[Prevalence of obstructive sleep apnea syndrome in Chilean adults. A sub-study of the national health survey, 2016/17]. / Estudio de prevalencia de síndrome de apneas obstructivas del sueño en la población adulta chilena. Subestudio de la Encuesta Nacional de Salud, 2016/17.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) affects approximately 10%-20% of adults and is associated with obesity, hypertension and metabolic syndrome. AIM: To assess the prevalence and risk factors associated with OSAS in Chilean adults. MATERIAL AND METHODS: A standardized sleep questionnaire and respiratory polygraphy at home were conducted on adults aged 18 years or more, residing in the Metropolitan Region and enrolled in the 2016/17 National Health Survey. RESULTS: Two-hundred and five people between 18 and 84 years old (46% men, mean age 50 years) underwent overnight respiratory polygraphy at home. The estimated obstructive sleep apnea prevalence was 49% (62% men, 31% women) considering an apnea-hypopnea index ≥ 5 respiratory events/hour, and 16% (21% men, 13% women) considering an apnea-hypopnea index ≥ 15 respiratory events/hour. The prevalence of obstructive sleep apnea continuously increased along with age for men and women, with a later onset for women. Age, gender, body mass index, cervical and waist circumference, snoring, reporting of apnea by proxies, self-reported cardiovascular and metabolic diseases such as hypertension, diabetes and dyslipidemia, were significantly associated with OSAS. No association was found with insomnia and daytime sleepiness. CONCLUSIONS: The prevalence and risk factors associated to obstructive sleep apnea syndrome were high among these adults.

Further in vivo evidence implying DNA apurinic/apyrimidinic endonuclease activity in Trypanosoma cruzi oxidative stress survival.

Trypanosoma cruzi is under the attack of reactive species produced by its mammalian and insect hosts. To survive, it must repair its damaged DNA. We have shown that a base excision DNA repair (BER)-specific parasite TcAP1 endonuclease is involved in the resistance to H2 O2 . However, a putative TcAP1 negative dominant form impairing TcAP1 activity in vitro did not show any in vivo effect. Here, we show that a negative dominant form of the human APE1 apurinic/apyrimidinic (AP) endonuclease (hAPE1DN) induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to H2 O2 . Those results confirm that TcAP1 AP endonuclease activity plays an important role in epimastigote and in infective metacyclic trypomastigote oxidative DNA damage resistance leading to parasite persistence in the insect and mammalian hosts. All along its biological cycle and in its different cellular forms, T. cruzi, the etiological parasite agent of Chagas' disease, is under the attack of reactive species produced by its mammalian and insect hosts. To survive, T. cruzi must repair their oxidative damaged DNA. We have previously shown that a specific parasite TcAP1 AP endonuclease of the BER is involved in the T. cruzi resistance to oxidative DNA damage. We have also demonstrated that epimastigotes and cell-derived trypomastigotes parasite forms expressing a putative TcAP1 negative dominant form (that impairs the TcAP1 activity in vitro), did not show any in vivo effect in parasite viability when exposed to oxidative stress. In this work, we show the expression of a negative dominant form of the human APE1 AP endonuclease fused to a green fluorescent protein (GFP; hAPE1DN-GFP) in T. cruzi epimastigotes. The fusion protein is found both in the nucleus and cytoplasm of noninfective epimastigotes but only in the nucleus in metacyclic and cell-derived trypomastigote infective forms. Contrarily to the TcAP1 negative dominant form, the ectopic expression of hAPE1DN-GFP induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to increasing H2 O2 concentrations. No such effect was evident in expressing hAPE1DN-GFP cell-derived trypomastigotes. Although the viability of both wild-type infective trypomastigote forms diminishes when parasites are submitted to acute oxidative stress, the metacyclic forms are more resistant to H2 O2 exposure than cell-derived trypomastigotes.Those results confirm that the BER pathway and particularly the AP endonuclease activity play an important role in epimastigote and metacyclic trypomastigote oxidative DNA damage resistance leading to parasite survival and persistence inside the mammalian and insect host cells.

[Performance of sleep questionnaires for the diagnosis of obstructive sleep apnea syndrome]. / Rendimiento de los cuestionarios de sueño en el diagnóstico de síndrome de apneas obstructivas del sueño en población chilena. Subestudio de la Encuesta Nacional de Salud, 2016/17.

Background The diagnosis of obstructive sleep apnea syndrome (OSAS) is based on nocturnal records: polysomnography or respiratory polygraphy. However, their high costs limit their use. Aim To examine the predictive value of three sleep questionnaires (STOP, STOP-Bang, Epworth Sleepiness Scale (ESS) in the screening of OSAS in Chilean adults. Material and Methods During the National Health Survey 2016/17, 205 adults aged 50.7 ± 15.0 years (46% males) living in the Metropolitan Region answered sleep questionnaires and underwent an ambulatory respiratory polygraphy. The sensitivity, specificity, positive and negative predictive values and receiver operating characteristic curves of sleep questionnaires were calculated. Results Fifty nine percent of participants had OSAS which was moderate to severe in 26%. The clinical variables associated with OSAS were age, male gender, hypertension, dyslipidemia, overweight, cervical and waist circumferences, history of regular snoring and witnessed apneas. Daytime somnolence, insomnia and unrefreshing sleep were not associated to OSAS risk. STOP, STOP-Bang and ESS questionnaires classified 64%, 71% and 12% of cases as high risk for OSAS, respectively. The STOP and STOP-Bang questionnaires had the highest sensitivity to predict OSAS (76% and 89%, respectively) while the ESS had the highest specificity (91%). Conclusions The sleep questionnaires allowed to identify the subjects at high risk for OSAS in this sample of adults from the Metropolitan Region.

The overexpression of TcAP1 endonuclease confers resistance to infective Trypanosoma cruzi trypomastigotes against oxidative DNA damage.

Trypanosoma cruzi, the causative agent of Chagas' disease survives to DNA damage generated by ROS/RNS inside to their different hosts. In recent eukaryotes, oxidative DNA damage is repaired mainly by the Base Excision Repair (BER) pathway, being essential the apurinic/apyrimidinic endonuclease activity. Using a pTREX-gfp vector, the nucleotide sequence that encodes T. cruzi AP endonuclease TcAP1 (orthologue of human APE1) and a putative TcAP1 dominant negative (TcAP1DN), were transfectedand expressed in T. cruzi epimastigotes. TcAP1-GFP and TcAP1DN-GFP were expressed in those modified epimastigotes and found in the parasite nucleus. The endonucleases were purified under native conditions and the AP endonuclease activity was evaluated. While TcAP1 presents the expected AP endonuclease activity TcAP1DN does not. Moreover, TcAP1DN partially inhibits in vitro TcAP1 enzymatic activity. Transfected epimastigotes expressing TcAP1-GFP and TcAP1DN-GFP were differentiated to infective trypomastigotes. The infective parasites maintained both proteins (TcAP1-GFP and TcAP1DN-GFP) in the nucleus. The overexpression of TcAP1-GFP in epimastigotes and trypomastigotes increases the viability of both parasite forms when exposed to oxidative stress while the expression of TcAP1DN-GFP did not show any in vivo inhibitory effect, suggesting that endogenous TcAP1 constitutive expression overcomes the TcAP1DN inhibitory activity. Our results show that TcAP1 is important for trypomastigote survival under oxidative conditions similar to those found in infected mammalian cells, then increasing its permanence in the infected cells and the possibility of development of Chagas disease.

A Flap Endonuclease (TcFEN1) Is Involved in Trypanosoma cruzi Cell Proliferation, DNA Repair, and Parasite Survival.

FLAP endonucleases (FEN) are involved both in DNA replication and repair by processing DNA intermediaries presenting a nucleotide flap using its phosphodiesterase activity. In spite of these important functions in DNA metabolism, this enzyme was not yet studied in Trypanosomatids. Trypanosoma cruzi, the ethiological agent of Chagas disease, presents two dividing cellular forms (epimastigote and amastigote) and one non-proliferative, infective form (trypomastigote). The parasite survives DNA damage produced by reactive species generated in its hosts. The activity of a T. cruzi FLAP endonuclease (TcFEN1) was determined in the three cellular forms of the parasite using a DNA substrate generated by annealing three different oligonucleotides to form a double-stranded DNA with a 5' flap in the middle. This activity showed optimal pH and temperature similar to other known FENs. The substrate cut by the flap endonuclease activity could be ligated by the parasite generating a repaired DNA product. A DNA flap endonuclease coding sequence found in the T. cruzi genome (TcFEN1) was cloned, inserted in parasite expression vectors and transfected to epimastigotes. The purified native recombinant protein showed DNA flap endonuclease activity. This endonuclease was found located in the parasite nucleus of transfected epimastigotes and its over-expression increased both parasite proliferation and survival to H2 O2 . The presence of a flap endonuclease activity in T. cruzi and its nuclear location are indicative of the participation of this enzyme in DNA processing of flap fragments during DNA replication and repair in this parasite of ancient evolutive origin. J. Cell. Biochem. 118: 1722-1732, 2017. © 2016 Wiley Periodicals, Inc.

Nontuberculous mycobacteria in cystic fibrosis patients on the Island of Gran Canaria. A population study.

OBJECTIVE: To determine the prevalence of nontuberculous mycobacteria (NTM) colonization and disease in cystic fibrosis (CF) patients. PATIENTS AND METHODS: All the CF patients followed-up from 2002 to 2012 with three acid-fast bacilli (AFB) cultures were included. The American Thoracic Society (ATS) criteria for NTM lung disease were applied. RESULTS: Forty-four of the 53 patients being followed-up were included. The mean time of follow-up was 7.0 years. A total of 18 patients (40.9%) were NTM positive. The NTN mean annual prevalence was 14.1%. The risk of Mycobacterium abscessus complex was higher in the group of 10-14 years-old (p < 0.001). Ten patients (22.7% of the entire cohort) met the ATS microbiological criteria. The mean annual prevalence of NTM disease was 10.4%. Seven patients (four with Mycobacterium simiae and three with M. abscessus complex) with multiple positive cultures, positive AFB smears and clinical worsening were treated. Three patients with M. simiae and none of those with M. abscessus were cured. CONCLUSIONS: Overall NTM prevalence of colonization and disease were high in our CF patients. Patients <15 years old had a higher risk of M. abscessus complex colonization. Multiple positive cultures or positive AFB smears were associated with disease.

Initial studies on mechanism of action and cell death of active N-oxide-containing heterocycles in Trypanosoma cruzi epimastigotes in vitro.

Chagas disease, endemic in 21 countries across Latin America, kills more people in the region each year than any other parasite-borne disease. Therapeutic options have problems ranging from toxicity, poor efficacy, drug resistance and high cost. Thus, cheaper and less toxic treatments are necessary. From our in-house chemical library of agents against Trypanosoma cruzi the most relevant N-oxide-containing heterocycles were selected for mode of action and type of death studies. Also included in these studies were two active nitrofuranes. Epimastigotes of T. cruzi were used as the biological model in this study. The metabolic profile was studied by 1H NMR in association with the MTT assay. Excreted catabolites data, using 1H NMR spectroscopy, showed that most of the studied N-oxides were capable of decreasing both the release of succinate and acetate shedding, the compounds therefore possibly acting on mitochondria. Only quinoxalines and the nitrofurane Nf1 showed significant mitochondrial dehydrogenase inhibitions, but with different dose-time profiles. In the particular case of quinoxaline Qx2 the glucose uptake study revealed that the integrity of some pathways into the glycosome could be affected. Optic, fluorescence (TUNEL and propidium iodide) and transmission electron microscopy (TEM) were employed for type of death studies. These studies were complemented with 1H NMR to visualize mobile lipids. At low concentrations none of the selected compounds showed a positive TUNEL assay. However, both quinoxalines, one furoxan and one benzofuroxan showed a necrotic effect at high concentrations. Curiously, one furoxan, Fx1, one benzofuroxan, Bfx1, and one nitrofurane, Nf1, caused a particular phenotype, with a big cytoplasmatic vacuole being observed while the parasite was still alive. Studies of TEM and employing a protease inhibitor (3-methyladenine) suggested an autophagic phenotype for Bfx1 and Nf1 and a 'BigEye' phenotype for Fx1.

Phospholipase C gamma and ERK1/2 mitogen activated kinase pathways are differentially modulated by Trypanosoma cruzi during tissue invasion in human placenta.

Chagas' disease is caused by the haemophlagelated protozoan Trypanosoma cruzi (T. cruzi). During congenital transmission the parasite breaks down the placental barrier, however studies about the physiopathology of this process are scarce. Different signal transduction pathways are involved during cell invasion of the parasite. However, the possible role of those processes during tissue infection has not been studied. In the present study we analyzed the modulation of two signal transduction pathways, PLC-γ and ERK1/2 MAPK, during ex vivo infection of human placental chorionic villi explants. Chorionic villi from healthy woman placentas were incubated in the presence or absence of 10(5) or 10(6)T. cruzi trypomastigotes (DM28c strain) with or without specific inhibitors for each pathway. Effective infection was tested determining parasite DNA by PCR. The activation of PLC-γ and ERK1/2 MAPK signaling pathways was determined by western blotting and immunofluorescence. The low concentration of T. cruzi trypomastigotes activates both signaling pathways; however, the high concentration of parasite induces a modest activation of the PLC-γ pathway and impairs the ERK1/2 MAPK pathway activation. Interestingly, inhibition of any of those signaling pathways did not prevent parasite infection, as it was previously shown in cell cultures. We conclude that both signal transduction pathways are modulated during ex vivo T. cruzi infection of human placental chorionic villi explants.

Zoonotic Cryptosporidium meleagridis in urban invasive monk parakeets.

Zoonotic Cryptosporidium meleagridis was identified in invasive monk parakeets (Myiopsitta monachus) from Santiago metropolis in Chile. Oocysts were isolated from nestlings' faeces, and a fragment of 18S ribosomal RNA gene was amplified and sequenced. This finding emphasizes the importance of permanent pathogen monitoring in synanthropic species with wide global distribution.

Study of Trypanosoma cruzi epimastigote cell death by NMR-visible mobile lipid analysis.

Cell death mechanisms in Trypanosoma cruzi have not been disclosed in detail though different conventional techniques have been used in the classification of parasite-cell death type. Nuclear magnetic resonance (NMR) has successfully been used as a tool to evaluate the onset of apoptosis in a number of higher eukaryote-cell models analysing the ratio of CH(2)/CH(3) integration from the visible mobile lipids (VML). Surprisingly, this versatile non-invasive spectroscopy technique has never been employed with this purpose in T. cruzi. In the present study it is shown that under different parasite death-conditions the ratio CH(2)/CH(3) varied drastically. Thus, T. cruzi epimastigotes in apoptotic conditions increase significantly this ratio while in necrotic as well as in autophagic situations the parasites maintain the VML, CH(2)/CH(3) ratio, in normal values. Additionally, other VML markers commonly used in these studies, such as the change in the region of methyl-choline moiety, -N(+)(CH(3))(3), exhibited different particular patterns according to the type of cell death. Our results suggest that the (1)H NMR-VML technique is an adequate tool to discriminate different T. cruzi death pathways.

Novel Proteoliposome-Based Vaccine against E. coli: A Potential New Tool for the Control of Bovine Mastitis.

Escherichia coli is an important causative agent of clinical mastitis in cattle. Current available vaccines have shown limited protection. We evaluated the efficacy of a novel vaccine based on bacterial proteoliposomes derived from an E. coli field strain. Female BALB/c mice were immunized subcutaneously with two doses of the vaccine, 3 weeks apart. Between days 5 and 8 after the first inoculation, the females were mated. At 5-8 days postpartum, the mice were intramammary challenged with the same E. coli strain. Two days after bacterial infection, mice were euthanized, and the mammary glands were examined and removed to evaluate the efficacy and safety of the vaccine as well as the immune response generated by the new formulation. The vaccinated mice showed mild clinical symptoms and a lower mammary bacterial load as compared to non-vaccinated animals. The vaccination induced an increase in levels of IgG, IgG1 and IgG2a against E. coli in blood and mammary glands that showed less inflammatory infiltration and tissue damage, as compared to the control group. In summary, the vaccine based on bacterial proteoliposomes is safe, immunogenic, and effective against E. coli, constituting a new potential tool for mastitis control.

Discrete Typing Units of Trypanosoma cruzi Identified by Real-Time PCR in Peripheral Blood and Dejections of Triatoma infestans Used in Xenodiagnosis Descriptive Study.

Chagas disease (ChD) is a vector zoonosis native to the American continent caused by the protozoan parasite Trypanosoma cruzi; the biological vectors are multiple species of hematophagous insects of the family Triatominae. A relevant aspect in the host-parasite relationship is the identification of the various genotypes of T. cruzi called discrete typing units (DTU) that circulate in mammals and vectors. In Chile, it has been described that the DTUs TcI, TcII, TcV, and TcVI circulate in infected humans, vectors, and wild animals. Identifying DTUs has acquired clinical importance, since it has been suggested that different genotypes could cause distinct pathologies, circulate in different geographical areas, and present different sensitivities to trypanocidal drugs. In this study, circulating T. cruzi DTUs in peripheral blood and Triatoma infestans dejections used in xenodiagnosis (XD) were amplified by qPCR in 14 Chilean patients with chronic ChD from highly endemic areas. More positive samples were detected by XD compared to peripheral blood samples, and 64.28% of the cases were simple infections and 35.72% mixed, with a statistically significant difference in the frequency of TcV DTU. This study would suggest that T. infestans from Chile is more competent to amplify one DTU over others, probably due to a process of co-evolution.

Bovine (Bos taurus) humoral immune response against Echinococcus granulosus and hydatid cyst infertility.

Echinococcus granulosus, the agent of hydatid disease, presents an indirect life cycle, with canines (mainly dogs) as definitive hosts, and herbivores and human as intermediary ones. In intermediary hosts fertile and infertile cysts develop, but only the first ones develop protoscoleces, the parasite form infective to definitive hosts. We report the presence of bovine IgGs in the germinal layer from infertile cysts (GLIC), in an order of magnitude greater than in the germinal layer from fertile cysts (GLFC). When extracted with salt solutions, bovine IgGs from GLIC are associated with low or with high affinity (most likely corresponding to non specific and antigen specific antibodies, respectively). Specific IgGs penetrate both the cells of the germinal layer and HeLa cultured cells and recognize parasitic proteins. These results, taken together with previous ones from our laboratory, showing induction of apoptosis in the germinal layer of infertile hydatid cysts, provide the first coherent explanation of the infertility process. They also offer the possibility of identifying the parasite antigens recognized, as possible targets for immune modulation.

Urban public squares as potential hotspots of dog-human contact: A spatial analysis of zoonotic parasites detection in Gran Santiago, Chile.

Zoonotic parasites are a major public health problem globally, representing a hazard to human health. The infections occur through contact with different parasite forms. Public squares are a common meeting place for people and dogs, becoming a risk area for transmission to other dogs and humans. A spatial analysis of zoonotic parasite distribution in urban public squares in Gran Santiago, Chile was performed to elucidate its importance, exploring spatial aggregation on positivity rates at commune level. A total of 170 stool samples were analysed; 54 (31.7%) were positive for gastrointestinal-zoonotic parasites. Positivity was detected in 27 (79.4%) squares. Diversity of parasite species was variable across sites. Toxocara canis was the most frequently detected (27 samples, 21/34 squares), followed by Toxascaris leonina (13 samples, 10/34 squares), cestode eggs (10 samples, 9/34 squares), Giardia sp. (6 samples, 5/34 squares), Ancylostomatidae-like eggs (4 samples, 4/34 squares), Cryptosporidium spp. (2 samples, 2/34 squares) and Entamoeba spp. (1 sample, 1/34 square). Spatial autocorrelation (SA) was observed at commune level. The local indicators of the spatial association test showed statistical significance for cestode eggs (p < 0.001) with a negative SA (Moran's I Index = -0.2929, CI-95% = -0.3869--0.1989) and Ancylostomatidae (p = 0.046) with a positive SA (Moran's I Index = 0.1404, CI-95% = 0.0499-0.2309). Toxocara canis is the most prevalent geo-helminth in public places of Santiago, Chile. Spatial autocorrelation was detected for cestode eggs and Ancylostomatidae. Diagnosis and detection of parasites is key for the establishment of control/eradication of environmental dissemination following One Health guidelines.

Echinococcus granulosus calreticulin: molecular characterization and hydatid cyst localization.

Hydatidosis, whose etiological agent is the larval stage of the platyhelminth parasite Echinococcus granulosus, is a major zoonotic public health problem and causes great economic losses in many countries, affecting humans and livestock species. Calreticulin (CRT) is a multifunctional and conserved chaperone calcium-binding protein, present in every cell of higher organisms, except erythrocytes. In other parasites species, CRT emerges as a key modulator of several immunological aspects of their relationships with their vertebrate hosts. We report herein the cloning of two DNA segments coding for sequences of E. granulosus CRT (EgCRT). Together with another partial sequence available at the NCBI database, a complete EgCRT cDNA sequence is now proposed. EgCRT mRNA is equally expressed in fertile and infertile hydatid cysts germinal layers, as well as in protoscoleces. In hydatid cysts, EgCRT is a 50 kDa protein, expressed in germinal layer and protoscoleces, mainly in perinuclear and cytoplasmic zones.

DNA damage, RAD9 and fertility/infertility of Echinococcus granulosus hydatid cysts.

Hydatidosis, caused by the larval stage of the platyhelminth parasite Echinococcus granulosus, affects human and animal health. Hydatid fertile cysts are formed in intermediate hosts (human and herbivores) producing protoscoleces, the infective form to canines, at their germinal layers. Infertile cysts are also formed, but they are unable to produce protoscoleces. The molecular mechanisms involved in hydatid cysts fertility/infertility are unknown. Nevertheless, previous work from our laboratory has suggested that apoptosis is involved in hydatid cyst infertility and death. On the other hand, fertile hydatid cysts can resist oxidative damage due to reactive oxygen and nitrogen species. On these foundations, we have postulated that when oxidative damage of DNA in the germinal layers exceeds the capability of DNA repair mechanisms, apoptosis is triggered and hydatid cysts infertility occurs. We describe a much higher percentage of nuclei with oxidative DNA damage in dead protoscoleces and in the germinal layer of infertile cysts than in fertile cysts, suggesting that DNA repair mechanisms are active in fertile cysts. rad9, a conserved gene, plays a key role in cell cycle checkpoint modulation and DNA repair. We found that RAD9 of E. granulosus (EgRAD9) is expressed at the mRNA and protein levels. As it was found in other eukaryotes, EgRAD9 is hyperphosphorylated in response to DNA damage. Our results suggest that molecules involved in DNA repair in the germinal layer of fertile hydatid cysts and in protoscoleces, such as EgRAD9, may allow preserving the fertility of hydatid cysts in the presence of ROS and RNS.

Differential infectivity of two Trypanosoma cruzi strains in placental cells and tissue.

Congenital Chagas disease, caused by Trypanosoma cruzi (T. cruzi), has become epidemiologically relevant. The probability of congenital transmission depends on the maternal and developing fetal/newborn immune responses, placental factors and importantly, the virulence of the parasite. It has been proposed, that different genotypes of T. cruzi and their associated pathogenicity, virulence and tissue tropism may play an important role in congenital infection. Since there is no laboratory or animal model that recapitulates the complexities of vertical transmission in humans, here we studied parasite infectivity in human placental explants (HPE) as well as in the human trophoblast-derived cell line BeWo of the Y(DTU II) and the VD (TcVI) T. cruzi strains; the latter was isolated from a human case of congenital infection. Our results show that the VD strain is more infective and pathogenic than the Y strain, as demonstrated by qPCR and cell counting as well as by histopathological analysis. The present study constitutes the first approach to study the relationship between parasite two parasite strains from different genotypes and the infection efficiency in human placenta.

First description of Echinococcus ortleppi and cystic echinococcosis infection status in Chile.

Cystic echinococcosis (CE), a parasitic disease caused by the cestode Echinococcus granulosus sensu lato (s.l.), is a worldwide zoonotic infection. Although endemic in Chile, information on the molecular characteristics of CE in livestock remains scarce. Therefore we aimed to describe the status of infection with E. granulosus s.l. in cattle from central Chile and also to contribute to the study of the molecular epidemiology of this parasite. According to our results, the prevalence of CE is 18.84% in cattle, similar to previous reports from Chile, suggesting that the prevalence in Santiago Metropolitan area has not changed in the last 30 years. Most of the cysts were found only in lungs (51%), followed by concurrent infection in liver and lungs (30%), and only liver (19%). Molecular characterization of the genetic diversity and population structure of E. granulosus s.l. from cattle in central Chile was performed using a section of the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene. E. granulosus sensu stricto (s.s.) (G1-G3 genotypes) was confirmed by RFLP-PCR to be the dominant species affecting cattle (284 samples/290 samples); we also report for the first time in Chile the presence of E. ortleppi (G5 genotype) (2 samples/61 samples). The Chilean E. granulosus s.s. parsimony network displayed 1 main haplotype. Additional studies using isolates from many locations across Chile and different intermediate hosts will provide more data on the molecular structure of E. granulosus s.s. within this region. Likewise, investigations of the importance of E. ortleppi in human infection in Chile deserve future attention.

Effect of alpha lipoic acid on leukotriene A4 hydrolase.

Leukotriene A4 hydrolase is a soluble enzyme with epoxide hydrolase and aminopeptidase activities catalysing the conversion of leukotriene A4 to leukotriene B4 and the hydrolysis of the peptide proline-glycine-proline. Imbalances in leukotriene B4 synthesis are related to several pathologic conditions. Currently there are no available drugs capable to modulate the synthesis of leukotriene B4 or to block its receptors. Here we show the inhibitory profile of alpha lipoic acid on the activity of leukotriene A4 Hydrolase. Alpha lipoic acid inhibited both activities of the enzyme at concentrations lower than 10µM. The 5-lipoxygenase inhibitor zileuton, or the 5-lipoxygenase activating protein inhibitor MK-886, were unable to inhibit the activity of the enzyme. Acute promyelocytic leukaemia HL-60 cells were differentiated to leukotriene A4 hydrolase expressing neutrophil-like cells. Alpha lipoic acid inhibited the aminopeptidase activity of the cytosolic fraction from neutrophil-like cells but had no effect on the cytosolic fraction from undifferentiated cells. Docking and molecular dynamic approximations revealed that alpha lipoic acid participates in electrostatic interactions with K-565 and R-563, which are key residues for the carboxylate group recognition of endogenous substrates by the enzyme. Alpha lipoic acid is a compound widely used in clinical practice, most of its therapeutic effects are associated with its antioxidants properties, however, antioxidant effect alone is unable to explain all clinical effects observed with alpha lipoic acid. Our results invite to evaluate the significance of the inhibitory effect of alpha lipoic acid on the catalytic activity of leukotriene A4 hydrolase using in vivo models.

Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response.

Trypanosoma cruzi is a protozoan parasite and the causative agent of Chagas disease. Like most living organisms, it is susceptible to oxidative stress, and must adapt to distinct environments. Hence, DNA repair is essential for its survival and the persistence of infection. Therefore, we studied whether T. cruzi has a homolog counterpart of the MutY enzyme (TcMYH), important in the DNA Base Excision Repair (BER) mechanism. Analysis of T. cruzi genome database showed that this parasite has a putative MutY DNA glycosylase sequence. We performed heterologous complementation assays using this genomic sequence. TcMYH complemented the Escherichia coli MutY- strain, reducing the mutation rate to a level similar to wild type. In in vitro assays, TcMYH was able to remove an adenine that was opposite to 8-oxoguanine. We have also constructed a T. cruzi lineage that overexpresses MYH. Although in standard conditions this lineage has similar growth to control cells, the overexpressor is more sensitive to hydrogen peroxide and glucose oxidase than the control, probably due to accumulation of AP sites in its DNA. Localization experiments with GFP-fused TcMYH showed this enzyme is present in both nucleus and mitochondrion. QPCR and MtOX results reinforce the presence and function of TcMYH in these two organelles. Our data suggest T. cruzi has a functional MYH DNA glycosylase, which participates in nuclear and mitochondrial DNA Base Excision Repair.