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.

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.

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.

Factors influencing fertility-sparing treatment for gynecologic malignancies: A survey of Society of Gynecologic Oncology members.

OBJECTIVES: This study aims to examine practice patterns of gynecologic oncologists (GO) regarding fertility-sparing treatments (FST) for gynecology malignancies and explores attitudes toward collaboration with reproductive endocrinologists (RE). METHODS: An anonymous 23-question survey was sent to 1087 GO with a 14.0% completion rate. Descriptive statistics, Fisher's exact test, and Chi-square tests were used for data analysis. RESULTS: The majority of GOs offer FST for gynecologic malignancies. Providers seeing larger numbers of reproductive age women were more likely to consider cancer prognosis (p<0.03) and cancer stage (p<0.01) as key factors. Providers in the Midwestern US considered socioeconomic status more often when offering FST than those in the South (p<0.04). Those practicing in urban settings were more likely to feel that collaborating with a RE prior to treatment could improve treatment planning for women considering FST (p<0.02). Finally, providers in urban or suburban areas more often felt collaboration with a RE improves pregnancy outcomes in women who pursue FST (p<0.01, p<0.02) compared to rural practitioners. CONCLUSIONS: While FST offers women the chance to pursue pregnancy after cancer, there are minimal data on factors that influence whether FST is offered and if collaboration with a RE is sought in the management of these patients. The number of reproductive age women seen, geographic location, and practice setting are important variables that may influence current practice. Understanding these factors can help identify opportunities to improve oncologic and reproductive outcomes of this patient population.

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.

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.

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.].

Pyruvate:ferredoxin oxidoreductase (PFO) is a surface-associated cell-binding protein in Trichomonas vaginalis and is involved in trichomonal adherence to host cells.

The Trichomonas vaginalis 120 kDa protein adhesin (AP120) is induced under iron-rich conditions and has sequence homology with pyruvate:ferredoxin oxidoreductase A (PFO A), a hydrogenosomal enzyme that is absent in humans. This homology raises the possibility that, like AP120, PFO might be localized to the parasite surface and participate in cytoadherence. Here, the cellular localization and function of PFO that was expressed under various iron concentrations was investigated using a polyclonal antibody generated against the 50 kDa recombinant C-terminal region of PFO A (anti-PFO50). In Western blot assays, this antibody recognized a 120 kDa protein band in total protein extracts, and proteins with affinity to the surface of HeLa cells from parasites grown under iron-rich conditions. In addition to localization that is typical of hydrogenosomal proteins, PFOs that were expressed under iron-rich conditions were found to localize at the surface. This localization was demonstrated using immunofluorescence and co-localization assays, as well as immunogold transmission electron microscopy. In addition to describing its enzyme activity, we describe a novel function in trichomonal host interaction for the PFO localized on the parasite surface. The anti-PFO50 antibody reduced the levels of T. vaginalis adherence to HeLa cell monolayers in a concentration-dependent manner. Thus, T. vaginalis PFO is an example of a surface-associated cell-binding protein that lacks enzyme activity and that is involved in cytoadherence. Additionally, PFO behaves like AP120 in parasites grown under iron-rich conditions. Therefore, these data suggest that AP120 and PFO A are encoded by the same gene, namely pfo a.

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.

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.

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.

Immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis.

Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad-active degradome, and the immunoproteome were obtained by using 2-DE, 2-D-zymograms, 2-D-Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty-nine silver-stained spots were detected in the region of 200-21 kDa of parasite protease-resistant extracts. A similar proteolytic pattern was observed in the 2-D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4-like, TvCP12, TvCPT, TvLEGU-1, and another legumain-like CP). The major reactive spots to T. vaginalis-positive patient sera by 2-D-WB corresponded to four papain-like (TvCP2, TvCP4, TvCP4-like, TvCPT), and one legumain-like (TvLEGU-1) CPs. The genes of TvCP4, TvCPT, and TvLEGU-1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture-positive patient sera in 1-D-WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.

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.

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.

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)

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.

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.

Trichomonas vaginalis cathepsin D-like aspartic proteinase (Tv-CatD) is positively regulated by glucose and degrades human hemoglobin.

Trichomonas vaginalis genome encodes ∼440 proteases, six of which are aspartic proteases (APs). However, only one belongs to a clan AA (EC 3.4.23.5), family A1 (pepsin A), cathepsin D-like protease. This AP is encoded by an 1113-bp gene (tv-catd), which translates into a 370-aa residues zymogen of 40.7-kDa and a theoretical pI of 4.6, generating a ∼35 kDa active enzyme after maturation (Tv-CatD). The goal of this study was to identify and analyze the effect of glucose on the expression of Tv-CatD at the transcript and protein levels, subcellular localization, and proteolytic activity. The qRT-PCR assays showed a ∼2-fold increase in tv-catd mRNA under high-glucose (HG) conditions compared to glucose-restriction (GR) conditions. We amplified, cloned, and expressed the tv-catd gene, and purified the recombinant precursor enzyme (Tv-CatDr) to generate a polyclonal antibody (anti-Tv-CatDr). Western blot (WB) and immunolocalization assays showed that glucose increases the amount of Tv-CatD in different subcellular localizations and in in vitro secretions. Additionally, Tv-CatD proteolytic activity was detected in protease-resistant extracts (PREs) using a synthetic fluorogenic peptide specific for cathepsin D/E APs at different pHs and in the presence of AP inhibitors. In a two-dimensional (2-DE) WB analysis of a PRE from parasites grown under GR and HG conditions, an anti-Tv-CatDr antibody detected a 35-kDa protein spot at pI 5.0 identified as the mature Tv-CatD form by mass spectrometry that showed proteolytic activity in 2-DE zymograms copolymerized with hemoglobin under both glucose conditions. Thus, Tv-CatD could be involved in trichomonal hemolysis.