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.

Utilidad de la neuromonitorización vagal continua en cirugía tiroidea: experiencia y resultados en un hospital de tercer nivel / The role of continuous vagal intraoperative nerve monitoring in thyroid surgery: experience and results in a third-level hospital

Resumen Objetivo: La lesión del nervio laríngeo recurrente es una grave complicación en cirugía tiroidea. El propósito del presente estudio es analizar la utilidad de la neuromonitorización vagal continua intraoperatoria en un hospital terciario. Materiales y Método: Estudio observacional, analítico y retrospectivo que recoge pacientes intervenidos de cirugía tiroidea con neuromonitorización en un período de 14 meses. La pérdida de señal se define como amplitud final nerviosa < 100 ^V, realizándose laringoscopia postquirúrgica ante la sospecha de lesión nerviosa. El análisis estadístico se realizó con el programa SPSS® V25,0, con p < 0,05. Resultados: Se incluyeron 120 pacientes intervenidos, registrándose en el 24,2% pérdida de señal. Factores de riesgo para lesión fueron bocio intratorácico (OR 5,31; IC 95% 1,56-17,99; p = 0,007), cirugía cervical previa (OR 5,76; IC 95% 0,64-51,97; p = 0,119) y patología maligna (OR 1,44; IC 95% 0,16-12,79; p = 0,743). Fue posible el cambio de estrategia quirúrgica en 7 casos. En el seguimiento posterior se cuantificó parálisis recurrencial transitoria en 27 pacientes y permanente en 4. Discusión: La neuromonitorización parece reducir la incidencia de parálisis laríngea porque aumenta la seguridad en la identificación del nervio recurrente y reduce su manipulación durante la cirugía. Conclusiones: La neuromonitorización intraoperatoria es útil para identificar el nervio laríngeo recurrente y advierte del riesgo potencial de lesión, permitiendo cambiar la estrategia quirúrgica para evitar la parálisis bilateral de cuerdas vocales.

Aim: Recurrent laryngeal nerve injury is a serious complication in thyroid surgery. The purpose of the present study is to analyze the use of intraoperative continuous vagal neuromonitoring in a tertiary hospital. Materials and Method: Observational, analytical and retrospective study that includes patients who underwent thyroid surgery with neuromonitoring in a period of 14 months. Loss of signal is defined as final nerve amplitude < 100 ^V, and postsurgical laryngoscopy is performed due to suspicion of nerve injury. Statistical analysis was performed with the SPSS® V25.0 program, with p < 0.05. Results: 120 operated patients were included, registering loss of signal in 24.2%. Risk factors for injury were intrathoracic goiter (OR 5.31; 95% CI 1.56-17.99; p = 0.007), previous cervical surgery (OR 5.76; 95% CI 0.64-51.97; p = 0.119) and malignant pathology (OR 1.44; 95% CI 0.16-12.79; p = 0.743). A change in surgical strategy was possible in 7 cases. In the subsequent follow-up, transient recurrent paralysis was quantified in 27 patients and permanent in 4. Discussion: Neuromonitoring seems to reduce the incidence of laryngeal paralysis because it increases the security in the identification of the recurrent nerve and reduces its manipulation during surgery. Conclusions: Intraoperative neuromonitoring is useful to identify the recurrent laryngeal nerve and warns of the potential risk of injury, allowing to change the surgical strategy to avoid bilateral vocal cord paralysis.

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.

Functional insight into the glycosomal peroxiredoxin of Leishmania.

Glycosomes of trypanosomatids are peroxisome-like organelles comprising unique metabolic features, among which the lack of the hallmark peroxisomal enzyme catalase. The absence of this highly efficient peroxidase from glycosomes is presumably compensated by other antioxidants, peroxidases of the peroxiredoxin (PRX) family being the most promising candidates for this function. Here, we follow on this premise and investigate the product of a Leishmania infantum gene coding for a putative glycosomal PRX (LigPRX). First, we demonstrate that LigPRX localizes to glycosomes, resorting to indirect immunofluorescence analysis. Second, we prove that purified recombinant LigPRX is an active peroxidase in vitro. Third, we generate viable LigPRX-depleted L. infantum promastigotes by classical homologous recombination. Surprisingly, phenotypic analysis of these knockout parasites revealed that promastigote survival, replication, and protection from oxidative and nitrosative insults can proceed normally in the absence of LigPRX. Noticeably, we also witness that LigPRX-depleted parasites can infect and thrive in mice to the same extent as wild type parasites. Overall, by disclosing the dispensable character of the glycosomal peroxiredoxin in L. infantum, this work excludes this enzyme from being a key component of the glycosomal hydroperoxide metabolism and contemplates alternative players for this function.

Role of human leukocyte antigen, killer-cell immunoglobulin-like receptors, and cytokine gene polymorphisms in leptospirosis.

Leptospirosis is an emerging zoonotic disease caused by pathogenic species of the genus Leptospira. It has a broad range of clinical presentations in humans. Although progress has been made in the characterization of the host immune system factors that may affect disease progression and outcome, to date few reports have addressed the role of genetic polymorphisms in the susceptibility to leptospirosis. In this work a group of patients with a history of leptospiral infection and a control group were compared for polymorphisms in the human leukocyte antigen (HLA), in killer-cell immunoglobulin-like receptors (KIR), and in cytokine genes. Alleles in the HLA-A and -B loci were associated with susceptibility, as were the class I haplotype A*01-B*08-Cw*07 and the 8.1 ancestral haplotype (A*01-B*08-Cw*07-DRB1*03-DQB1*02). Single nucleotide polymorphisms in the interleukin (IL)-4 and IL-4Ralpha genes also had significantly higher frequencies in the patient group. No association was reported between KIR gene profile and leptospirosis. This work highlights the importance of using genetic polymorphisms to better understand the mechanisms involved in the immune response to leptospirosis.

Purification, crystallization and preliminary X-ray diffraction analysis of the glyoxalase II from Leishmania infantum.

In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of the two enzymes involved in the glyoxalase pathway, glyoxalase I and glyoxalase II, the latter shows absolute specificity towards trypanothione thioester, making this enzyme an excellent model to understand the molecular basis of trypanothione binding. Cloned glyoxalase II from Leishmania infantum was overexpressed in Escherichia coli, purified and crystallized. Crystals belong to space group C222(1) (unit-cell parameters a = 65.6, b = 88.3, c = 85.2 angstroms) and diffract beyond 2.15 angstroms using synchrotron radiation. The structure was solved by molecular replacement using the human glyoxalase II structure as a search model. These results, together with future detailed kinetic characterization using lactoyltrypanothione, should shed light on the evolutionary selection of trypanothione instead of glutathione by trypanosomatids.

Quantitative assessment of the glyoxalase pathway in Leishmania infantum as a therapeutic target by modelling and computer simulation.

The glyoxalase pathway of Leishmania infantum was kinetically characterized as a trypanothione-dependent system. Using time course analysis based on parameter fitting with a genetic algorithm, kinetic parameters were estimated for both enzymes, with trypanothione derived substrates. A K(m) of 0.253 mm and a V of 0.21 micromol.min(-1).mg(-1)for glyoxalase I, and a K(m) of 0.098 mm and a V of 0.18 micromol.min(-1).mg(-1) for glyoxalase II, were obtained. Modelling and computer simulation were used for evaluating the relevance of the glyoxalase pathway as a potential therapeutic target by revealing the importance of critical parameters of this pathway in Leishmania infantum. A sensitivity analysis of the pathway was performed using experimentally validated kinetic models and experimentally determined metabolite concentrations and kinetic parameters. The measurement of metabolites in L. infantum involved the identification and quantification of methylglyoxal and intracellular thiols. Methylglyoxal formation in L. infantum is nonenzymatic. The sensitivity analysis revealed that the most critical parameters for controlling the intracellular concentration of methylglyoxal are its formation rate and the concentration of trypanothione. Glyoxalase I and II activities play only a minor role in maintaining a low intracellular methylglyoxal concentration. The importance of the glyoxalase pathway as a therapeutic target is very small, compared to the much greater effects caused by decreasing trypanothione concentration or increasing methylglyoxal concentration.