Overoxidation and Oligomerization of Trypanosoma cruzi Cytosolic and Mitochondrial Peroxiredoxins.

Peroxiredoxins (Prxs) have been shown to be important enzymes for trypanosomatids, counteracting oxidative stress and promoting cell infection and intracellular survival. In this work, we investigate the in vitro sensitivity to overoxidation and the overoxidation dynamics of Trypanosoma cruzi Prxs in parasites in culture and in the infection context. We showed that recombinant m-TXNPx, in contrast to what was observed for c-TXNPx, exists as low molecular mass forms in the overoxidized state. We observed that T. cruzi Prxs were overoxidized in epimastigotes treated with oxidants, and a significant proportion of the overoxidized forms were still present at least 24 h after treatment suggesting that these forms are not actively reversed. In in vitro infection experiments, we observed that Prxs are overoxidized in amastigotes residing in infected macrophages, demonstrating that inactivation of at least part of the Prxs by overoxidation occurs in a physiological context. We have shown that m-TXNPx has a redox-state-dependent chaperone activity. This function may be related to the increased thermotolerance observed in m-TXNPx-overexpressing parasites. This study suggests that despite the similarity between protozoan and mammalian Prxs, T. cruzi Prxs have different oligomerization dynamics and sensitivities to overoxidation, which may have implications for their function in the parasite life cycle and infection process.

On the occurrence of a glutaredoxin-like small protein in the anaerobic protozoan parasite Entamoeba histolytica.

BACKGROUND: Entamoeba histolytica, an intestinal parasitic protozoan that usually lives and multiplies within the human gut, is the causative agent of amoebiasis. To date, de novo glutathione biosynthesis and its associated enzymes have not been identified in the parasite. Cysteine has been proposed to be the main intracellular thiol. METHODS: Using bioinformatics tools to search for glutaredoxin homologs in the E. histolytica genome database, we identified a coding sequence for a putative Grx-like small protein (EhGLSP) in the E. histolytica HM-1:IMSS genome. We produced the recombinant protein and performed its biochemical characterization. RESULTS: Through in vitro experiments, we observed that recombinant EhGLSP could bind GSH and L-Cys as ligands. However, the protein exhibited very low GSH-dependent disulfide reductase activity. Interestingly, via UV-Vis spectroscopy and chemical analysis, we detected that recombinant EhGLSP (freshly purified from Escherichia coli cells by IMAC) was isolated together with a redox-labile [FeS] bio-inorganic complex, suggesting that this protein could have some function linked to the metabolism of this cofactor. Western blotting showed that EhGLSP protein levels were modulated in E. histolytica cells exposed to exogenous oxidative species and metronidazole, suggesting that this protein cooperates with the antioxidant mechanisms of this parasite. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our findings support the existence of a new metabolic actor in this pathogen. To the best of our knowledge, this is the first report on this protein class in E. histolytica.

Biochemical characterization of GAF domain of free-R-methionine sulfoxide reductase from Trypanosoma cruzi.

Trypanosoma cruzi is the causal agent of Chagas Disease and is a unicellular parasite that infects a wide variety of mammalian hosts. The parasite exhibits auxotrophy by L-Met; consequently, it must be acquired from the extracellular environment of the host, either mammalian or invertebrate. Methionine (Met) oxidation produces a racemic mixture (R and S forms) of methionine sulfoxide (MetSO). Reduction of L-MetSO (free or protein-bound) to L-Met is catalyzed by methionine sulfoxide reductases (MSRs). Bioinformatics analyses identified the coding sequence for a free-R-MSR (fRMSR) enzyme in the genome of T. cruzi Dm28c. Structurally, this enzyme is a modular protein with a putative N-terminal GAF domain linked to a C-terminal TIP41 motif. We performed detailed biochemical and kinetic characterization of the GAF domain of fRMSR in combination with mutant versions of specific cysteine residues, namely, Cys12, Cys98, Cys108, and Cys132. The isolated recombinant GAF domain and full-length fRMSR exhibited specific catalytic activity for the reduction of free L-Met(R)SO (non-protein bound), using tryparedoxins as reducing partners. We demonstrated that this process involves two Cys residues, Cys98 and Cys132. Cys132 is the essential catalytic residue on which a sulfenic acid intermediate is formed. Cys98 is the resolutive Cys, which forms a disulfide bond with Cys132 as a catalytic step. Overall, our results provide new insights into redox metabolism in T. cruzi, contributing to previous knowledge of L-Met metabolism in this parasite.

Consideraciones clínicas y desenlaces quirúrgicos del cáncer asociado a implante mamario: una enfermedad emergente de interés

El cáncer de mama sigue siendo una de las principales prioridades en salud global y salud pública y permanece como la neoplasia maligna más frecuente y mortal en mujeres en el mundo. El linfoma anaplásico de células grandes asociado a implante mamario (LACG-AIM) consiste en un linfoma no-Hodgkin de tipo raro, del cual se desconoce mucho sobre su patogenia y fisiopatología, pero que se ve cada vez con mayor frecuencia, debido al aumento de procedimientos estéticos. A la fecha, existen limitaciones en cuanto al conocimiento sobre el comportamiento clínico y se manifiesta de muchas formas, con un tiempo de evolución variable, y desenlaces quirúrgicos inciertos a mediano y largo plazo. Con base en lo anterior, el objetivo de esta revisión consiste en resumir evidencia sobre las consideraciones clínicas y desenlaces quirúrgicos del cáncer asociado a implante mamario, que faciliten la identificación y abordaje de esta condición. Se realizó una búsqueda bibliográfica en los motores de búsqueda y bases de datos PubMed, ScienceDirect, Embase, EBSCO y MEDLINE. Dentro de las consideraciones clínicas y quirúrgicas, se debe tener en cuenta el tipo de implante utilizado (texturizado), el tiempo del antecedente del implante, la severidad de las manifestaciones y la estadificación, para poder determinar la oportunidad de intervención quirúrgica y terapia neoadyuvante e intentar garantizar la supervivencia y evitar recurrencia. Aquellos pacientes sometidos a capsulectomía completa acompañado de radioterapia tienen mejores desenlaces.

Breast cancer continues to be one of the main priorities in global health and public health, and remains the most frequent and deadly malignant neoplasm in women worldwide. Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare type of Non-Hodgkin's lymphoma, whose pathogenesis and pathophysiology are not well known, but which is seen with increasing frequency due to the increase in cosmetic procedures. To date, there are limitations in terms of knowledge about the clinical behavior of the disease, which can manifest itself in many forms, with a variable evolution time and uncertain surgical outcomes in the medium- and long-term. Based on the above, the aim of this review is to summarize evidence on the clinical considerations and surgical outcomes of breast implant-associated cancer to facilitate the identification and management of this condition. A bibliographic search was performed in the search engines and databases pubmed, sciencedirect, embase, ebsco and medline. Within the clinical and surgical considerations, the type of implant used (textured), the time of the implant history, the severity of the manifestations, and the staging, must be taken into account in order to determine the opportunity for surgical intervention and neoadjuvant therapy, and to try to guarantee survival and avoid recurrence. Patients who undergo complete capsulectomy with radiotherapy have better outcomes.

Functional and structural characterization of an endo-ß-1,3-glucanase from Euglena gracilis.

Endo-ß-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading ß-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-ß-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg ml-1,kcat 1.20 s-1) and also, but with less enzymatic efficiency, paramylon (KM 20.2 mg ml-1,kcat 0.23 ml mg-1 s-1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified ß-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg ml-1, kcat 0.88 ml mg-1 s-1). This latter result, combined with interfacial kinetic analysis evidenced a protein's greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the ß-1,3/ß-1,6-glucan. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 µM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing ß-glucans.

PRO score: predictive scoring system for visual outcomes after rhegmatogenous retinal detachment repair.

BACKGROUND/AIMS: To compare risk factors for poor visual outcomes in patients undergoing primary rhegmatogenous retinal detachment (RRD) repair and to develop a scoring system. METHODS: Analysis of the Primary Retinal detachment Outcomes (PRO) study, a multicentre interventional cohort of consecutive primary RRD surgeries performed in 2015. The main outcome measure was a poor visual outcome (Snellen VA ≤20/200). RESULTS: A total of 1178 cases were included. The mean preoperative and postoperative logMARs were 1.1±1.1 (20/250) and 0.5±0.7 (20/63), respectively. Multivariable logistic regression identified preoperative risk factors predictive of poor visual outcomes (≤20/200), including proliferative vitreoretinopathy (PVR) (OR 1.26; 95% CI 1.13 to 1.40), history of antivascular endothelial growth factor (VEGF) injections (1.38; 1.11 to 1.71), >1-week vision loss (1.17; 1.08 to 1.27), ocular comorbidities (1.18; 1.00 to 1.38), poor presenting VA (1.06 per initial logMAR unit; 1.02 to 1.10) and age >70 (1.13; 1.04 to 1.23). The data were split into training (75%) and validation (25%) and a scoring system was developed and validated. The risk for poor visual outcomes was 8% with a total score of 0, 17% with 1, 29% with 2, 47% with 3, and 71% with 4 or higher. CONCLUSIONS: Independent risk factors were compared for poor visual outcomes after RRD surgery, which included PVR, anti-VEGF injections, vision loss >1 week, ocular comorbidities, presenting VA and older age. The PRO score was developed to provide a scoring system that may be useful in clinical practice.

Associations between MDMA/ecstasy, classic psychedelics, and suicidal thoughts and behaviors in a sample of U.S. adolescents.

Suicide is one of the leading causes of death amongst adolescents and decades of research have failed to curb suicide rates within this population. There is thus a need to better understand factors that correlate with adolescent suicidal thoughts and behaviors (STBs). MDMA/ecstasy and classic psychedelics represent two areas for exploration, as use of these substances has been associated with both increased and lowered odds of STBs. Thus, the goal of this study was to test the associations between MDMA/ecstasy and classic psychedelics (psilocybin, peyote, mescaline, LSD) and STBs in a nationally representative sample of U.S. adolescents. We tested these associations in a sample of adolescents aged 12-17 years old from the National Survey on Drug Use and Health (2004-2019) (N = 262,617) using survey-weighted multivariable logistic regression models. Lifetime psilocybin use was associated with lowered odds of lifetime suicidal thinking, planning, and attempts (aOR range 0.77-0.85). Conversely, LSD was associated with increased odds of these same outcomes (aOR range 1.20-1.35). MDMA/ecstasy, peyote, and mescaline did not share associations with STBs. Our study demonstrates that individual classic psychedelics share varying relationships to STBs among adolescents. Future cross-sectional and longitudinal studies are needed to further elucidate the link between classic psychedelic use and STBs in youth.

Effects of Kilohertz Electrical Stimulation of the Trigeminal Nerve on Motor Learning.

Neurological disorders such as stroke remain leading causes of disability worldwide. A current thrust in the neurorehabilitation of such disorders involves exogenous neuromodulation of cranial nerves in order to enhance neuro-plasticity and maximize recovery of function. Here we present preliminary results on the effects of kilohertz range electrical stimulation of the trigeminal nerve (TNS) on motor learning, using an upper extremity visuomotor adaptation paradigm. Twenty-five (25) healthy adult subjects were randomly assigned to 2 groups: 3kHz stimulation ( n=13) and sham ( n=12). Participants performed a visuomotor rotation task that involved center-out reaching movements to eight vertically arranged targets. Four blocks of trials were performed: two baseline blocks with veridical visual feedback, one adaptation block involving a 30° CCW rotation of hand visual feedback, and one washout block with no rotation. TNS was applied for 20 minutes before the 2nd baseline block using two electrodes targeting the ophthalmic branches of the trigeminal nerve. Early in the rotation block, learning rates were similar between the 3kHz and sham groups but gradually diverged, with the 3kHz group demonstrating slightly faster rates than sham later in the rotation block. The results provide new information on the potential use of TNS in neurorehabilitation.

Functional characterization of monothiol and dithiol glutaredoxins from Leptospira interrogans.

Thiol redox proteins and low molecular mass thiols have essential functions in maintaining cellular redox balance in almost all living organisms. In the pathogenic bacterium Leptospira interrogans, several redox components have been described, namely, typical 2-Cys peroxiredoxin, a functional thioredoxin system, glutathione synthesis pathway, and methionine sulfoxide reductases. However, until now, information about proteins linked to GSH metabolism has not been reported in this pathogen. Glutaredoxins (Grxs) are GSH-dependent oxidoreductases that regulate and maintain the cellular redox state together with thioredoxins. This work deals with recombinant production at a high purity level, biochemical characterization, and detailed kinetic and structural study of the two Grxs (Lin1CGrx and Lin2CGrx) identified in L. interrogans serovar Copenhageni strain Fiocruz L1-130. Both recombinant LinGrxs exhibited the classical in vitro GSH-dependent 2-hydroxyethyl disulfide and dehydroascorbate reductase activity. Strikingly, we found that Lin2CGrx could serve as a substrate of methionine sulfoxide reductases A1 and B from L. interrogans. Distinctively, only recombinant Lin1CGrx contained a [2Fe2S] cluster confirming a homodimeric structure. The functionality of both LinGrxs was assessed by yeast complementation in null grx mutants, and both isoforms were able to rescue the mutant phenotype. Finally, our data suggest that protein glutathionylation as a post-translational modification process is present in L. interrogans. As a whole, our results support the occurrence of two new redox actors linked to GSH metabolism and iron homeostasis in L. interrogans.

The Cough Cold and Fever Clinic: A Model for the Safe Management of Ambulatory Patients With Suspected COVID-19.

BACKGROUND AND OBJECTIVE: When the coronavirus disease-2019 (COVID-19) pandemic swept through New York City, hospital systems became quickly overwhelmed and ambulatory strategies were needed. We designed and implemented an innovative program called the Cough Cold and Fever (CCF) Clinic to safely triage, evaluate, treat, and follow up patients with symptoms concerning for COVID-19. METHODS: The CCF Clinic was launched on March 13, 2020, in the ambulatory internal medicine office of New York Presbyterian-Weill Cornell Medicine. Patients with symptoms suspicious for COVID-19 were first triaged via telemedicine to determine necessity of in-person evaluation. Clinic workspaces and workflows were fashioned to minimize risk of viral transmission and to conserve COVID-19 testing supplies and personal protective equipment. Protocols containing the most recent COVID-19 practice guidelines were created, updated regularly, and communicated through twice-daily huddles and as a shareable online document. Discharged patients were followed up for at least 7 days through telemedicine. Patient outcomes, including admission to the emergency department (ED), hospitalization, and death, were tracked to ensure clinical quality. RESULTS: We report on the first 620 patients seen at CCF between March 13, 2020, and June 19, 2020. Telemedicine follow-up was achieved for 500 (81%). We tested 347 (56%) patients for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with 119 (34%) testing positive. Forty-seven (8%) patients were sent to the ED directly from the CCF Clinic and 42 (89%) of these were admitted. Of the patients discharged home from CCF, 15 (3%) were later admitted to a hospital. Twelve (2%) patients in total died. CONCLUSION: The vast majority of patients, over 90%, seen in CCF were discharged home, with only a small percentage (3%) later requiring admission to a hospital. Of the patients sent directly to the ED from CCF, close to 90% were admitted, verifying the accuracy of our triage. Overall mortality was low (2%), especially when compared with mortality rates in New York City during the pandemic peak. Telemedicine was effective in identifying patients in need of in-person evaluation and in tracking and follow-up. Workflows and protocols were adaptable to reflect rapidly changing resources and clinical guidelines. Frequent communication through a diversity of methods was critical. Through these strategies, we were able to create a safe and effective outpatient program for patients with potential COVID-19.

Full-thickness macular holes after surgical repair of primary rhegmatogenous retinal detachments: incidence, clinical characteristics, and outcomes.

PURPOSE: Full-thickness macular hole (FTMH) formation following rhegmatogenous retinal detachment (RRD) repair may limit post-operative visual acuity and often requires a return to the operating room, but little is known about this phenomenon. METHODS: This study included all patients with a FTMH that developed after RRD repair from January 1, 2015-July 31, 2020. The main outcome was the rate of FTMH formation following RRD repair as well as characteristics of FTMH following RRD repair that spontaneously close. RESULTS: There were 470 eyes with a diagnosis of both a FTMH and a RRD during the study period. Of these, 27 (0.28%) developed a FTMH following RRD repair. The median time to FTMH diagnosis was 91 days (25th, 75th quartiles 40, 204 days). The mean minimum hole diameter was 514.5 ± 303.6 microns. There were 4 FTMHs (14.8%) that spontaneously closed without surgical intervention. The spontaneous closure was noted from 4 to 12 weeks after the initial diagnosis of the FTMH. These holes were smaller than the holes that did not close spontaneously (mean minimum diameter 161.8 ± 85.2 vs 588.7 ± 279.3 microns, p = 0.0058). Of the 27 post-operative FTMHs, there were 23 eyes (85%) that underwent surgical intervention with pars plana vitrectomy and internal limiting membrane peeling. Nineteen eyes (83%) closed with one surgery, 20 eyes (87%) ultimately closed, while 3 eyes (11.1%) did not close. CONCLUSIONS: FTMH is relatively uncommon to occur following RRD repair with a prevalence of 0.28% in our series with 87% of these holes achieving closure following surgery or spontaneously. Approximately 15% of FTMHs following RRD repair closed spontaneously and these holes were significantly smaller.

Elucidating carbohydrate metabolism in Euglena gracilis: Reverse genetics-based evaluation of genes coding for enzymes linked to paramylon accumulation.

Euglena gracilis is a eukaryotic single-celled and photosynthetic organism grouped under the kingdom Protista. This phytoflagellate can accumulate the carbon photoassimilate as a linear ß-1,3-glucan chain called paramylon. This storage polysaccharide can undergo degradation to provide glucose units to obtain ATP and reducing power both in aerobic and anaerobic growth conditions. Our group has recently characterized an essential enzyme for accumulating the polysaccharide, the UDP-glucose pyrophosphorylase (Biochimie vol 154, 2018, 176-186), which catalyzes the synthesis of UDP-glucose (the substrate for paramylon synthase). Additionally, the identification of nucleotide sequences coding for putative UDP-sugar pyrophosphorylases suggests the occurrence of an alternative source of UDP-glucose. In this study, we demonstrate the active involvement of both pyrophosphorylases in paramylon accumulation. Using techniques of single and combined knockdown of transcripts coding for these proteins, we evidenced a substantial decrease in the polysaccharide synthesis from 39 ± 7 µg/106 cells determined in the control at day 21st of growth. Thus, the paramylon accumulation in Euglena gracilis cells decreased by 60% and 30% after a single knockdown of the expression of genes coding for UDP-glucose pyrophosphorylase and UDP-sugar pyrophosphorylase, respectively. Besides, the combined knockdown of both genes resulted in a ca. 65% reduction in the level of the storage polysaccharide. Our findings indicate the existence of a physiological dependence between paramylon accumulation and the partitioning of sugar nucleotides into other metabolic routes, including the Leloir pathway's functionality in Euglena gracilis.

Effects of Contrast-Enhanced Ultrasound of Indeterminate Renal Masses on Patient Clinical Management: Retrospective Analysis From 2 Institutions.

OBJECTIVES: To investigate the long-term impact of contrast-enhanced ultrasound (CEUS) on the treatment of patients with indeterminate renal masses. METHODS: In this retrospective study, consecutive charts of all patients receiving renal CEUS at 1 of 2 academic medical centers between January 1, 2014, and December 31, 2018, were reviewed. Patients were included in the study if they had documented chronic renal disease (estimated glomerular filtration rate < 60 mL/min/1.73 m2 ) or prior nephrectomy and received CEUS for a previously untreated renal mass. RESULTS: A total of 215 lesions in 157 patients were used for analysis. Contrast-enhanced ultrasound provided a final treatment recommendation in 71.6% of lesions (154 of 215). Of these 154 lesions, 7.8% (12 of 154) were lost to follow-up despite CEUS suggesting malignancy; 15.6% (24 of 154) went directly for surgical intervention, with malignancy confirmed by pathologic results in 87.5% (21 of 24) of these cases; and the remaining 76.6% (118 of 154) were deemed benign and required no additional follow-up. Of the 118 lesions diagnosed by CEUS as benign and requiring no follow-up, none showed evidence of later renal cell carcinoma development and, only 5.1% (6 of 118) of the total population was referred for further cross-sectional imaging of the mass in question. In 28.4% of all lesions (61 of 215), CEUS resulted in a recommendation for surveillance imaging at a 6- to 12-month interval, and less than 10% (6 of 61) of these underwent additional cross-sectional imaging within the recommended 6 months after CEUS. CONCLUSIONS: These findings highlight the impact of CEUS on clinical treatment of indeterminate renal masses, including reducing the use of the potentially nephrotoxic contrast agents and providing a direct pathway to transplant.

Functional characterization of methionine sulfoxide reductases from Leptospira interrogans.

BACKGROUND: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. METHODS: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. RESULTS: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. CONCLUSIONS AND GENERAL SIGNIFICANCE: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.

Trypanothione Metabolism as Drug Target for Trypanosomatids.

Chagas Disease, African sleeping sickness, and leishmaniasis are neglected diseases caused by pathogenic trypanosomatid parasites, which have a considerable impact on morbidity and mortality in poor countries. The available drugs used as treatment have high toxicity, limited access, and can cause parasite drug resistance. Long-term treatments, added to their high toxicity, result in patients that give up therapy. Trypanosomatids presents a unique trypanothione based redox system, which is responsible for maintaining the redox balance. Therefore, inhibition of these essential and exclusive parasite's metabolic pathways, absent from the mammalian host, could lead to the development of more efficient and safe drugs. The system contains different redox cascades, where trypanothione and tryparedoxins play together a central role in transferring reduced power to different enzymes, such as 2-Cys peroxiredoxins, non-selenium glutathione peroxidases, ascorbate peroxidases, glutaredoxins and methionine sulfoxide reductases, through NADPH as a source of electrons. There is sufficient evidence that this complex system is essential for parasite survival and infection. In this review, we explore what is known in terms of essentiality, kinetic and structural data, and the development of inhibitors of enzymes from this trypanothione-based redox system. The recent advances and limitations in the development of lead inhibitory compounds targeting these enzymes have been discussed. The combination of molecular biology, bioinformatics, genomics, and structural biology is fundamental since the knowledge of unique features of the trypanothione-dependent system will provide tools for rational drug design in order to develop better treatments for these diseases.

On the functionality of a methionine sulfoxide reductase B from Trypanosoma cruzi.

BACKGROUND: Methionine is an amino acid susceptible to be oxidized to give a racemic mixture of R and S forms of methionine sulfoxide (MetSO). This posttranslational modification has been reported to occur in vivo under either normal or stress conditions. The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductases (MSRs), thiol-dependent enzymes present in almost all organisms. These enzymes can reduce specifically one or another of the isomers of MetSO (free and protein-bound). This redox modification could change the structure and function of many proteins, either concerned in redox or other metabolic pathways. The study of antioxidant systems in Trypanosoma cruzi has been mainly focused on the involvement of trypanothione, a specific redox component for these organisms. Though, little information is available concerning mechanisms for repairing oxidized methionine residues in proteins, which would be relevant for the survival of these pathogens in the different stages of their life cycle. METHODS: We report an in vitro functional and in vivo cellular characterization of methionine sulfoxide reductase B (MSRB, specific for protein-bound MetSO R-enantiomer) from T. cruzi strain Dm28c. RESULTS: MSRB exhibited both cytosolic and mitochondrial localization in epimastigote cells. From assays involving parasites overexpressing MSRB, we observed the contribution of this protein to increase the general resistance against oxidative damage, the infectivity of trypomastigote cells, and intracellular replication of the amastigote stage. Also, we report that epimastigotes overexpressing MSRB exhibit inhibition of the metacyclogenesis process; this suggesting the involvement of the proteins as negative modulators in this cellular differentiation. CONCLUSIONS AND GENERAL SIGNIFICANCE: This report contributes to novel insights concerning redox metabolism in T. cruzi. Results herein presented support the importance of enzymatic steps involved in the metabolism of L-Met and in repairing oxidized macromolecules in this parasite.

Conjunctival Melanoma: Features and Outcomes Based on the Fitzpatrick Skin Type in 540 Patients at a Single Ocular Oncology Center.

PURPOSE: To determine the association of Fitzpatrick skin type (FST) with conjunctival melanoma. METHODS: Retrospective case series of 540 patients with conjunctival melanoma to assess clinical features and outcomes per FST. RESULTS: The FST was Type I (n = 126, 23%), II (n = 337, 62%), III (n = 56, 10%), IV (n = 8, 2%), V (n = 12, 2%), and VI (n = 1, <1%). A comparison (FST I vs. II vs. III, IV, V, and VI) revealed Types I and II associated with older mean patient age (63.9 vs. 60.7 vs. 51.1 years, p < 0.001), greater percentage of female patients (68% vs. 44% vs. 42%, p < 0.001), lower frequency of complexion associated melanosis (1% vs. 2% vs. 13%, p < 0.001), smaller tumor thickness (2.1 vs. 2.8 vs. 3.6 mm, p = 0.01), and less eyelid involvement (13% vs. 13% vs. 28%, p = 0.02). Kaplan-Meier estimates for 5-year risk showed no difference by Types for visual acuity loss ≥3 lines, local tumor recurrence, exenteration, metastasis, or death. CONCLUSION AND RELEVANCE: Most patients with conjunctival melanoma show FST I or II, and this demonstrated no association with 5-year rate of vision loss, tumor recurrence, exenteration, metastasis, or death.

Biological activity of esters of quinoxaline-7-carboxylate 1,4-di-N-oxide against E. histolytica and their analysis as potential thioredoxin reductase inhibitors.

Amoebiasis is caused by the protozoan Entamoeba histolytica that affects millions of people throughout the world. The standard treatment is metronidazole, however, this drug causes several side effects, and is also mutagenic and carcinogenic. Therefore, the search for therapeutic alternatives is necessary. Quinoxaline 1,4-di-N-oxides (QdNOs) derivatives have been shown to exhibit activity against different protozoan. In the present study, the effects of esters of quinoxaline-7-carboxylate 1,4-di-N-oxide (7-carboxylate QdNOs) derivatives on E. histolytica proliferation, morphology, ultrastructure, and oxidative stress were evaluated, also their potential as E. histolytica thioredoxin reductase (EhTrxR) inhibitors was analyzed. In vitro tests showed that 12 compounds from n-propyl and isopropyl series, were more active (IC50 = 0.331 to 3.56 µM) than metronidazole (IC50 = 4.5 µM). The compounds with better biological activity have a bulky, trifluoromethyl and isopropyl group at R1-, R2-, and R3-position, respectively. The main alterations found in trophozoites treated with some of these compounds included changes in chromatin, cell granularity, redistribution of vacuoles with cellular debris, and an increase in reactive oxygen species. Interestingly, docking studies suggested that 7-carboxylate QdNOs derivatives could interact with amino acid residues of the NADPH-binding domain and/or the redox-active site of EhTrxR. Enzymatic assays demonstrated that selected 7-carboxylate QdNOs inhibits EhTrxR disulfide reductase activity, and diaphorase activity shows that these compounds could act as electron acceptor substrates for the enzyme. Taken together, these data indicate that among the mechanisms involved in the antiamoebic effect of the 7-carboxylate QdNOs derivatives studied, is the induction of oxidative stress and the inhibition of EhTrxR activity.