The cytosolic hyperoxidation-sensitive and -robust Leishmania peroxiredoxins cPRX1 and cPRX2 are both dispensable for parasite infectivity.

Typical two-cysteine peroxiredoxins (2-Cys-PRXs) are H2O2-metabolizing enzymes whose activity relies on two cysteine residues. Protists of the family Trypanosomatidae invariably express one cytosolic 2-Cys-PRX (cPRX1). However, the Leishmaniinae sub-family features an additional isoform (cPRX2), almost identical to cPRX1, except for the lack of an elongated C-terminus with a Tyr-Phe (YF) motif. Previously, cytosolic PRXs were considered vital components of the trypanosomatid antioxidant machinery. Here, we shed new light on the properties, functions and relevance of cPRXs from the human pathogen Leishmania infantum. We show first that LicPRX1 is sensitive to inactivation by hyperoxidation, mirroring other YF-containing PRXs participating in redox signaling. Using genetic fusion constructs with roGFP2, we establish that LicPRX1 and LicPRX2 can act as sensors for H2O2 and oxidize protein thiols with implications for signal transduction. Third, we show that while disrupting the LicPRX-encoding genes increases susceptibility of L. infantum promastigotes to external H2O2in vitro, both enzymes are dispensable for the parasites to endure the macrophage respiratory burst, differentiate into amastigotes and initiate in vivo infections. This study introduces a novel perspective on the functions of trypanosomatid cPRXs, exposing their dual roles as both peroxidases and redox sensors. Furthermore, the discovery that Leishmania can adapt to the absence of both enzymes has significant implications for our understanding of Leishmania infections and their treatment. Importantly, it questions the conventional notion that the oxidative response of macrophages during phagocytosis is a major barrier to infection and the suitability of cPRXs as drug targets for leishmaniasis.

Structure-Activity Relationship Studies of 9-Alkylamino-1,2,3,4-tetrahydroacridines against Leishmania (Leishmania) infantum Promastigotes.

Leishmaniasis is one of the most neglected diseases in modern times, mainly affecting people from developing countries of the tropics, subtropics and the Mediterranean basin, with approximately 350 million people considered at risk of developing this disease. The incidence of human leishmaniasis has increased over the past decades due to failing prevention and therapeutic measures-there are no vaccines and chemotherapy, which is problematic. Acridine derivatives constitute an interesting group of nitrogen-containing heterocyclic compounds associated with numerous bioactivities, with emphasis to their antileishmanial potential. The present work builds on computational studies focusing on a specific enzyme of the parasite, S-adenosylmethionine decarboxylase (AdoMet DC), with several 1,2,3,4-tetrahydro-acridines emerging as potential inhibitors, evidencing this scaffold as a promising building block for novel antileishmanial pharmaceuticals. Thus, several 1,2,3,4-tetrahydroacridine derivatives have been synthesized, their activity against Leishmania (Leishmania) infantum promastigotes evaluated and a structure-activity relationship (SAR) study was developed based on the results obtained. Even though the majority of the 1,2,3,4-tetrahydroacridines evaluated presented high levels of toxicity, the structural information gathered in this work allowed its application with another scaffold (quinoline), leading to the obtention of N1,N12-bis(7-chloroquinolin-4-yl)dodecane-1,12-diamine (12) as a promising novel antileishmanial agent (IC50 = 0.60 ± 0.11 µM, EC50 = 11.69 ± 3.96 µM and TI = 19.48).

Leishmania infantum Enhances Migration of Macrophages via a Phosphoinositide 3-Kinase γ-Dependent Pathway.

Leishmania infantum (L. infantum) and Leishmania major (L. major) are phylogenetically related protozoan parasites that cause different pathologies in humans (visceral and cutaneous infections, respectively). Here, we report on how these obligatory intracellular pathogens differentially affect the migration of macrophages. Resorting to gap closure assays of infected murine bone marrow derived macrophages, we observed that L. infantum enhances the mobility of these cells. This is not the case of L. major, whose impact on macrophage migration is null. Resorting to kinase inhibition assays, we witnessed that chemical inhibition of phosphoinositide 3-kinase-γ (PI3Kγ) critically impairs cell mobility in all experimental conditions. Importantly, the blockade of tyrosine kinases with dasatinib also slows down naïve and L. major-parasitized cells but not macrophages exposed to L. infantum. The dasatinib-resistant phenotype of L. infantum-infected macrophages aligns with the hypothesis that this parasite invokes a tyrosine kinase-independent pathway to increase the PI3Kγ activity of macrophages and enhance migration.

Folate pathway modulation in Rhipicephalus ticks in response to infection.

Folate pathways components were demonstrated to be present in RNA-sequencing data obtained from uninfected and pathogen-infected Rhipicephalus ticks. Here, PCR and qPCR allowed the identification of folate-related genes in Rhipicephalus spp. ticks and in the tick cell line IDE8. Genes coding for GTP cyclohydrolase I (gch-I), thymidylate synthase (ts) and 6-pyrovoyltetrahydropterin (ptps) were identified. Differential gene expression was evaluated by qPCR between uninfected and infected samples of four biological systems, showing significant upregulation and largest fold-change for the gch-I gene in the majority of the biological systems, supporting the selection for functional analysis by RNAi silencing. Efficient knockdown of the gch-I gene in uninfected and Ehrlichia canis-infected IDE8 cells showed no detectable impact on the capacity of the bacteria to invade or replicate in the tick cells. Overall, this work demonstrated an increase in the expression of some folate-related genes, though not always statistically significantly, in the presence of infection, suggesting gene expression modulation of these pathways, either as a tick response to an invader or manipulation of the tick cell machinery by the pathogens to their advantage. This discovery points to folate pathways as interesting targets for further studies.

Cyanobacterial Allelochemicals But Not Cyanobacterial Cells Markedly Reduce Microbial Community Diversity.

The freshwater cyanobacterium Phormidium sp. LEGE 05292 produces allelochemicals, including the cyclic depsipeptides portoamides, that influence the growth of heterotrophic bacteria, cyanobacteria, and eukaryotic algae. Using 16S rRNA gene amplicon metagenomics, we show here that, under laboratory conditions, the mixture of metabolites exuded by Phormidium sp. LEGE 05292 markedly reduces the diversity of a natural planktonic microbial community. Exposure of the same community to the portoamides alone resulted in a similar outcome. In both cases, after 16 days, alpha-diversity estimates for the allelochemical-exposed communities were less than half of those for the control communities. Photosynthetic organisms, but also different heterotrophic-bacteria taxa were found to be negatively impacted by the allelochemicals. Intriguingly, when Phormidium sp. LEGE 05292 was co-cultured with the microbial community, the latter remained stable and closer to non-exposed than to allelochemical-exposed communities. Overall, our observations indicate that although under optimal growth conditions Phormidium sp. LEGE 05292 is able to synthesize potent allelochemicals that severely impact different microorganisms, its allelopathic effect is not pronounced when in contact with a complex microbial community. Therefore, under ecologically relevant conditions, the allelopathic behavior of this cyanobacterium may be regulated by nutrient availability or by interactions with the surrounding microbiota.

[Congenital Heart Disease in Children with Down Syndrome: What Has Changed in the Last Three Decades?] / Cardiopatia Congénita em Crianças com Síndrome de Down: O que Mudou nas Últimas Três Décadas?

INTRODUCTION: The prevalence of Down syndrome has increased in the last 30 years; 55% of these children have congenital heart disease. MATERIAL AND METHODS: A retrospective longitudinal cohort study; clinical data from 1982 to 2013 databases with the diagnosis of Down syndrome or trisomy 21 in a reference hospital in pediatric cardiology and cardiac surgery. OBJECTIVE: to assess the progress in the last three decades of cardiological care given to children with Down syndrome and congenital heart disease. RESULTS: We studied 102 patients with Down syndrome and congenital heart disease subjected to invasive therapy: corrective or palliative cardiac surgery and therapeutic catheterization. The referral age was progressively earlier in patients referred in the first year of life. The most frequent diagnosis was complete atrioventricular sptal defect (41%). There was a trend towards increasingly early corrective surgery in patients under 12 months (p < 0.001). Since 2000, the large majority of patients were operated before reaching six months of age. The main cardiac complications were rhythm dysfunction and low output. More frequent noncardiac complications were pulmonary and infectious. The 30-day mortality rate was 3/102 cases (2.9%). Of patients in follow-up, 89% are in NYHA class I. DISCUSSION AND CONCLUSION: The early surgical correction seen over the past 15 years follows the approach suggested in the literature. The observed 30-day mortality rate is overlapping international results. Patients with Down syndrome subjected to corrective surgery of congenital heart disease have an excellent long-term functional capacity.

Introdução: A prevalência da síndrome de Down tem aumentado nos últimos 30 anos; 55% destas crianças apresentam cardiopatia congénita. Material e Métodos: Estudo retrospetivo longitudinal de coorte; dados clínicos obtidos em bases de dados de 1982 a 2013 com o diagnóstico de síndrome de Down ou trissomia 21 num hospital de referência em cardiologia pediátrica e cirurgia cardíaca. Objetivo: Avaliar a evolução, nas últimas três décadas, dos cuidados cardiológicos prestados às crianças com síndrome de Down ecardiopatia congénita.Resultados: Estudámos 102 doentes com síndrome de Down e cardiopatia congénita submetidos a terapêutica invasiva: cirurgiacardíaca corretiva, paliativa e cateterismo terapêutico. Em doentes referenciados no primeiro ano de vida, a referenciação foi cada vez mais precoce. O diagnóstico mais frequente foi o defeito completo do septo aurículo-ventricular (41%). Verificou-se uma tendência para cirurgia corretiva cada vez mais precoce em doentes abaixo dos 12 meses (p < 0,001). A partir de 2000, a grande maioria dos doentes foi operada antes dos seis meses de idade. As principais complicações cardíacas foram alterações de ritmo e baixo débito e as principais não cardíacas foram pulmonares e infeciosas. A taxa de mortalidade a 30 dias foi de 3/102 casos (2,9%). Dos doentes em follow-up, 89% estão em classe funcional I da NYHA. Discussão e Conclusão: A correção cirúrgica mais precoce verificada nos últimos 15 anos vai ao encontro do proposto na literatura. A taxa de mortalidade a 30 dias verificada é sobreponível aos resultados internacionais. Os doentes com síndrome de Down submetidos a cirurgia corretiva de cardiopatia congénita apresentam uma excelente capacidade funcional a longo prazo.

Biofilm and fluoroquinolone resistance of canine Escherichia coli uropathogenic isolates.

BACKGROUND: Escherichia coli is the most common uropathogen involved in urinary tract infection (UTI). Virulence of strains may differ, and may be enhanced by antimicrobial resistance and biofilm formation, resulting in increased morbidity and recurrent infections. The aim of this study was to evaluate the in vitro biofilm forming capacity of E. coli isolates from dogs with UTI, by using fluorescent in situ hybridization, and its association with virulence genes and antimicrobial resistance. FINDINGS: The proportion of biofilm-producing isolates significantly increased with the length of incubation time (P < 0.05). Biofilm production was significantly associated with fluoroquinolone resistance at all incubation time points and was independent of the media used (P < 0.05). Biofilm production was not associated with cnf1, hly, pap and sfa genes (P > 0.05), but was significantly associated with afa, aer and the ß-lactamase genes (P < 0.05). CONCLUSIONS: To the best of our knowledge, this is the first report showing significant association between biofilm production and fluoroquinolone resistance in E. coli isolates from dogs with UTI. Biofilm formation may contribute to UTI treatment failure in dogs, through the development of bacterial reservoirs inside bladder cells, allowing them to overcome host immune defenses and to establish recurrent infections.