Reconstitution of a biofilm adhesin system from a sulfate-reducing bacterium in Pseudomonas fluorescens.

Biofilms of sulfate-reducing bacterium (SRB) like Desulfovibrio vulgaris Hildenborough (DvH) can facilitate metal corrosion in various industrial and environmental settings leading to substantial economic losses. Although the mechanisms of biofilm formation by DvH are not yet well understood, recent studies indicate the large adhesin, DvhA, is a key determinant of biofilm formation. The dvhA gene neighborhood resembles the biofilm-regulating Lap system of Pseudomonas fluorescens but is curiously missing the c-di-GMP-binding regulator LapD. Instead, DvH encodes an evolutionarily unrelated c-di-GMP-binding protein (DVU1020) that we hypothesized is functionally analogous to LapD. To study this unusual Lap system and overcome experimental limitations with the slow-growing anaerobe DvH, we reconstituted its predicted SRB Lap system in a P. fluorescens strain lacking its native Lap regulatory components (ΔlapGΔlapD). Our data support the model that DvhA is a cell surface-associated LapA-like adhesin with a N-terminal "retention module" and that DvhA is released from the cell surface upon cleavage by the LapG-like protease DvhG. Further, we demonstrate DVU1020 (named here DvhD) represents a distinct class of c-di-GMP-binding, biofilm-regulating proteins that regulates DvhG activity in response to intracellular levels of this second messenger. This study provides insight into the key players responsible for biofilm formation by DvH, thereby expanding our understanding of Lap-like systems.

Emergence of cefiderocol resistance during ceftazidime/avibactam treatment caused by a large genomic deletion, including ampD and piuCD genes, in Pseudomonas aeruginosa.

We report the emergence of cefiderocol resistance during the treatment of a ST312 Pseudomonas aeruginosa respiratory infection with ceftazidime/avibactam. whole genome sequencing (WGS) revealed that resistance was caused by a large genomic deletion, including PiuDC (iron transport system) and AmpD (ampC negative regulator), driven by the integration of phage DNA. Thus, our findings alert that this type of deletion could be an efficient (two mechanisms in one step) specific cefiderocol resistance mechanism that might occur nonspecifically upon treatment with ß-lactams that select for AmpC overexpression.

Mechanisms leading to in vivo ceftazidime/avibactam resistance development during treatment of GES-5-producing Pseudomonas aeruginosa infections.

The mechanisms underlying ceftazidime/avibactam resistance development in four ceftazidime/avibactam susceptible/resistant pairs of GES-5-producing ST235 Pseudomonas aeruginosa clinical isolates were investigated. In three of the cases, ceftazidime/avibactam resistance was driven by a single mutation leading to GES-27 (P162Q), GES-29 (P162A), or the novel GES-60 (N136S), as confirmed through cloning experiments. Moreover, these mutations were associated with increased cefiderocol MICs but reduced carbapenem, particularly imipenem/relebactam, resistance. Understanding the complexity of resistance mechanisms to the growing repertoire of antipseudomonal ß-lactams is crucial to guide optimized treatments and antimicrobial stewardship measures.

Impact of transferable ß-lactamases and intrinsic AmpC amino acid substitutions on the activity of cefiderocol against wild-type and iron uptake-deficient mutants of Pseudomonas aeruginosa.

OBJECTIVES: We aimed to analyse the interplay between impaired iron uptake and ß-lactamases on cefiderocol resistance in Pseudomonas aeruginosa. METHODS: Thirty-one transferable ß-lactamases and 16 intrinsic P. aeruginosa AmpC (PDC) variants were cloned and expressed in wild-type (PAO1) and iron uptake-deficient (PAO ΔpiuC) P. aeruginosa backgrounds. MICs of cefiderocol and antipseudomonal ß-lactams were determined by reference broth microdilution. RESULTS: Relative to PAO1, deletion of piuC caused a specific 16-fold decrease in cefiderocol activity but negligible effects on the activity of other ß-lactams. Among transferable ß-lactamases, SHV-12, KPC Ω-loop mutants, NDMs and OXA-15 showed cefiderocol MIC values above the clinical breakpoint (2 mg/L) when expressed in PAO1. When expressed in PAO ΔpiuC, these and the transformants harbouring PER-1, VEB-1, KPC-2, KPC-3, VIM-1, CMY-2, OXA-2 and OXA-14 showed increased MIC values from 16 to >256 mg/L. The PDC variants carrying the Ω-loop changes ΔP215-G222 (PDC-577), E219K (PDC-221 and PDC-558) and the H10 helix change L293P (PDC-219) had the greatest impact on cefiderocol resistance, with MICs of 2-4 mg/L in PAO1 and of up to 32-64 mg/L in PAO ΔpiuC. Widespread enzymes such as GES, CTX-M-9, CTX-M-15, VIM-2-like enzymes, IMPs, DHA-1, FOX-4, OXA-10, OXA-48 and the other PDC variants tested had weaker effects on cefiderocol resistance. CONCLUSION: We add evidence about the effect of the interplay between iron uptake and ß-lactamases on the acquisition of cefiderocol resistance in P. aeruginosa. These findings may help to anticipate the emergence of resistance and optimize the use of cefiderocol against P. aeruginosa infections.

Formulation Studies with Cyclodextrins for Novel Selenium NSAID Derivatives.

Commercial cyclodextrins (CDs) are commonly used to form inclusion complexes (ICs) with different molecules in order to enhance their water solubility, stability, and bioavailability. Nowadays, there is strong, convincing evidence of the anticancer effect of selenium (Se)-containing compounds. However, pharmaceutical limitations, such as an unpleasant taste or poor aqueous solubility, impede their further evaluation and clinical use. In this work, we study the enhancement of solubility with CD complexes for a set of different nonsteroidal anti-inflammatory drug (NSAID) derivatives with Se as selenoester or diacyl diselenide chemical forms, with demonstrated antitumoral activity. The CD complexes were analyzed via nuclear magnetic resonance (NMR) spectroscopic techniques. In order to obtain additional data that could help explain the experimental results obtained, 3D models of the theoretical CD-compound complexes were constructed using molecular modeling techniques. Among all the compounds, I.3e and II.5 showed a remarkable increase in their water solubility, which could be ascribed to the formation of the most stable interactions with the CDs used, in agreement with the in silico studies performed. Thus, the preliminary results obtained in this work led us to confirm the selection of ß and γ-CD as the most suitable for overcoming the pharmaceutical drawbacks of these Se derivatives.

In vitro dynamics and mechanisms of cefiderocol resistance development in wild-type, mutator and XDR Pseudomonas aeruginosa.

OBJECTIVES: To analyse the dynamics and mechanisms of stepwise resistance development to cefiderocol in Pseudomonas aeruginosa. METHODS: Cefiderocol resistance evolution was analysed in WT PAO1, PAOMS (mutS mutator derivate) and three XDR clinical isolates belonging to ST111, ST175 and ST235 clones. Strains were incubated in triplicate experiments for 24 h in iron-depleted CAMHB with 0.06-128 mg/L cefiderocol. Tubes from the highest antibiotic concentration showing growth were reinoculated into fresh medium containing concentrations up to 128 mg/L for 7 consecutive days. Two colonies per strain and experiment were characterized by determining the susceptibility profiles and WGS. RESULTS: Evolution of resistance was significantly enhanced in PAOMS, but was variable for the XDR strains, including levels similar to PAOMS (ST235), similar to PAO1 (ST175) or even below PAO1 (ST111). WGS revealed 2-5 mutations for PAO1 lineages and 35-58 for PAOMS. The number of mutations in the XDR clinical strains ranged from 2 to 4 except for one of the ST235 experiments in which a mutL lineage was selected, thus increasing the number of mutations. The most frequently mutated genes were piuC, fptA and pirR, related to iron uptake. Additionally, an L320P AmpC mutation was selected in multiple lineages and cloning confirmed its major impact on cefiderocol (but not ceftolozane/tazobactam or ceftazidime/avibactam) resistance. Mutations in CpxS and PBP3 were also documented. CONCLUSIONS: This work deciphers the potential resistance mechanisms that may emerge upon the introduction of cefiderocol into clinical practice, and highlights that the risk of resistance development might be strain-specific even for XDR high-risk clones.

Determinants and Trends of the Use of Intravenous Thrombolysis for Minor Stroke: A Population-Based Study, 2016 to 2020.

BACKGROUND: We analyzed the main factors associated with intravenous thrombolysis (IVT) in patients with minor ischemic stroke. METHODS: Data were obtained from a prospective, government-mandated, population-based registry of stroke code patients in Catalonia (6 Comprehensive Stroke Centers, 8 Primary Stroke Centers, and 14 TeleStroke Centers). We selected patients diagnosed with ischemic stroke and National Institutes of Health Stroke Scale (NIHSS) ≤5 at hospital admission from January 2016 to December 2020. We excluded patients with a baseline modified Rankin Scale score of ≥3, absolute contraindication for IVT, unknown stroke onset, or admitted to hospital beyond 4.5 after stroke onset. The main outcome was treatment with IVT. We performed univariable and binary logistic regression analyses to identify the most important factors associated with IVT. RESULTS: We included 2975 code strokes; 1433 (48.2%) received IVT of which 30 (2.1%) had a symptomatic hemorrhagic transformation. Patients treated with IVT as compared to patients who did not receive IVT were more frequently women, had higher NIHSS, arrived earlier to hospital, were admitted to a Comprehensive Stroke Centers, and had large vessel occlusion. After binary logistic regression, NIHSS score 4 to 5 (odds ratio, 40.62 [95% CI, 31.73-57.22]; P<0.001) and large vessel occlusion (odds ratio, 16.39 [95% CI, 7.25-37.04]; P<0.001) were the strongest predictors of IVT. Younger age, female sex, baseline modified Rankin Scale score of 0, earlier arrival to hospital (<120 minutes after stroke onset), and the type of stroke center were also independently associated with IVT. The weight of large vessel occlusion on IVT was higher in patients with lower NIHSS. CONCLUSIONS: Minor stroke female patients, with higher NIHSS, arriving earlier to the hospital, presenting with large vessel occlusion and admitted to a Comprehensive Stroke Centers were more likely to receive intravenous thrombolysis.

Comparative analysis of in vitro dynamics and mechanisms of ceftolozane/tazobactam and imipenem/relebactam resistance development in Pseudomonas aeruginosa XDR high-risk clones.

OBJECTIVES: To analyse the dynamics and mechanisms of stepwise resistance development to ceftolozane/tazobactam and imipenem/relebactam in XDR Pseudomonas aeruginosa clinical strains. METHODS: XDR clinical isolates belonging to ST111 (main resistance mechanisms: oprD-, dacB-, CARB-2), ST175 (oprD-, ampR-G154R) and ST235 (oprD-, OXA-2) high-risk clones were incubated for 24 h in Müeller-Hinton Broth with 0.125-64 mg/L of ceftolozane + tazobactam 4 mg/L or imipenem + relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated into fresh medium containing concentrations up to 64 mg/L for 7 consecutive days. Two colonies per strain from each of the triplicate experiments were characterized by determining the susceptibility profiles, whole genome sequencing (WGS), and in vitro fitness through competitive growth assays. RESULTS: Resistance development occurred more slowly and reached a lower level for imipenem/relebactam than for ceftolozane/tazobactam in all tested XDR strains. Moreover, resistance development to imipenem/relebactam remained low even for ST175 isolates that had developed ceftolozane/tazobactam resistance during therapy. Lineages evolved in the presence of ceftolozane/tazobactam showed high-level resistance, imipenem/relebactam hypersusceptibility and low fitness cost, whereas lineages evolved in the presence of imipenem/relebactam showed moderate (borderline) resistance, no cross-resistance to ceftolozane/tazobactam and high fitness cost. WGS evidenced that ceftolozane/tazobactam resistance was mainly caused by mutations in the catalytic centres of intrinsic (AmpC) or acquired (OXA) ß-lactamases, whereas lineages evolved in imipenem/relebactam frequently showed structural mutations in MexB or in ParS, along with some strain-specific mutations. CONCLUSIONS: Imipenem/relebactam could be a useful alternative for the treatment of XDR P. aeruginosa infections, potentially reducing resistance development during therapy.

Examination of multiple Trypanosoma cruzi targets in a new drug discovery approach for Chagas disease.

Chagas disease (CD) is a centenarian neglected parasitosis caused by the protozoan Trypanosoma cruzi (T. cruzi). Despite the continuous efforts of many organizations and institutions, CD is still an important human health problem worldwide. A lack of a safe and affordable treatment has led drug discovery programmes to focus, for years, on the search for molecules enabling interference with enzymes that are essential for T. cruzi survival. In this work, the authors want to offer a brief overview of the different validated targets that are involved in diverse parasite pathways: glycolysis, sterol synthesis, the de novo biosynthesis of pyrimidine nucleotides, the degradative processing of peptides and proteins, oxidative stress damage and purine salvage and nucleotide synthesis and metabolism. Their structural aspects, function, active sites, etc. were studied and considered with the aim of defining molecular bases in the search for new effective treatments for CD. This review also compiles, as much as possible, all the inhibitors reported to date against these T. cruzi targets, serving as a reference for future research in this field.

In Vivo Evolution of GES ß-Lactamases Driven by Ceftazidime/Avibactam Treatment of Pseudomonas aeruginosa Infections.

The mechanisms underlying an in vivo switch in the resistance phenotype of P. aeruginosa after ceftazidime-avibactam treatment was investigated. The initial isolate (a blood culture) was resistant to meropenem but remained susceptible to antipseudomonal cephalosporins and combinations with ß-lactamase inhibitors. One week after ceftazidime-avibactam therapy, a subsequent isolate (a rectal swab) recovered from the same patient showed the opposite phenotype. Whole-genome sequence analysis revealed a single SNP difference between both (ST235) isolates, leading to a P162S change in blaGES-5, creating blaGES-15. Thus, blaGES-1, blaGES-5, and blaGES-15 were cloned and expressed in the wild-type strain PAO1. Susceptibility profiles confirmed the P162S substitution reverted the carbapenemase phenotype determined by the G170S change of GES-5 back into the ESBL phenotype of GES-1.

Emergence of Resistance to Novel Cephalosporin-ß-Lactamase Inhibitor Combinations through the Modification of the Pseudomonas aeruginosa MexCD-OprJ Efflux Pump.

A ceftolozane-tazobactam- and ceftazime-avibactam-resistant Pseudomonas aeruginosa isolate was recovered after treatment (including azithromycin, meropenem, and ceftolozane-tazobactam) from a patient that had developed ventilator-associated pneumonia after COVID-19 infection. Whole-genome sequencing revealed that the strain, belonging to ST274, had acquired a nonsense mutation leading to truncated carbapenem porin OprD (W277X), a 7-bp deletion (nt213Δ7) in NfxB (negative regulator of the efflux pump MexCD-OprJ), and two missense mutations (Q178R and S133G) located within the first large periplasmic loop of MexD. Through the construction of mexD mutants and complementation assays with wild-type nfxB, it was evidenced that resistance to the novel cephalosporin-ß-lactamase inhibitor combinations was caused by the modification of MexD substrate specificity.

PATJ Low Frequency Variants Are Associated With Worse Ischemic Stroke Functional Outcome.

RATIONALE: Ischemic stroke is among the leading causes of adult disability. Part of the variability in functional outcome after stroke has been attributed to genetic factors but no locus has been consistently associated with stroke outcome. OBJECTIVE: Our aim was to identify genetic loci influencing the recovery process using accurate phenotyping to produce the largest GWAS (genome-wide association study) in ischemic stroke recovery to date. METHODS AND RESULTS: A 12-cohort, 2-phase (discovery-replication and joint) meta-analysis of GWAS included anterior-territory and previously independent ischemic stroke cases. Functional outcome was recorded using 3-month modified Rankin Scale. Analyses were adjusted for confounders such as discharge National Institutes of Health Stroke Scale. A gene-based burden test was performed. The discovery phase (n=1225) was followed by open (n=2482) and stringent joint-analyses (n=1791). Those cohorts with modified Rankin Scale recorded at time points other than 3-month or incomplete data on previous functional status were excluded in the stringent analyses. Novel variants in PATJ (Pals1-associated tight junction) gene were associated with worse functional outcome at 3-month after stroke. The top variant was rs76221407 (G allele, ß=0.40, P=1.70×10-9). CONCLUSIONS: Our results identify a set of common variants in PATJ gene associated with 3-month functional outcome at genome-wide significance level. Future studies should examine the role of PATJ in stroke recovery and consider stringent phenotyping to enrich the information captured to unveil additional stroke outcome loci.

Progression of Large Vessel Disease in Patients With Giant Cell Arteritis-Associated Ischemic Stroke: The Role of Vascular Imaging: A Case Series.

OBJECTIVE: Giant cell arteritis (GCA) can cause ischemic stroke (IS) due to the involvement of the internal carotid and vertebral arteries. The aim of our study is to describe the pattern of stroke recurrence in patients with GCA-related IS and the role of vascular imaging in the follow-up of these patients. METHODS: We conducted an observational study of 2417 consecutive patients diagnosed with IS and admitted to our hospital from January 2012 to December 2018. We reviewed patients with GCA-related IS and the relationship of erythrocyte sedimentation rate, C-reactive protein, vascular status, and clinical course. RESULTS: We found 4 patients with GCA-related IS among 2417 IS patients: 1 woman (25%); median age, 77.3 years (67-85 years). Mean follow-up was 3.6 years. Initial vascular workup showed vertebral artery stenosis in all of them and internal carotid artery stenosis in 2 patients. All patients were started on treatment with full-dose prednisone, associated with methotrexate in 2 cases. Follow-up color-coded duplex sonography disclosed progression of arterial stenoses in 3 patients who suffered a recurrent IS (days after index stroke; mean, 27.67 [SD, 10.97]) despite normal C-reactive protein and erythrocyte sedimentation rate values. CONCLUSIONS: Vascular imaging, especially with color-coded duplex sonography, could play a role in the follow-up of patients with GCA-related IS and identify those patients with higher risk of recurrent stroke.

In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa.

OBJECTIVES: We analysed the dynamics and mechanisms of resistance development to imipenem alone or combined with relebactam in Pseudomonas aeruginosa WT (PAO1) and mutator (PAOMS; ΔmutS) strains. METHODS: PAO1 or PAOMS strains were incubated for 24 h in Mueller-Hinton Broth with 0.125-64 mg/L of imipenem ± relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 mg/L of imipenem ± relebactam for 7 days. Two colonies per strain, replicate experiment and antibiotic from early (Day 1) and late (Day 7) cultures were characterized by determining the susceptibility profiles, WGS and determination of the expression of ampC and efflux-pump-coding genes. Virulence was studied in a Caenorhabditis elegans infection model. RESULTS: Relebactam reduced imipenem resistance development for both strains, although resistance emerged much faster for PAOMS. WGS indicated that imipenem resistance was associated with mutations in the porin OprD and regulators of ampC, while the mutations in imipenem/relebactam-resistant mutants were located in oprD and regulatoras of MexAB-OprM. High-level imipenem/relebactam resistance was only documented in the PAOMS strain and was associated with an additional specific (T680A) mutation located in the catalytic pocket of ponA (PBP1a) and with reduced virulence in the C. elegans model. CONCLUSIONS: Imipenem/relebactam could be a useful alternative for the treatment of MDR P. aeruginosa infections, potentially reducing resistance development during treatment. Moreover, this work deciphers the potential resistance mechanisms that may emerge upon the introduction of this novel combination into clinical practice.

First report of Tomato brown rugose fruit virus in tomato in Spain.

In October 2019, tomato (Solanum lycopersicum L.) plants showing chlorosis and brown necrosis in apical leaflets and rugose surface in fruits were observed in a greenhouse in Vicar, Almería, Spain. A total of 0.5% of the tomato plants in the greenhouse (1,38 ha) showed these symptoms. The presence of tomato brown rugose fruit virus (ToBRFV) was suspected. A total of 5 symptomatic and 2 symptomless leaf samples were collected and analyzed by double-antibody sandwich (DAS)-ELISA with antibodies for ToBRFV, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) (Loewe Biochemica, Germany). Symptomatic samples tested positive by DAS-ELISA only for ToBRFV. Therefore, one sample was selected and analyzed by reverse transcription (RT)-PCR with specific primers ToBRFV-F5722/ToBRFV-R6179 for ToBRFV (Panno et al. 2019a) which amplified a 458 bp fragment of the coat protein gene. The sequence obtained by Sanger sequencing from the amplicon showed 99.7% nt identity with ToBRFV isolate from United Kingdom (Acc. No. MN182533) and was deposited in the GenBank database under the accession number MT211630. Further surveys were performed in Vicar and El Egido (Almería, Spain) on plants showing viral-like symptoms. A total of 50 tomato and two pepper leaf samples from 28 greenhouses were collected and analyzed by DAS-ELISA for ToBRFV, TMV, ToMV, pepino mosaic virus (PepMV) , tomato spotted wilt virus (TSWV) and tomato yellow leaf curl virus (TYLCV) with respective antibodies (Loewe Biochemica, Germany). Five tomato plants (four from Vicar and one from El Ejido) tested positive for ToBRFV. PepMV, ToMV and TYLCV were also detected in one, three and five tomato plants, respectively. The ELISA positive results for ToBRFV were confirmed by end point RT-PCR with primer pairs ToBRFV-F5722/ToBRFV-R6179 and ToBRFV-F/ToBRFV-R (Alkouni et al., 2019) and by real-time RT-PCR with Taqman probe ToB-probe and specific primers ToB5520F/ToB5598R (Panno et al., 2019b). Tomato seeds used for plantation in these greenhouses were also analyzed, but ToBRFV was not detected. Eradication measures have been undertaken to prevent the virus spread and to control this outbreak. Official seed analysis by DAS-ELISA and real time RT-PCR (ISF, 2019) are being conducted on the tomato and pepper imported seeds to prevent the appearance of new sources of ToBRFV inoculum in Spain. References: Alkowni, R., et al. 2019. J. Plant Pathol. 101: 719. doi: 10.1007/s42161-019-00240-7. ISF, 2019. International Seed Federation. Version 1.3, September 2019. Available at https://www.worldseed.org/wp-content/uploads/2019/09/Tomato-ToBRFV_2019.09.pdf Panno, S., et al. 2019a. Plant Dis. 103: 1443. https://doi.org/10.1094/PDIS-12-18-2254-PDN Panno, S. et al. 2019b. PeerJ 7:e7928 DOI 10.7717/peerj.7928.

An N-Terminal Retention Module Anchors the Giant Adhesin LapA of Pseudomonas fluorescens at the Cell Surface: a Novel Subfamily of Type I Secretion Systems.

LapA of Pseudomonas fluorescens Pf0-1 belongs to a diverse family of cell surface-associated bacterial adhesins that are secreted via the type I secretion system (T1SS). We previously reported that the periplasmic protease LapG cleaves the N terminus of LapA at a canonical dialanine motif to release the adhesin from the cell surface under conditions unfavorable to biofilm formation, thus decreasing biofilm formation. Here, we characterize LapA as the first type I secreted substrate that does not follow the "one-step" rule of T1SS. Rather, a novel N-terminal element, called the retention module (RM), localizes LapA at the cell surface as a secretion intermediate. Our genetic, biochemical, and molecular modeling analyses support a model wherein LapA is tethered to the cell surface through its T1SS outer membrane TolC-like pore, LapE, until LapG cleaves LapA in the periplasm. We further demonstrate that this unusual retention strategy is likely conserved among LapA-like proteins, and it reveals a new subclass of T1SS ABC transporters involved in transporting this group of surface-associated LapA-like adhesins. These studies demonstrate a novel cell surface retention strategy used throughout the Proteobacteria and highlight a previously unappreciated flexibility of function for T1SS.IMPORTANCE Bacteria have evolved multiple secretion strategies to interact with their environment. For many bacteria, the secretion of cell surface-associated adhesins is key for initiating contact with a preferred substratum to facilitate biofilm formation. Our work demonstrates that P. fluorescens uses a previously unrecognized secretion strategy to retain the giant adhesin LapA at its cell surface. Further, we identify likely LapA-like adhesins in various pathogenic and commensal proteobacteria and provide phylogenetic evidence that these adhesins are secreted by a new subclass of T1SS ABC transporters.

GRECOS Project (Genotyping Recurrence Risk of Stroke): The Use of Genetics to Predict the Vascular Recurrence After Stroke.

BACKGROUND AND PURPOSE: Vascular recurrence occurs in 11% of patients during the first year after ischemic stroke (IS) or transient ischemic attack. Clinical scores do not predict the whole vascular recurrence risk; therefore, we aimed to find genetic variants associated with recurrence that might improve the clinical predictive models in IS. METHODS: We analyzed 256 polymorphisms from 115 candidate genes in 3 patient cohorts comprising 4482 IS or transient ischemic attack patients. The discovery cohort was prospectively recruited and included 1494 patients, 6.2% of them developed a new IS during the first year of follow-up. Replication analysis was performed in 2988 patients using SNPlex or HumanOmni1-Quad technology. We generated a predictive model using Cox regression (GRECOS score [Genotyping Reurrence Risk of Stroke]) and generated risk groups using a classification tree method. RESULTS: The analyses revealed that rs1800801 in the MGP gene (hazard ratio, 1.33; P=9×10-03), a gene related to artery calcification, was associated with new IS during the first year of follow-up. This polymorphism was replicated in a Spanish cohort (n=1.305); however, it was not significantly associated in a North American cohort (n=1.683). The GRECOS score predicted new IS (P=3.2×10-09) and could classify patients, from low risk of stroke recurrence (1.9%) to high risk (12.6%). Moreover, the addition of genetic risk factors to the GRECOS score improves the prediction compared with previous Stroke Prognosis Instrument-II score (P=0.03). CONCLUSIONS: The use of genetics could be useful to estimate vascular recurrence risk after IS. Genetic variability in the MGP gene was associated with vascular recurrence in the Spanish population.

Novel Heteroaryl Selenocyanates and Diselenides as Potent Antileishmanial Agents.

A series of new selenocyanates and diselenides bearing interesting bioactive scaffolds (quinoline, quinoxaline, acridine, chromene, furane, isosazole, etc.) was synthesized, and their in vitro leishmanicidal activities against Leishmania infantum amastigotes along with their cytotoxicities in human THP-1 cells were determined. Interestingly, most tested compounds were active in the low micromolar range and led us to identify four lead compounds (1h, 2d, 2e, and 2f) with 50% effective dose (ED50) values ranging from 0.45 to 1.27 µM and selectivity indexes of >25 for all of them, much higher than those observed for the reference drugs. These active derivatives were evaluated against infected macrophages, and in order to gain preliminary knowledge about their possible mechanism of action, the inhibition of trypanothione reductase (TryR) was measured. Among these novel structures, compounds 1h (3,5-dimethyl-4-isoxazolyl selenocyanate) and 2d [3,3'-(diselenodiyldimethanediyl)bis(2-bromothiophene)] exhibited good association between TryR inhibitory activity and antileishmanial potency, pointing to 1h, for its excellent theoretical ADME (absorption, distribution, metabolism, and excretion) properties, as the most promising lead molecule for leishmancidal drug design.

Assessment of ß-lapachone loaded in lecithin-chitosan nanoparticles for the topical treatment of cutaneous leishmaniasis in L. major infected BALB/c mice.

Patients affected by cutaneous leishmaniasis need a topical treatment which cures lesions without leaving scars. Lesions are produced not only by the parasite but also by an uncontrolled and persistent inflammatory immune response. In this study, we proposed the loading of ß-lapachone (ß-LP) in lecithin-chitosan nanoparticles (NP) for targeting the drug to the dermis, where infected macrophages reside, and promote wound healing. Although the loading of ß-LP in NP did not influence the drug antileishmanial activity it was critical to achieve important drug accumulation in the dermis and permeation through the skin. When topically applied in Leishmania major infected BALB/c mice, ß-LP NP achieved no parasite reduction but they stopped the lesion progression. Immuno-histopathological assays in CL lesions and quantitative mRNA studies in draining lymph nodes confirmed that ß-LP exhibited anti-inflammatory activity leading to the down-regulation of IL-1ß and COX-2 expression and a decrease of neutrophils infiltrate. FROM THE CLINICAL EDITOR: Cutaneous leishmaniasis often leaves patients with unsightly scars due to the body's inflammatory response to the infection. The authors in this paper described topical treatment using ß-lapachone (ß- LP) loaded in lecithin-chitosan nanoparticles (NP) in an animal model. Results confirmed the reduction of inflammatory response without affecting the parasite killing efficacy. These findings would pave way for further clinical testing in the near future.