough to ensure development of XDR-TB from a multidrug resistant strain. Moreover, in Lisboa3 cluster it was possible to determine that the development of kanamycin low-level resistance mediated by eis promoter mutations was at the origin of independent emergence of several XDRTB strains that can be discriminated within Lisboa3 genetic cluster by tlyA mutations

Tribenuron-methyl metabolism and the rare Pro197Phe double mutation together with 2,4-D metabolism and reduced absorption can evolve in Papaver rhoeas with multiple and cross herbicide resistance to ALS inhibitors and auxin mimics

Multiple resistance mechanisms to ALS inhibitors and auxin mimics in two Papaver rhoeas populations were investigated in wheat fields from Portugal. Dose-response trials, also with malathion (a cytochrome P450 inhibitor), cross-resistance patterns for ALS inhibitors and auxin mimics, alternative herbicides tests, 2,4-D and tribenuron-methyl absorption, translocation and metabolism experiments, together with ALS activity, gene sequencing and enzyme modelling and ligand docking were carried out. Results revealed two different resistant profiles: one population (R1) multiple resistant to tribenuron-methyl and 2,4-D, the second (R2) only resistant to 2,4-D. In R1, several target-site mutations in Pro197 and enhanced metabolism (cytochrome P450-mediated) were responsible of tribenuron-methyl resistance. For 2,4-D, reduced transport was observed in both populations, while cytochrome P450-mediated metabolism was also present in R1 population. Moreover, this is the first P. rhoeas population with enhanced tribenuron-methyl metabolism. This study reports the first case for P. rhoeas of the amino acid substitution Pro197Phe due to a double nucleotide change. This double mutation could cause reduced enzyme sensitivity to most ALS inhibitors according to protein modelling and ligand docking. In addition, this study reports a P. rhoeas population resistant to 2,4-D, apparently, with reduced transport as the sole resistance mechanism.This work has been supported by the Asociacion de Agroquimicos y Medio Ambiente, Spain. Joel Torra acknowledges support from the Spanish Ministry of Science, Innovation, and Universities (grant Ramon y Cajal RYC2018–023866-I) and by the Spanish State Research Agency, Spain (AEI) and the European Regional Development Fund, EU (ERDF) through the projects AGL2017-83325-C4-2-R and PID2020-113229RB- C42. The field surveys made in Portugal were supported by Foundation for Science and Technology through the project UIDB/05064/2020 (VALORIZA). Jorge Lozano-Juste group is funded by grants RYC2020- 029097-I and PID2021-128826OA-I00 from Ministerio de Ciencia e Innovaci ́on (MCIN, Spain), AEI and the ERDF

Block copolymer-based magnetic mixed matrix membranes-effect of magnetic field on protein permeation and membrane fouling

In this study, we report the impact of the magnetic field on protein permeability through magnetic-responsive, block copolymer, nanocomposite membranes with hydrophilic and hydrophobic characters. The hydrophilic nanocomposite membranes were composed of spherical polymeric nanoparticles (NPs) synthesized through polymerization-induced self-assembly (PISA) with iron oxide NPs coated with quaternized poly(2-dimethylamino)ethyl methacrylate. The hydrophobic nanocomposite membranes were prepared via nonsolvent-induced phase separation (NIPS) containing poly (methacrylic acid) and meso-2, 3-dimercaptosuccinic acid-coated superparamagnetic nanoparticles (SPNPs). The permeation experiments were carried out using bovine serum albumin (BSA) as the model solute, in the absence of the magnetic field and under permanent and cyclic magnetic field conditions OFF/ON (strategy 1) and ON/OFF (strategy 2). It was observed that the magnetic field led to a lower reduction in the permeate fluxes of magnetic-responsive membranes during BSA permeation, regardless of the magnetic field strategy used, than that obtained in the absence of the magnetic field. Nevertheless, a comparative analysis of the effect caused by the two cyclic magnetic field strategies showed that strategy 2 allowed for a lower reduction of the original permeate fluxes during BSA permeation and higher protein sieving coefficients. Overall, these novel magneto-responsive block copolymer nanocomposite membranes proved to be competent in mitigating biofouling phenomena in bioseparation processes

Silica-Based Nanocoating Doped by Layered Double Hydroxides to Enchance the Paperboard Barrier Properties

Paperboard is an environment -friendly multi -layer material widely used for packaging applic a- tions. However, for food packaging paperboard lacks essential barrier properties towards oxygen and water vapor. Conventional solutions to enhance these barrier properties (e.g. paperboard film coating with synthetic polymers) require special manufacturing facilities and difficult the end -of - life disposal and recycling of the paperboard. Paperboard coating with silica -based formulations is an eco -friendly alternative hereby disclosed. Silica -nanocoating s were prepared by sol -gel synth e- sis, with or without the addition of Zn(2) -Al -NO 3 layered double hydroxides (LDHs), and applied on the surface (ca 2 g/m 2 ) of industrial paperboard samples by a roll -to -roll technique. The ph y- sicochemical features of silica -nanocoatings were studied by FTIR -ATR, SEM/EDS, XRD analysis and surface energy measurements. The barrier properties of uncoated and silica -coated pape r- board were accessed by water vapor transmission rate (WVTR) and oxygen permeability ( J O 2 ) measurements. The best barrier results were obtained for paperboard coated with a mixture of tetraethoxysilane (TEOS) and 3 -aminopropyltriethoxysilane (APTES), with and without the in- corporation of LDHs

CD4+ T Cell-Dependent Macrophage Activation Modulates Sustained PS Exposure on Intracellular Amastigotes of Leishmania amazonensis

Leishmania amazonensis amastigotes can make use of surface-exposed phosphatidylserine (PS) molecules to promote infection and non-classical activation of macrophages (MΦ), leading to uncontrolled intracellular proliferation of the parasites. This mechanism was quoted as apoptotic mimicry. Moreover, the amount of PS molecules exposed on the surface of amastigotes correlates with the susceptibility of the host. In this study, we tested whether host cellular responses influence PS expression on intracellular amastigotes. We found that the level of PS exposure on intracellular amastigotes was modulated by CD4+ T cell and MΦ activation status in vitro and in vivo. L. amazonensis infection generated a Th1/Th2-mixed cytokine profile, providing the optimal MΦ stimulation that favored PS exposure on intracellular amastigotes. Maintenance of PS exposed on the parasite was dependent on low, but sustained, levels of nitric oxide and polyamine production. Amastigotes obtained from lymphopenic nude mice did not expose PS on their surface, and adoptive transfer of CD4+ T cells reversed this phenotype. In addition, histopathological analysis of mice treated with anti-PS antibodies showed increased inflammation and similarities to nude mouse lesions. Collectively, our data confirm the role of pathogenic CD4+ T cells for disease progression and point to PS as a critical parasite strategy to subvert host immune responses