Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras.

Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.

Same day service: A genetic testing station model to improve germline genetic testing in patients with ovarian cancer.

OBJECTIVE: Genetic testing for ovarian cancer (OC) patients is essential to consideration of PARP inhibitor therapy. To improve access, we piloted a Genetic Testing Station (GTS) allowing patients to have a same-day genetic testing visit facilitated by Genetic Counselor Assistants (GCAs) under the supervision of Genetic Counselors (GCs). METHODS: The GTS was implemented December 2018 and operated through February 2020. Gynecologic Oncologists offered ovarian cancer patients a same-day GTS visit with a GCA. The patient received education via videos designed by GCs and then provided consent, a brief family history, and a sample for a standardized 133-gene panel. Results were provided by a GC. Patients were retrospectively identified by querying the medical record for OC patients seen 12 months prior to and 18 months after GTS implementation. RESULTS: A total of 482 patients pre-GTS were compared to 625 patients post-GTS. Genetic testing increased from 68.5% to 75.4% (p = 0.012) after implementation, primarily in patients with epithelial histologies (80% vs 89% in pre-GTS vs post-GTS, p = 0.005). Time from referral for genetic testing to obtaining results was evaluated in the post-GTS cohort, comparing patients who had traditional counseling to those who utilized the GTS. Time to obtaining results was 21 days in the GTS group (95% CI [10, 34]) compared to 56 days (95% CI [41,76]) in the traditional genetic counseling group. CONCLUSIONS: The GTS reduces barriers to care and facilitates discussion of precision treatment within a timely fashion while optimizing GC clinic time. Access improvement remains integral to improving uptake of genetic testing.

Rehabilitación cardíaca integral en un caso de paciente con miocardiopatía por SARS-COV-2 / Comprehensive Cardiac Rehabilitation in a Case of a Patient with SARS-CoV-2 Cardiomyopathy

Introducción: Una complicación reportada en la infección por SARS-CoV-2 es la miocardiopatía. Se ha descrito que los pacientes pueden presentar un deterioro significativo de la clase funcional, que limita el desempeño en las actividades de la vida diaria, sociales, familiares y laborales. La rehabilitación cardíaca como componente integral es una alternativa de tratamiento no farmacológica en el manejo de secuelas cardiovasculares ocasionadas por SARS-CoV-2. Objetivo: Describir el impacto de la rehabilitación cardíaca integral en un caso de paciente con miocardiopatía por SARS-CoV-2. Caso clínico: Se presenta el caso de un hombre de 56 años de edad con miocardiopatía por SARS-CoV-2 quien realizó rehabilitación cardíaca integral. Conclusiones: La rehabilitación cardíaca con un componente de intervención integral multidisciplinar permitió al paciente mejorar la capacidad aeróbica, sintomatología, calidad de vida relacionada con la salud y reincorporarse a las actividades sociales, familiares y laborales(AU)

Introduction: A reported complication of SARS-CoV-2 infection is cardiomyopathy. It has been described that patients may present with significant impairment of functional class, limiting performance in activities of daily living, social, family and work. Cardiac rehabilitation as an integral component is a non-pharmacological treatment alternative in the management of cardiovascular sequelae caused by SARS-CoV-2. Objective: To describe the impact of comprehensive cardiac rehabilitation in a case of a patient with cardiomyopathy due to SARS-CoV-2. Case report: We present the case of a 56-year-old patient with SARS-CoV-2 cardiomyopathy who underwent comprehensive cardiac rehabilitation. Conclusions: Cardiac rehabilitation with a comprehensive multidisciplinary intervention component allowed the patient to improve aerobic capacity, symptomatology, health-related quality of life and reincorporation to social, family and work activities(AU)

Understanding the Professional Care Experience of Patients with Stroke: A Qualitative Study Using In-Depth Interviews.

Background: Professional support and communication stimulates the professional-patient relationship and supports the recovery of stroke patients. Objectives: To describe the perspectives of patients with stroke regarding communication, professional support, and their ability to participate in processes and integrated care with health providers. Methods: A qualitative study was conducted. A purposeful sampling and snowball-technique were used. Patients diagnosed with moderate or severe stroke in the post-acute or chronic stage of the disease were included. Data collection consisted of in-depth interviews and researcher field notes. A thematic analysis was performed. Results: Thirty-one patients were included. Three themes were identified: 1) Providing support, with four categories, professional behavior, personalized attention, the heart of the professional and building a bond with the patient; 2) Facilitating communication, with three categories, the patient as the recipient, the content of the message and the channel, and the professional as the person that conveys the message; and 3) Promoting participation, with two categories, barriers, and incentives to participate. Conclusions: When providing support, professionals should consider communicating information and encouraging the participation of stroke patients for integrated care.

Person-centred care in individuals with stroke: a qualitative study using in-depth interviews.

BACKGROUND: Person-centred care (PCC) has considerable effects on the clinical practice of health professionals. The purpose of this study was to describe the perspectives and perceived barriers and enablers of individuals with stroke regarding the PCC model in stroke rehabilitation. METHODS: A qualitative exploratory study was conducted based on an interpretive framework. Participants were recruited using non-probabilistic purposeful sampling and a snowball-technique strategy. The inclusion criteria consisted of: (a) individuals > 18 years, (b) diagnosed with moderate or severe stroke according to the National Institutes of Health Stroke Scale and (c) in the post-acute or chronic stage of the disease. In total, 31 individuals with stroke were included. In-depth interviews and researchers' field notes were used to collect the data. A thematic analysis was performed. Also, credibility, transferability, dependability and confirmability techniques were followed to establish trustworthiness of the data. RESULTS: Thirty-one individuals with stroke (11 women) were included. Three main themes were identified: (a) The person behind the "patient" label, recognizing the person beyond their illness and valuing their identity and individual characteristics, (b) The person at the centre of care, considering themselves as an active agent in their own care and respecting their preferences and expectations for their care process and (c) Training for PCC, providing health professionals with tools to achieve professional skills for the implementation and development of the PCC model. CONCLUSIONS AND SIGNIFICANCE: This paper describes relevant aspects that health professionals should consider when providing PCC in the context of the rehabilitation of individuals with stroke. Key messagesThe individuals' perspective regarding person-centred care (PCC) has considerable effects on the clinical practice of health professionals.Individuals with stroke describe how there is a person behind the "patient" label, with identity, needs and desire to participate in decision making.Training in the PCC model helps healthcare professionals identify the needs of individuals with stroke during rehabilitation.

Recomendaciones para la rehabilitación pulmonar en pacientes con COVID-19 / Recommendations for pulmonary rehabilitation in patients with COVID-19

Introducción: La COVID-19 es una enfermedad que desencadena infecciones respiratorias graves como el síndrome respiratorio agudo severo, que puede requerir atención prolongada intra y extrahospitalaria. Objetivo: Brindar recomendaciones y pautas para una correcta evaluación y tratamiento del paciente con COVID-19 mediante la rehabilitación pulmonar. Materiales y métodos: Revisión de la literatura en las bases de datos MEDLINE, PEDRO, Scielo y en Google Scholar, a partir de las palabras clave: COVID-19, Síndrome de Dificultad Respiratoria del Adulto, Enfermedades pulmonares, Rehabilitación, pruebas de función respiratoria, Unidad de cuidado intensivo; seleccionando, aquellos artículos que presentaran información sobre rehabilitación en pacientes con COVID-19 y otras alteraciones con compromiso similar como el Síndrome Respiratorio Agudo y las Enfermedades Pulmonares Intersticiales Difusas. Resultados: Para realizar una intervención en paciente con COVID-19 u otra alteración respiratoria como Síndrome de Dificultad Respiratoria del Adulto y Enfermedad Pulmonar Intersticial Difusa, es necesario realizar un adecuado diagnóstico de la enfermedad, una minuciosa evaluación integral y una intervención basada en las necesidades de cada individuo. Conclusiones: El paciente intrahospitalario puede recibir intervención enfocada a evitar el deterioro funcional y una vez son dados de alta la rehabilitación pulmonar extrahospitalaria podría ser una alternativa eficaz en el tratamiento para los pacientes.

Introduction: COVID-19 is a disease that triggers serious respiratory infections such as severe acute respiratory syndrome, which requires treatment on an inpatient and outpatient basis. Objective: To provide recommendations and guidelines for the appropriate assessment and treatment of COVID-19 patients through pulmonary rehabilitation. Materials and methods: A literature review using MEDLINE, PEDRO, Scielo and Google Scholar databases and the keywords: COVID-19, Adult Respiratory Distress Syndrome, Pulmonary diseases, Rehabilitation, Respiratory function tests, Intensive Care Unit. Articles that described information regarding rehabilitation in COVID-19 patients and other similar dysfunctions such as Acute Respiratory Syndrome and Diffuse Interstitial Lung Diseases. Results: It is necessary to carry out an appropriate disease diagnosis, a detailed integral assessment, and an intervention based on the needs of each patient in order to intervene with either COVID-19 patients or cases with other respiratory dysfunctions such as Adult Respiratory Distress Syndrome and Diffuse Interstitial Lung Disease. Conclusions: Inpatients can be subjected to an intervention focused on avoiding functional failure. Once they are discharged, outpatient pulmonary rehabilitation could be an effective treatment alternative for these patients.

Corrigendum: The sole DNA ligase in Entamoeba histolytica is a high-fidelity DNA ligase involved in DNA damage repair.

[This corrects the article DOI: 10.3389/fcimb.2018.00214.].

Survival impact of bowel resection at the time of interval cytoreductive surgery for advanced ovarian cancer.

OBJECTIVES: To evaluate the impact of bowel resection at the time of interval cytoreductive surgery on survival. METHODS: We identified patients with advanced ovarian cancer who underwent neoadjuvant chemotherapy and interval cytoreductive surgery between 2008 and 2018 from a single-institution tumor registry. Kaplan-Meier survival analysis and Cox proportional hazards models were performed comparing patients who underwent bowel resection to those who did not. RESULTS: Of 158 patients, 43 (27%) underwent bowel resection. Rates of optimal (95%) and sub-optimal (5%) resection did not differ with bowel resection. Patients that required bowel resection had worse three-year survival (43% vs. 63%), even after adjusting for confounding variables of age, stage, number of neoadjuvant cycles, R0 resection, and ASA score (HR 2.27, p < 0.01). Adjusted progression-free survival did not differ between groups (HR 0.92, p = 0.72). Patients who underwent bowel resection were more likely to require blood transfusion (p < 0.01), and have a longer hospital stay (5 days vs 7.5 days, p < 0.01). CONCLUSIONS: Bowel resection at the time of interval cytoreduction confers a greater than 2-fold increased risk of mortality and does not impact progression-free survival. Long-term sequelae of the peri-operative morbidity of bowel resection may contribute to increased mortality, and bowel resection may be a surrogate for disease biology with poor prognosis.

Differential activity on trypanosomatid parasites of a novel recombinant defensin type 1 from the insect Triatoma (Meccus) pallidipennis.

Defensins are one of the major families of antimicrobial peptides (AMPs) that are widely distributed in insects. In Triatomines (Hemiptera: Reduviidae) vectors of Trypanosoma cruzi the causative agent of Chagas disease, two large groups of defensin isoforms have been described: type 1 and type 4. The aim of this study was to analyze the trypanocidal activity of a type 1 recombinant defensin (rDef1.3) identified in Triatoma (Meccus) pallidipennis, an endemic specie from México. The trypanocidal activity of this defensin was evaluated in vitro, against the parasites T. cruzi, T. rangeli, and two species of Leishmania (L. mexicana and L. major) both causative agents of cutaneous leishmaniasis. Our data demonstrated that the defensin was active against all the parasites although in different degrees. The defensin altered the morphology, reduced the viability and inhibited the growth of T.cruzi. When tested against T. rangeli (a parasite that infects a variety of mammalian species), stronger morphological effects where observed. Surprisingly the greatest effects were observed against the two Leishmania species, of which L. major was the parasite most affected with 50% of dead cells or with damaged membranes, in addition of a reduction in its proliferative capacity in culture. These results suggest that rDef1.3 has an important antimicrobial effect against trypanosomatids which cause some of the more important neglected tropical diseases transmitted by insect vectors.

Validación del índice de gravedad del síndrome de Cushing en pacientes con síndrome de Cushing iatrógénico / Validity of the Cushing's Syndrome Severity Index in Patients with Iatrogenic Cushing's Syndrome

Introducción: Actualmente no se cuenta con instrumentos clinimétricos para la medición de la gravedad del síndrome de Cushing iatrógeno (SCI). Sonino et al. crearon un índice clínico de gravedad del síndrome de Cushing endógeno (IGC), pero no ha sido aplicado a pacientes con SCI. Objetivo: Validar y determinar la utilidad del IGC y su correlación con variables clínicas en pacientes reumatológicos con uso de glucocorticoides (GC) de forma continua. Sujetos y métodos: Se incluyó a pacientes con antecedentes de uso de GC sistémicos de forma continua indicados para el tratamiento de la enfermedad reumatológica, por lo menos durante 4 semanas. Todos los pacientes rellenaron un cuestionario sobre datos sociodemográficos, características de los GC empleados, la forma de uso y la presencia o ausencia de eventos adversos. Se aplicó el IGC por 2 observadores de forma independiente. Se calculó la consistencia, la concordancia interobservador y un análisis de componentes principales. Resultados: Se estudió a 32 pacientes con edad promedio de 35,72±12,8 años; 29 eran mujeres (90,6%). La calificación promedio del IGC por el primer observador fue de 3,50±2,02 y la del segundo observador fue de 2,31±1,75 (p=0,004). La concordancia interobservador fue baja en los ítems con definiciones imprecisas, por lo cual se modificaron las definiciones para mejorar su desempeño. Los puntajes del IGC correlacionaron con la presencia de efectos adversos y el tipo de dosis utilizada. Conclusiones: El IGC tiene una adecuada correlación con manifestaciones clínicas en pacientes con uso crónico de GC. Las características clinimétricas del cuestionario mejoraron al estandarizar las definiciones de variables clínicas subjetivas.(AU)

Introduction: Currently there are no clinimetric instruments for the measurement of the severity of iatrogenic Cushing's syndrome (ICS). Sonino et al. created a clinical severity index of endogenous Cushing's disease (CSI) but it has not been applied to patients with ICS. Objective: To validate and determine the utility of the CSI and its correlation with clinical variables in rheumatological patients with continuous use of glucocorticoids (GC). Subjects and methods: Patients with a history of continuous systemic GC use (for at least 4 weeks) indicated for treatment of rheumatological disease were included. All the patients filled out a questionnaire on sociodemographic data, characteristics of the CG used; the way of use and the presence or absence of adverse events. The CSI was applied by 2 observers independently. Consistency, interobserver concordance and principal component analysis were calculated. Results: A total of 32 patients with an average age of 35.72±12.8 years were studied; 29 were women (90.6%). The average CSI score by the first observer was 3.50±2.02, and by the second observer was 2.31±1.75 (p=.004). The interobserver concordance was low in the items with imprecise definitions; for which modifications were made in the definitions to improve their performance. The CSI scores correlated with the presence of adverse effects and the type of dose used. Conclusions: The CSI has an adequate correlation with clinical manifestations in patients with chronic use of GC. The clinimetric characteristics of the questionnaire improved by standardising the definitions of subjective clinical variables.(AU)

A Bisphosphonate With a Low Hydroxyapatite Binding Affinity Prevents Bone Loss in Mice After Ovariectomy and Reverses Rapidly With Treatment Cessation.

Bisphosphonates (BPs) are a mainstay of osteoporosis treatment; however, concerns about bone health based on oversuppression of remodeling remain. Long-term bone remodeling suppression adversely affects bone material properties with microdamage accumulation and reduced fracture toughness in animals and increases in matrix mineralization and atypical femur fractures in patients. Although a "drug holiday" from BPs to restore remodeling and improve bone quality seems reasonable, clinical BPs have long functional half-lives because of their high hydroxyapatite (HAP) binding affinities. This places a practical limit on the reversibility and effectiveness of a drug holiday. BPs with low HAP affinity and strong osteoclast inhibition potentially offer an alternative approach; their antiresorptive effect should reverse rapidly when dosing is discontinued. This study tested this concept using NE-58025, a BP with low HAP affinity and moderate osteoclast inhibition potential. Young adult female C57Bl/6 mice were ovariectomized (OVX) and treated with NE-58025, risedronate, or PBS vehicle for 3 months to test effectiveness in preventing long-term bone loss. Bone microarchitecture, histomorphometry, and whole-bone mechanical properties were assessed. To test reversibility, OVX mice were similarly treated for 3 months, treatment was stopped, and bone was assessed up to 3 months post-treatment. NE-58025 and RIS inhibited long-term OVX-induced bone loss, but NE-58025 antiresorptive effects were more pronounced. Withdrawing NE-58025 treatment led to the rapid onset of trabecular resorption with a 200% increase in osteoclast surface and bone loss within 1 month. Cessation of risedronate treatment did not lead to increases in resorption indices or bone loss. These results show that NE-58025 prevents OVX-induced bone loss, and its effects reverse quickly following cessation treatment in vivo. Low-HAP affinity BPs may have use as reversible, antiresorptive agents with a rapid on/off profile, which may be useful for maintaining bone health with long-term BP treatment. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Validity of the Cushing's Syndrome Severity Index in Patients with Iatrogenic Cushing's Syndrome. / Validación del índice de gravedad del síndrome de Cushing en pacientes con síndrome de Cushing iatrógénico.

INTRODUCTION: Currently there are no clinimetric instruments for the measurement of the severity of iatrogenic Cushing's syndrome (ICS). Sonino et al. created a clinical severity index of endogenous Cushing's disease (CSI) but it has not been applied to patients with ICS. OBJECTIVE: To validate and determine the utility of the CSI and its correlation with clinical variables in rheumatological patients with continuous use of glucocorticoids (GC). SUBJECTS AND METHODS: Patients with a history of continuous systemic GC use (for at least 4 weeks) indicated for treatment of rheumatological disease were included. All the patients filled out a questionnaire on sociodemographic data, characteristics of the CG used; the way of use and the presence or absence of adverse events. The CSI was applied by 2 observers independently. Consistency, interobserver concordance and principal component analysis were calculated. RESULTS: A total of 32 patients with an average age of 35.72±12.8 years were studied; 29 were women (90.6%). The average CSI score by the first observer was 3.50±2.02, and by the second observer was 2.31±1.75 (p=.004). The interobserver concordance was low in the items with imprecise definitions; for which modifications were made in the definitions to improve their performance. The CSI scores correlated with the presence of adverse effects and the type of dose used. CONCLUSIONS: The CSI has an adequate correlation with clinical manifestations in patients with chronic use of GC. The clinimetric characteristics of the questionnaire improved by standardising the definitions of subjective clinical variables.

Delivery of Antisense DNA into Pathogenic Parasite Trypanosoma cruzi Using Virus-Like Protein-Based Nanoparticles.

Trypanosoma cruzi, which causes Chagas disease, is one of the most lacerating parasites in terms of health and social impacts. New approaches for its study and treatment are urgently needed since in more than 50 years only two drugs have been approved. Genetic approaches based on antisense oligonucleotides (AONs) are promising; however, to harness their full potential the development of effective carriers is paramount. Here, we report the use of an engineered virus-like protein C-BK12 to transfect AONs into T. cruzi. Using gel electrophoresis, Dynamic Light Scattering, and atomic force microscopy, we found that C-BK12 binds AONs and forms 10-25 nm nanoparticles (NPs), which are very stable when incubated in biological media, only releasing up to 25% of AON. Fluorescence microscopy and qPCR revealed that the NPs successfully delivered AONs into epimastigotes and reduced the expression of a target gene down to 68%. Importantly, the protein did not show cytotoxicity. The combination of high stability and capability to transfect and knock down gene expression without causing cell damage and death makes the protein C-BK12 a promising starting point for the further development of safe and effective carriers to deliver AONs into T. cruzi for biological studies.

Lysosomal Membrane Stability in Hemocytes and Micronuclei in Gills of Perumytilus purpuratus Lamarck 1819 (Bivalvia: Mytilidae) Exposed to Copper.

The aim of this study was to determine the cytotoxic and genotoxic effects of copper on the bivalve Perumytilus purpuratus. The individuals were exposed to three copper concentrations: 1, 30 and 45 µg L-1 for 24, 48 and 96 h. Lysosomal membrane stability in hemocytes was determined through the neutral red retention time (NRRT) and micronucleus (MN) frequency tests in hemocytes and gills. The results show that the NRRT decreased significantly at 30 µg L-1 after 48 h of exposure. The frequency of MN was significantly greater in gills after 24 h in all concentrations tested. Copper is cytotoxic from 30 µg L-1 and genotoxic from 1 µg L-1. The use of these biomarkers of effects in P. purpuratus is proposed as an early warning tool for monitoring in environmental assessment of coastal ecosystems impacted by mining activities.

Glucose-restriction increases Trichomonas vaginalis cellular damage towards HeLa cells and proteolytic activity of cysteine proteinases (CPs), such as TvCP2.

Trichomonas vaginalis induces cellular damage to the host cells (cytotoxicity) through the proteolytic activity of multiple proteinases of the cysteine type (CPs). Some CPs are modulated by environmental factors such as iron, zinc, polyamines, etc. Thus, the goal of this study was to assess the effect of glucose on T. vaginalis cytotoxicity, proteolytic activity and the particular role of TvCP2 (TVAG_057000) during cellular damage. Cytotoxicity assays showed that glucose-restriction (GR) promotes the highest HeLa cell monolayers destruction (~95%) by trichomonads compared to those grown under high glucose (~44%) condition. Zymography and Western blot using different primary antibodies showed that GR increased the proteolytic activity, amount and secretion of certain CPs, including TvCP2. We further characterized the effect of glucose on TvCP2. TvCP2 increases in GR, localized in vesicles close to the plasma membrane and on the surface of T. vaginalis. Furthermore, pretreatment of GR-trichomonads with an anti-TvCP2r polyclonal antibody specifically reduced the levels of cytotoxicity and apoptosis induction to HeLa cells in a concentration-dependent manner. In conclusion, our data show that GR, as a nutritional stress condition, promotes trichomonal cytotoxicity to the host cells, increases trichomonad proteolytic activity and amount of CPs, such as TvCP2 involved in cellular damage.

Telomeric Repeat-Binding Factor Homologs in Entamoeba histolytica: New Clues for Telomeric Research.

Telomeric Repeat Binding Factors (TRFs) are architectural nuclear proteins with critical roles in telomere-length regulation, chromosome end protection and, fusion prevention, DNA damage detection, and senescence regulation. Entamoeba histolytica, the parasite responsible of human amoebiasis, harbors three homologs of human TRFs, based on sequence similarities to their Myb DNA binding domain. These proteins were dubbed EhTRF-like I, II and III. In this work, we revealed that EhTRF-like I and II share similarity with human TRF1, while EhTRF-like III shares similarity with human TRF2 by in silico approach. The analysis of ehtrf-like genes showed they are expressed differentially under basal culture conditions. We also studied the cellular localization of EhTRF-like I and III proteins using subcellular fractionation and western blot assays. EhTRF-like I and III proteins were enriched in the nuclear fraction, but they were also present in the cytoplasm. Indirect immunofluorescence showed that these proteins were located at the nuclear periphery co-localizing with Lamin B1 and trimethylated H4K20, which is a characteristic mark of heterochromatic regions and telomeres. We found by transmission electron microscopy that EhTRF-like III was located in regions of more condensed chromatin. Finally, EMSA assays showed that EhTRF-like III forms specific DNA-protein complexes with telomeric related sequences. Our data suggested that EhTRF-like proteins play a role in the maintenance of the chromosome ends in this parasite.

Characterization of a novel endogenous cysteine proteinase inhibitor, trichocystatin-3 (TC-3), localized on the surface of Trichomonas vaginalis.

Trichomonas vaginalis is a flagellated protist responsible for human trichomoniasis. T. vaginalis has three genes encoding for endogenous cysteine proteinase (CP) inhibitors, known as trichocystatin-1 through trichocystatin-3 (TC-1, TC-2, and TC-3). These inhibitors belong to the cystatin family. In this study, we characterized trichocystatin-3 (TC-3), an endogenous cysteine proteinase (CP) inhibitor of T. vaginalis. TC-3 possesses a signal peptide in the N-terminus and two putative glycosylation sites (typical of family 2, cystatins) but lacks the PW motif and cysteine residues (typical of family 1, stefins). Native TC-3 was recognized as an ∼18 kDa protein band in a T. vaginalis protein extract. By confocal microscopy, endogenous TC-3 was found in the Golgi complex, cytoplasm, large vesicles, and the plasma membrane. These localizations are consistent with an in silico prediction. In addition, the purified recombinant protein (TC-3r) functions as an inhibitor of cathepsin L CPs, such as human liver cathepsin L and trichomonad CPs, present in a proteinase-resistant extract (PRE). Via a pull-down assay using TC-3r as bait and PRE, we identified several trichomonad CPs targeted by TC-3, primarily TvCP3. These CP-TC-3 interactions occur in vesicles, in the cytoplasm, and on the parasite surface. In addition, TC-3r showed a protective effect on HeLa cell monolayers by inhibiting trichomonad surface CPs involved in cellular damage. Our results show that the endogenous inhibitor TC-3 plays a key role in the regulation of endogenous CP proteolytic activity.

The Sole DNA Ligase in Entamoeba histolytica Is a High-Fidelity DNA Ligase Involved in DNA Damage Repair.

The protozoan parasite Entamoeba histolytica is exposed to reactive oxygen and nitric oxide species that have the potential to damage its genome. E. histolytica harbors enzymes involved in DNA repair pathways like Base and Nucleotide Excision Repair. The majority of DNA repairs pathways converge in their final step in which a DNA ligase seals the DNA nicks. In contrast to other eukaryotes, the genome of E. histolytica encodes only one DNA ligase (EhDNAligI), suggesting that this ligase is involved in both DNA replication and DNA repair. Therefore, the aim of this work was to characterize EhDNAligI, its ligation fidelity and its ability to ligate opposite DNA mismatches and oxidative DNA lesions, and to study its expression changes and localization during and after recovery from UV and H2O2 treatment. We found that EhDNAligI is a high-fidelity DNA ligase on canonical substrates and is able to discriminate erroneous base-pairing opposite DNA lesions. EhDNAligI expression decreases after DNA damage induced by UV and H2O2 treatments, but it was upregulated during recovery time. Upon oxidative DNA damage, EhDNAligI relocates into the nucleus where it co-localizes with EhPCNA and the 8-oxoG adduct. The appearance and disappearance of 8-oxoG during and after both treatments suggest that DNA damaged was efficiently repaired because the mainly NER and BER components are expressed in this parasite and some of them were modulated after DNA insults. All these data disclose the relevance of EhDNAligI as a specialized and unique ligase in E. histolytica that may be involved in DNA repair of the 8-oxoG lesions.

Dataset of cathepsin L-like CP inhibition of Naegleria fowleri and Acanthamoeba castellanii by ppTvCP4r from Trichomonas vaginalis.

The recombinant TvCP4 prepro region (ppTvCP4r) acts as an exogenous inhibitor of cathepsin L-like CPs from Trichomonas vaginalis (Cárdenas-Guerra et al., 2015 [1]). Here, we present the dataset of the trichomonad ppTvCP4r inhibitory effect against the CP proteolytic activities from other microorganisms, such as Naegleria fowleri and Acanthamoeba castellanii free-living amoeba. The proteolytic activity inhibition of total crude extracts (TCEs) of N. fowleri and A. castellanii was determined and recorded using a fluorogenic substrate specific for cathepsin L CPs without or with a ppTvCP4r treatment at different concentrations and pH.