Molecular, immunological, and physiological evidences of a sphingosine-activated plasma membrane Ca2+-channel in Trypanosoma equiperdum.

The hemoparasite Trypanosoma equiperdum belongs to the Trypanozoon subgenus and includes several species that are pathogenic to animals and humans in tropical and subtropical areas across the world. As with all eukaryotic organisms, Ca2+ is essential for these parasites to perform cellular processes thus ensuring their survival across their life cycle. Despite the established paradigm to study proteins related to Ca2+ homeostasis as potential drug targets, so far little is known about Ca2+ entry into trypanosomes. Therefore, in the present study, the presence of a plasma membrane Ca2+-channel in T. equiperdum (TeCC), activated by sphingosine and inhibited by verapamil, is described. The TeCC was cloned and analyzed using bioinformatic resources, which confirmed the presence of several domains, motifs, and a topology similar to the Ca2+ channels found in higher eukaryotes. Biochemical and confocal microscopy assays using antibodies raised against an internal region of human L-type Ca2+ channels indicate the presence of a protein with similar predicted molar mass to the sequence analyzed, located at the plasma membrane of T. equiperdum. Physiological assays based on Fura-2 signals and Mn2+ quenching performed on whole parasites showed a unidirectional Ca2+ entry, which is activated by sphingosine and blocked by verapamil, with the distinctive feature of insensitivity to nifedipine and Bay K 8644. This suggests a second Ca2+ entry for T. equiperdum, different from the store-operated Ca2+ entry (SOCE) previously described. Moreover, the evidence presented here for the TeCC indicates molecular and pharmacological differences with their mammal counterparts, which deserve further studies to evaluate the potential of this channel as a drug target.

Seroprevalence of trypanosomosis and associated risk factors in cattle from coast and amazonian provinces of Ecuador.

Trypanosomosis is a tropical disease caused by various protozoan haemoparasites, which affects wild and domestic animals, the latter ones related to worldwide livestock production systems. Species such as Trypanosoma vivax and Trypanosoma evansi have been described using serological and molecular tools in several countries from South and Central America. However, Ecuador presents a relevant knowledge gap in the associated general epidemiology and risk factors of the disease. Therefore, the objective of this study was to determine the seroprevalence of trypanosomosis in cattle from different regions of Ecuador. 745 serum samples from 7 Coastal and 3 Amazon provinces were screened for IgG anti-Trypanosoma spp. antibodies, using an in-house indirect ELISA. The seropositivity was explored and associated with several variables such as sex, age, breed, region, management, and province, using statistical tools. The general seroprevalence of trypanosomosis was 19.1% (95% CI: 16.30-22.1%). The Amazonian provinces of Sucumbíos and Napo and the Coastal province of Esmeraldas presented the highest seroprevalence values of 36.7% (95% CI: 27.67-46.47%), 23.64% (95% CI: 16.06-32.68%) and 25% (95% CI: 15.99-35.94%), respectively. Statistical significance was found for the region, province, and management variables, indicating as relevant risk factors the extensive management and Amazon location of the cattle analyzed. Specific actions should be taken to identify the exact species on reservoirs and susceptible hosts, evaluate the implication of farm management and cattle movement as risk factors, and implement surveillance and treatment plans for affected herds.

Molecular identification of Trypanosoma theileri in cattle from the Ecuadorian Amazon.

Trypanosoma theileri is a cosmopolitan opportunistic haemoparasite described in wild and domestic ruminants, and also in arthropod vectors. The presence of this parasite has been reported in several South American countries, including Amazonian regions. Despite the importance of livestock production, Ecuador possesses scarce studies about trypanosomosis and no T. theileri reports in its territory. Here, we showed molecular evidences of the presence of T. theileri in cattle from a province located in the Ecuadorian Amazon. Bovine blood samples were collected from 2014 to 2019, during campaigns to detect haemoparasites in the Ecuadorian provinces of Orellana and Sucumbíos. DNA was extracted from the buffy coat and used in PCR assays with three different molecular markers, ITS1, 18S and Cathepsin L-like. T. theileri was detected only in the Sucumbíos province, with a specific molecular prevalence of 8.6% (3/35) using the three primers and an additional animal detected as positive (11.4% prevalence) only by the ITS1 marker. DNA sequences derived from the generated amplicons were subjected to phylogenetics maximum parsimony and maximum likelihood analysis, which indicate the presence of TthI and TthII genotypes circulating in the evaluated animals. Molecular surveillance should be continually implemented in Ecuador in order to deepen the epidemiological and evolutionary knowledge about T. theileri as well other haemoparasites in the amazon parts of the country.

Active compounds and distinctive sensory features provided by American ginseng (Panax quinquefolius L.) extract in a new functional milk beverage.

American ginseng (Panax quinquefolius L.) has recognized neurocognitive effects, and a ginsenoside-rich extract of the root of the plant has been shown to improve cognitive functions in young adults. This study aimed at assessing the chemical and sensory profiles of a UHT-treated, low-lactose functional milk containing American ginseng. Individual ginsenosides in the milk were analyzed by HPLC. Descriptive sensory analysis was performed by a trained panel to quantitatively document sensory changes resulting from the addition of ginseng and the UHT process on flavored and unflavored milks. Consumer acceptance of the product was also investigated. Total ginsenoside content in the UHT-treated milk enriched with the ginseng extract after UHT process treatment was 7.52 mg/100 g of milk, corresponding to a recovery of 67.6% compared with the content in the unprocessed extract. The intake of 150 to 300 mL of this ginseng-enriched milk provides the amount of total ginsenosides (11.5 to 23 mg) necessary to improve cognitive function after its consumption. Both the presence of ginsenosides and their thermal treatment affected some sensory properties of the milk, most notably an increase in bitterness and metallic taste, the appearance of a brownish color, and a decrease in milky flavor. Levels of brown color, bitterness, and metallic taste were highest in the industrially processed ginseng-enriched milk. The bitterness attributable to ginseng extract was reduced by addition of vanilla flavor and sucralose. A consumer exploratory study revealed that a niche of consumers exists who are willing to consume this type of product.

Novel probiotic Bifidobacterium bifidum CECT 7366 strain active against the pathogenic bacterium Helicobacter pylori.

Helicobacter pylori is considered one of the major risk factors underlying the development of gastritis and gastric and duodenal ulcers. Moreover, 50% of the population carries this bacterium, and consequently, when it is detected, eradication of H. pylori is strongly recommended. Regarding the use of probiotics as functional agents, several studies have shown that there is a direct relationship between the addition of certain probiotic bacteria and in vitro inhibition of H. pylori; however, in vivo studies showing bifidobacterial activity against H. pylori remain scarce. In this study, a Bifidobacterium bifidum strain which proved active in vitro against H. pylori has been isolated, with inhibition levels reaching 81.94% in the case of the supernatant and even 94.77% inhibition for supernatant purified by cationic exchange followed by an inverse phase. In vivo studies using a BALB/c mouse model have proved that this strain partially relieves damage to gastric tissues caused by the pathogen and also decreases the H. pylori pathogenicity ratio. This novel strain fulfills the main properties required of a probiotic (resistance to gastrointestinal juices, biliary salts, NaCl, and low pH; adhesion to intestinal mucus; and sensitivity to antibiotics). Furthermore, the absence of undesirable metabolites has been demonstrated, and its food safety status has been confirmed by acute ingestion studies in mice. In summary, the results presented here demonstrate that Bifidobacterium bifidum CECT 7366 can be considered a probiotic able to inhibit H. pylori both in vitro and in vivo.

Trypanosoma evansi: a comparative study of four diagnostic techniques for trypanosomosis using rabbit as an experimental model.

The goal of this study was to compare two parasitological diagnostic techniques, such as by Micro-Haematocrit Centrifugation Technique (MHCT) and Direct Microscopic Examination (DME) with a serological method (iELISA), and a molecular procedure PCR, in rabbits experimentally infected with Trypanosoma evansi, in order to determine their sensitivity throughout the course of disease. The parasitological methods were not able of detecting the presence of the parasite during the phases of low parasitemia, the prepatency period and the chronic phase. In contrast, PCR detected T. evansi in the prepatency and chronic phase, when increase the amount of DNA from 100 to 300ng. 100% detection was observed with iELISA only in the chronic stage of the disease. In the acute phase, all samples were positively diagnosed using either MHCT or PCR, whereas only few samples were diagnosed by DME. Samples obtained from day 15 post infection were also detected by iELISA. The highest diagnostic register during the course of infection was achieved by the PCR technique (93.8%), followed by iELISA (71.1%), MHCT (59%) and DME (13.6%). Therefore, we recommend the use of PCR in epidemiological studies in order to implement sanitary control plans for the improvement of livestock productivity in the country.

Evaluation of five primer sets for molecular detection of Trypanosoma vivax by polymerase chain reaction (PCR) and their implementation for diagnosis in naturally infected ruminants from Venezuela.

Trypanosoma vivax is a protozoan parasite that causes trypanosomosis in ruminants and is widely distributed in tropical areas in the world. The control of this disease depends on the sensitivity and specificity of the diagnostic tests implemented for naturally infected samples, where parasitaemias are usually low. This study aimed to evaluate the analytical sensitivity and specificity of several primers for T. vivax detection in experimental infections and their implementation for the diagnosis of trypanosomosis in naturally infected bovine and ovine samples. Using a T. vivax Venezuelan isolate, five sets of primers were evaluated: TviSL1/2, ITS1CF/BR, TVMF/R, ILO1264/1265, TVWA/B. Additionally, we tested the PCR protocols using different DNA quantities. The best set of primers (ILO1264/1265) was used to detect T. vivax DNA from whole blood and buffy coat samples of 12 sheep (ovine) and 45 cattle (bovine) of small farms from Venezuela, and compared to the micro-haematocrite centrifugation technique (MHCT). The highest sensitivity was 0.0001 ng for ILO1264/1265 and TVWA/B primers. Using 100 ng of DNA extracted from the buffy coat and the ILO1264/1265 primers for trypanosomosis diagnosis from naturally infected samples, yielded 66.7% (8/12) and 35.7% (16/45) positives in ovine and bovine respectively. The percentage of positives samples increased to 83.3% (10/12) and 64.4% (29/45), with 300 ng in the assays. Contrary, using 300 ng of DNA extracted from the whole blood yielded only 50% (6/12) and 28.9% (13/45) of positives samples for T. vivax respectively. MHCT only detected the parasite in bovine samples with 17.8% (8/45) of positives. Based on our results, we recommend the use of the ILO1264/1265 primers and 300 ng of DNA extracted from the buffy coat for epidemiological studies of naturally infected animals. Moreover, detection of the parasite in ovine herds highlights a possible role of this host in the epidemiology of trypanosomosis in Venezuela.

[The effect of a fibre enriched dietary milk product in chronic primary idiopatic constipation]. / Efecto de la ingesta de un preparado lácteo con fibra dietética sobre el estreñimiento crónico primario idiopático.

BACKGROUND: fibre is effective in some types of constipation. Our objective was to determine if the administration of an enriched dairy preparation with a supplement of soluble fibre (Naturfibra) [inulin and digestion resistant maltodextrin (Fibersol 2)], improves primary chronic constipation. METHODS: Prospective, randomized, double blind clinical trial randomized with dietary intervention in 32 subjects with constipation according to the Rome II criteria. Thirty two subjects (men 4; women 28) with an average age of 47 +/- 15 years were randomly divided in two homogeneous groups. A type of milk (A or B) was assigned to each group. Group A received fibre enriched semi-skimmed milk; Group B received semi skimmed milk. The subjects drank half a litre of milk per day during 20 days, meaning that those who drank the enriched milk ingested 20 grams of fibre a day. RESULTS: The subjects that presented straining at defecation (p < 0.001), sensation of incomplete evacuation (p < 0.001), sensation of obstruction in the evacuation (p < 0.001) and days between bowel movements, diminished significantly after the intake of milk supplemented with fibre. CONCLUSION: The intake of a dairy preparation with a supplement of fibre containing 20 grams of soluble fibre (inulin and digestion resistant maltodextrin), improves the situation of idiopathic primary chronic constipation based on the Rome II criteria.

Molecular diagnosis of cattle trypanosomes in Venezuela: evidences of Trypanosoma evansi and Trypanosoma vivax infections.

In South America Trypanosoma evansi has been determined by molecular methods in cattle from Bolivia, Brazil, Colombia and Peru, reason for which the presence of this parasite is not excluded in Venezuelan livestock. Therefore, the aim of this study was to perform parasitological and molecular diagnosis of cattle trypanosomosis in small livestock units from two regions in this country. The parasitological diagnosis was carried out by MHCT and the molecular by PCR using genus-specific ITS1 primers that differentiate T. vivax and T. evansi infections. 47 cattle were evaluated in the "Laguneta de la Montaña" sector, Miranda State, where 3 animals were diagnosed as positive (6.4 %) by MHCT and 14 (30 %) by PCR as Trypanosoma spp., out of which 9 animals resulted positive for T. vivax, 3 for T. evansi and 2 with double infections. Whilst in the "San Casimiro" sector, State of Aragua, out of the 38 cattle evaluated 7 animals were diagnosed as positive (18.4 %) by MHCT and 19 (50 %) by PCR, determining only the presence of T. evansi in this locality. The molecular diagnosis by PCR using ITS1 primers allowed T. evansi detection in cattle field populations, which suggests the possible role of these animals as reservoirs in the epidemiology of the disease caused by T. evansi in Venezuela.

Rete mirabile of goat: in vitro effects of adrenergic stimulation.

The carotid rete of the goat, a network of small arteries, is placed in the pathway of the main arteries which supply the brain. This structure lies within the cavernous sinus on each side of the pituitary. The presence of a carotid rete in many mammals has led to frequent speculations about its physiological function. The present study was designed to examine comparatively the responsiveness of goat retial and cerebral arteries to direct or indirect adrenergic stimulation. The contractile response of isolated retial arteries (150-500 micron in external diameter) to norepinephrine (10(-8) - 10(-4)M), field electrical stimulation (2-16 c/s), and tyramine (10(-6) - 10(-3) M) was negligible. In contrast, cerebral arteries of 300-500 micron in external diameter exhibited dose- or frequency-dependent contractions qualitatively similar to those previously described in larger cerebral arteries. The norepinephrine content in the walls of retial arteries was about 13% of that measured in cerebral arteries. These results suggest that the role of the carotid rete in the regulation of resistance to blood flow during adrenergic stimulation is negligible or nonexistent.

Intrasellar granuloma. Case report.

A case of giant-cell granuloma of the pituitary in a 28-year-old woman is reported. Clinical complaints included headache and amenorrhea. Endocrinological studies showed hypopituitarism. X-ray films showed enlargement of the sella turcica.

A model of semiautomatic type-specific diagnosis of brain tumours by computed tomography: mathematical fundaments and practical application.

Applying Escudero's formula to Bayes' theorem of conditional probabilities, we have developed a mathematical model to diagnose type-specific brain tumours with great accuracy. We have transformed a multidimensional system of n characteristics on computed tomography into a linear system, making this model very easy to use and, thus, accessible to the clinician and radiologist. We tested this model in 177 brain tumours. In 86% of the cases we obtained a type-specific correct diagnosis, achieving better results than those previously reported. Suggestions are given as to how to improve this method further.

Gliomatosis cerebri: clinical and histological findings.

The clinical and pathological data of ten patients with gliomatosis cerebri are compared with 48 well documented cases from the literature. The most striking clinical findings were behavioural and mental changes, seizures, motor weakness and headaches. Though diagnostic techniques have gained in sophistication, the clinical diagnosis of gliomatosis cerebri remains difficult. Laboratory and radiograph tests are mostly unconclusive. Expectations that computed tomography might lead to an accurate diagnosis were not fulfilled. Histological examination disclosed a diffuse proliferation of glial elements infiltrating normal nervous tissue with destruction of myelin sheaths, but only slight damage to neurons and axons. In two cases, areas typical of oligodendroglioma were also present. Glial fibrillary acidic protein staining showed in seven cases that most of the neoplastic cells were of astrocytic origin. In addition, GFAP negative neoplastic cells with the appearance of oligodendroglia and intermediate elements between astroglia and oligodendroglia and irregularly shaped naked nuclei of unidentified nature were found. On the basis of the two-stage theory of carcinogenesis, it is suggested that this disease might be the result of propagation of initiated glial elements which have not yet undergone the process of tumor conversion.

[The magnetic resonance tomography of intracranial ependymomas. Their clinical appearance and comparison with computed tomography]. / Magnetische Resonanztomographie intrakranieller Ependymome. Erscheinungsbild und Vergleich mit der Computertomographie.

The MRT and CT images of histologically confirmed intracranial ependymomas in 10 patients have been compared. All tumours were demonstrated by CT and MRT. The solid portions of the tumours showed variation in density on CT (hypodense, isodense, hyperdense, or mixed), whereas there was uniform signal intensity on MRT (T1 weighted: hypointense, T2 weighted: hyperintense). Cystic portions of the tumours (6 patients) were shown equally well by both procedures, calcifications were only shown by CT. All ependymomas were related to the ventricles. On unenhanced CT only 3 tumours were clearly delineated, whereas MRT defined all tumours satisfactorily. The significant advantage of MRT is better topographic demonstration of the tumour.

Superficial siderosis of the central nervous system.

We report on three patients with superficial siderosis of the central nervous system. The main clinical findings were progressive dementia, hearing loss and ataxia in combination with repeated xanthochromia of the cerebrospinal fluid. Diagnosis was made in one patient with magnetic resonance imaging (MRI), which showed a hyperintense rim around the surface of the cerebellum and the spinal cord. In the two other cases necropsy disclosed superficial iron and hemosiderin deposits on the surface of the brain. The etiology either was idiopathic or secondary to chronic intracranial bleeding by an angioma or after multiple head injuries. Superficial siderosis should be taken into account as one reason for dementia.

Trypanosoma evansi: A clinical, parasitological and immunological evaluation of trypanosomosis using a chronic rabbit model.

We evaluated the clinical, parasitological and immunological effects of a Venezuelan strain of Trypanosoma evansi (T. evansi) throughout in experimentally inoculated rabbits over the course of infection and compared them with the same aspect in healthy animals. Body temperature was recorded in degrees Celsius, animal weight in kilograms, serum proteins in g/dl using a refractometer, haematocrit percentage by capillary centrifugation and the anti-T. evansi IgG titer by indirect ELISA immunoassay, from both infected animals and controls for 95 days. Infected animals showed a higher body temperature, total serum protein and anti- T. evansi antibody titer, and a lower haematocrit and weight gain than controls. These differences were related to the presence of the parasites in the blood as detected micro-haematocrit centrifugation technique (MHCT) and direct microscopic examination (DME). This study confirms the usefulness of rabbits as a model for the study of trypanosomosis; the clinical features of the disease can be observed and the three characteristic stages, prepatent period, acute and chronic phase clearly defined over the course of the infection.

A two-band model for superconductivity in the checkerboard lattice.

Motivated by the superconducting properties of the metallic oxide Cd(2)Re(2)O(7), whose crystal structure is of the pyrochlore type, we propose an electronic model on a checkerboard lattice, which can be viewed as a two-dimensional analog of the pyrochlore lattice. Including only charge degrees of freedom, we treat the model via a Bardeen-Cooper-Schrieffer (BCS) approximation, decoupling the interaction terms in real space. Going over to reciprocal space yields a BCS model with two coupled bands. Characteristic properties such as order parameters and specific heat as functions of temperature are obtained. We also discuss the symmetry properties of the superconducting gap in wavevector space and the behavior of the critical temperature as a function of the electronic doping for various values of the interaction strength.