Using AnABlast for intergenic sORF prediction in the Caenorhabditis elegans genome.

MOTIVATION: Short bioactive peptides encoded by small open reading frames (sORFs) play important roles in eukaryotes. Bioinformatics prediction of ORFs is an early step in a genome sequence analysis, but sORFs encoding short peptides, often using non-AUG initiation codons, are not easily discriminated from false ORFs occurring by chance. RESULTS: AnABlast is a computational tool designed to highlight putative protein-coding regions in genomic DNA sequences. This protein-coding finder is independent of ORF length and reading frame shifts, thus making of AnABlast a potentially useful tool to predict sORFs. Using this algorithm, here, we report the identification of 82 putative new intergenic sORFs in the Caenorhabditis elegans genome. Sequence similarity, motif presence, expression data and RNA interference experiments support that the underlined sORFs likely encode functional peptides, encouraging the use of AnABlast as a new approach for the accurate prediction of intergenic sORFs in annotated eukaryotic genomes. AVAILABILITY AND IMPLEMENTATION: AnABlast is freely available at http://www.bioinfocabd.upo.es/ab/. The C.elegans genome browser with AnABlast results, annotated genes and all data used in this study is available at http://www.bioinfocabd.upo.es/celegans. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Relationship between health-related quality of life, disease activity and disease damage in a prospective international multicenter cohort of childhood onset systemic lupus erythematosus patients.

Previously, we described associations between health-related quality of life (HRQOL) and disease-related factors among childhood onset systemic lupus erythematosus (cSLE) patients. Here we determined the relationship between HRQOL, disease activity and damage in a large prospective international cohort of cSLE. We compared HRQOL, disease activity and disease damage across different continents and examined the relationship between children's and parents' assessments of HRQOL. Patients with cSLE and their parents completed HRQOL measures at enrollment and ≥4 follow-up visits. Physicians assessed disease activity and damage. The multinational cohort ( n = 467) had relatively low disease activity and damage. Patient and parent HRQOL scores were significantly correlated. Asian and European patients had the highest HRQOL, while South and North American patients had lower HRQOL scores. Renal, CNS, skin and musculoskeletal systems exhibited the highest levels of damage. North and South American and Asian patients were more likely to have disease damage and activity scores above median values, compared with Europeans. Asians were more likely to use cyclophosphamide/rituximab. Female gender, high disease activity and damage, non-White ethnicity, and use of cyclophosphamide and/rituximab were related to lower HRQOL. HRQOL domain scores continue to emphasize that SLE has widespread impact on all aspects of children's and parents' lives.

Pharmacological approaches in either intermittent or permanent hypoxia: A tale of two exposures.

Hypoxia induces several responses at cardiovascular, pulmonary and reproductive levels, which may lead to chronic diseases. This is relevant in human populations exposed to high altitude (HA), in either chronic continuous (permanent inhabitants) or intermittent fashion (HA workers, tourists and mountaineers). In Chile, it is estimated that 1.000.000 people live at highlands and more than 55.000 work in HA shifts. Initial responses to hypoxia are compensatory and induce activation of cardioprotective mechanisms, such as those seen under intermittent hypobaric (IH) hypoxia, events that could mediate preconditioning. However, whenever hypoxia is prolonged, the chronic activation of cellular responses induces long-lasting modifications that may result in acclimatization or produce maladaptive changes with increase in cardiovascular risk. HA exposure during pregnancy induces hypoxia and oxidative stress, which in turn may promote cellular responses and epigenetic modifications resulting in severe impairment in growth and development. Sadly, this condition is accompanied with an increased fetal and neonatal morbi-mortality. Further, developmental hypoxia may program cardio-pulmonary circulations later in postnatal life, ending in vascular structural and functional alterations with augmented risk on pulmonary and cardiovascular failure. Additionally, permanent HA inhabitants have augmented risk and prevalence of chronic hypoxic pulmonary hypertension, right ventricular hypertrophy and cardiopulmonary remodeling. Similar responses are seen in adults that are intermittently exposed to chronic hypoxia (CH) such as shift workers in HA areas. The mechanisms involved determining the immediate, short and long-lasting effects are still unclear. For several years, the study of the responses to hypoxic insults and pharmacological targets has been the motivation of our group. This review describes some of the mechanisms underlying hypoxic responses and potential therapeutic approaches with antioxidants such as melatonin, ascorbate, omega 3 (Ω3) or compounds that increase the nitric oxide (NO) bioavailability.

Determination of marker pteridines in urine by HPLC with fluorimetric detection and second-order multivariate calibration using MCR-ALS.

A liquid chromatographic method has been developed, in combination with the multivariate curve resolution-alternating least squares algorithm (MCR-ALS), for the simultaneous determination of marker pteridines in urine samples. A central composite design has been applied to optimize the factors influencing the separation (buffer concentration, buffer pH, flow rate, oven temperature, mobile-phase composition). A set of 15 calibration samples were randomly prepared, in a concentration range of 0.5-10.5 ng mL(-1) for neopterin, biopterin, and pterin; 4.0-8.0 ng mL(-1) for xanthopterin; and 0.5-4.5 ng mL(-1) for isoxanthopterin. The validation was carried out with fortified urine samples from healthy adults. The optimized conditions were a mobile-phase composition of 10 mM citric buffer at pH 5.44 and acetonitrile (94.5/5.5, v/v), a flow rate of 1.0 mL min(-1), and an oven temperature of 25 °C. The detection system consisted of a fast-scanning spectrofluorimeter, which allows obtaining of second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. In this work, MCR-ALS was used to cope with coeluting interferences, on account of the second-order advantage inherent to this algorithm which, in addition, is able to handle data sets deviating from trilinearity, like the high-performance liquid chromatography data analyzed in the present report. The developed approach enabled us to determine five pteridines, some of them with overlapped profiles, reducing the experimental time and reagent consumption. Ratio values for pteridines/creatinine in urine, for infected children with different pathologies, are reported in this work.

Perinatal cardiopulmonary adaptation to the thin air of the Alto Andino by a native Altiplano dweller, the llama.

Most mammals have a poor tolerance to hypoxia, and prolonged O2 restriction can lead to organ injury, particularly during fetal and early postnatal life. Nevertheless, the llama (Lama Glama) has evolved efficient mechanisms to adapt to acute and chronic perinatal hypoxia. One striking adaptation is the marked peripheral vasoconstriction measured in the llama fetus in response to acute hypoxia, which allows efficient redistribution of cardiac output toward the fetal heart and adrenal glands. This strong peripheral vasoconstrictor tone is triggered by a carotid body reflex and critically depends on α-adrenergic signaling. A second adaptation is the ability of the llama fetus to protect its brain against hypoxic damage. During hypoxia, in the llama fetus there is no significant increase in brain blood flow. Instead, there is a fall in brain O2 consumption and temperature, together with a decrease of Na+-K+-ATPase activity and Na+ channels expression, protecting against seizures and neuronal death. Finally, the newborn llama does not develop pulmonary hypertension in response to chronic hypoxia. In addition to maintaining basal pulmonary arterial pressure at normal levels the pulmonary arterial pressor response to acute hypoxia is lower in highland than in lowland llamas. The protection against hypoxic pulmonary arterial hypertension and pulmonary contractile hyperreactivity is partly due to increased hemoxygenase-carbon monoxide signaling and decreased Ca2+ sensitization in the newborn llama pulmonary vasculature. These three striking physiological adaptations of the llama allow this species to live and thrive under the chronic influence of the hypobaric hypoxia of life at high altitude.

American tegumentary leishmaniasis: antigen-gene polymorphism, taxonomy and clinical pleomorphism.

Multi-locus enzyme electrophoresis is the current gold standard for the genetic characterisation of Leishmania. However, this method is time-consuming and, more importantly, cannot be directly applied to parasites present in host tissue. PCR-based methods represent an ideal alternative but, to date, a multi-locus analysis has not been applied to the same sample. This has now been achieved with a sample of 55 neotropical isolates (Leishmania (Viannia) braziliensis, L. (V.) peruviana, L. (V.) guyanensis, L. (V.) lainsoni and L. (L.) amazonensis), using five different genes as targets, four of which encoded major Leishmania antigens (gp63, Hsp70, H2B and Cpb). Our multi-locus approach strongly supports the current taxonomy and demonstrates a highly robust method of distinguishing different strains. Within L. (V.) braziliensis, we did not encounter so far specific genetic differences between parasites isolated from cutaneous and mucosal lesions. Interestingly, results provided by each of the different antigen-genes in the species considered, were different, suggesting different selective pressures. Our work emphasises the need for a multi-disciplinary approach to study the clinical pleomorphism of leishmaniasis.

Fetal prolactin levels respond to a maternal melatonin implant.

Seasonal PRL changes in adult sheep are controlled by photoperiod. The ability to detect photoperiod is mediated by the pineal gland through melatonin secretion. A rhythm in plasma melatonin has been described in fetal sheep. In this work we investigated whether the fetus responds to changes in circulating levels of melatonin. Fetal plasma PRL was measured every 2 h for 24 h, 7 days after the mother had received a sc Silastic implant containing approximately 1 g melatonin. Control fetuses received an empty implant. Melatonin is slowly released from implants, and it is known to cross the sheep placenta. Fetal plasma melatonin increased 10-fold after the implant. Plasma PRL in fetuses treated with melatonin was a third of that of control fetuses. Fetal plasma PRL concentrations were 56.6 +/- 5.7 ng/ml (mean +/- SE) in control and 18.1 +/- 2.7 ng/ml 7 days after the melatonin implant. We conclude from these data that the ability to respond to melatonin is present in sheep during fetal life.

The circadian variation of prolactin in fetal sheep is affected by the seasons.

Plasma PRL concentration shows a circadian variation in fetal and adult sheep. In the adult sheep the presence of this variation depends on the season. In this paper we investigated whether season affects the presence of the circadian variation of PRL in the fetal sheep. To that effect we measured plasma PRL concentration every 2 h for 24 h during summer, fall, and winter in three groups of fetal sheep whose gestational ages ranged from 125-133 days. Mean (+/- SEM) fetal plasma PRL concentrations were 352.8 +/- 65.0 ng/ml during summer (n = 6), 98.7 +/- 12.9 during fall (n = 8), and 10.5 +/- 2.6 during winter (n = 4). A 24-h variation of plasma PRL was detected during summer [PRL (ng/ml) = 352.8 + 85.2 cos 15 (t - 18.5); P = 0.007] and fall [PRL (ng/ml) = 98.7 + 26.6 cos 15 (t - 23.6); P = 0.041] but not during winter. The mesor and amplitude of the variation are higher in summer than in fall, and the acrophases differ by 5 h, taking place at dusk in summer and close to midnight in fall. These findings show that in fetal sheep, PRL responds to seasons in utero. The signal triggering this response is most likely photoperiod.

alpha-Melanocyte-stimulating hormone and adrenocorticotropin in the regulation of glucocorticoid secretion during the perinatal period in sheep.

To determine the role of other ACTH-like peptides in the regulation of glucocorticoid secretion in fetal sheep, we examined the responses of the adrenal gland of fetal and newborn sheep to comparable single doses of alpha MSH (75 micrograms) or ACTH (50 micrograms) during the last third of gestation and the first month of postnatal life. alpha MSH first increased the plasma glucocorticoid concentration at 121--130 days of gestation [from 16 +/- 1.5 to 36.9 +/- 9 (SE) ng/ml]; the response to alpha MSH persisted on days 131--140 of gestation and during the first month after birth. ACTH first increased the plasma glucocorticoid concentration at 131--140 days of gestation and increased it further in the first month after birth (from 18.9 +/- 3.6 to 97.0 +/- 10 ng/ml). The observations that the adrenal glands of fetuses and newborn lambs responded to alpha MSH at a dose comparable to that of ACTH and that the response to alpha MSH in the fetus preceded the response to ACTH may indicate that adrenal receptors mature during fetal development. These data also suggest that the regulation of the adrenal during fetal life may involve more than one tropic hormone.

Micro-evolutionary implications of allozymic and morphometric variations in sealworms Pseudoterranova sp. (Ascaridoidea: Anisakidae) among sympatric hosts from the southeastern Pacific Ocean.

We found significant morphometric and electrophoretic differences between sealworm larvae collected from four sympatric fish host species off the central coast of Chile. The South American sea lion, Otaria byronia, is a suitable host and most likely the only definitive host species in the study area. Morphological patterns of caudal papillae in adult males collected from sea lions and electrophoretic evidence from larvae and adults substantiate our conclusion that they belong to just one, new species yet to be described. The genetic and morphometric differences found between sealworm larvae from sympatric fish hosts may be due to selective pressures arising from the internal environment of the intermediate hosts, although they may serve only for passing sequential filters along the life cycle. The discussion deals with the roles that definitive and intermediate hosts may play in the micro-evolutionary processes of sealworms.

Carotid blood flow changes with behavioral states in the late gestation llama fetus in utero.

This study tested the hypothesis that in the llama fetus changes in cerebral blood flow are closely associated with changes in cerebral oxidative metabolism such as occur during transitions between electrocortical states. For the first time reported in any species, instantaneous changes in common carotid blood flow, employed as a continuous index of cerebrovascular perfusion, were related to instantaneous changes in electrocortical activity. Three late gestation fetal llamas were surgically prepared under general anesthesia with vascular catheters, a tracheal and amniotic catheter, and with electrodes implanted to monitor the fetal electrocorticogram (ECoG). In addition, Transonic flow probes were placed around a common carotid artery and a femoral artery. At least 4 days after surgery fetal arterial blood, amniotic and tracheal pressures, carotid and femoral blood flows and the fetal ECoG were recorded continuously. Our results suggest a close association between increases in common carotid blood flow and low voltage ECoG in the llama fetus. Close coupling between instantaneous changes in carotid blood flow and electrocortical states together with the lack of an increase in brain blood flow without increased cerebral oxygen extraction during hypoxemia in the llama fetus supports a fall in cerebral oxidative metabolism in this species during hypoxemic episodes.

Characterization of Leishmania species from Peru.

Twenty-six isolates of Leishmania parasites of Peruvian origin were studied by isoenzyme electrophoresis of four marker enzymes (ASAT, ALAT, G6PD and GPI), kinetoplast DNA hybridization and monoclonal antibody binding and compared with marker strains of the New World organisms L. b. braziliensis, L. b. guyanensis, L. m. mexicana and L. m. amazonensis. 12 of the isolates studied were of Andean origin; 11 of these were isolated from patients with Andean cutaneous leishmaniasis. The organisms originating from the Peruvian Amazonian forest were isolated from patients with cutaneous (12 cases) or mucocutaneous (2 cases) leishmaniasis. One of the Andean isolates was obtained from an infected phlebotomine vector. 25 of the new isolates were identified as L. braziliensis ssp. according to the three techniques employed. The results of monoclonal antibody binding showed that 23 of the isolates were indistinguishable from L. b. braziliensis. Two isolates identified as L. braziliensis ssp. according to their isoenzyme profiles and k-DNA hybridization patterns could not be classified at the subspecies level. The isolate obtained from the phlebotomine vector could not be identified. No evidence of the existence of parasites of the L. mexicana complex in Peruvian territory was found in this study. The results obtained show a remarkable similarity between Leishmania of Andean origin and L. b. braziliensis.

Insulin homeostasis in the extremely low birth weight infant.

The need to improve the nutritional status of extremely low birth weight infants has resulted in a higher incidence of problems related to glucose intolerance. The inability of the newborn to inhibit gluconeogenesis in response to a glucose infusion has been postulated as an important determinant of the hyperglycemia observed in extremely low birth weight infants. The 2 proposed mechanisms to explain this finding include inappropriate secretion of insulin by the pancreas and decrease sensitivity of the liver to the gluco-regulatory effect of insulin. The capacity of extremely low birth weight infants to oxidize glucose at higher rates, and the positive effect that insulin may have in glucose utilization and tolerance, support the use of insulin in the prevention and treatment of hyperglycemia. Continuous infusion of insulin appears to be safe for the treatment of hyperglycemia, based on the available studies. However, the effectiveness of insulin treatment needs to be critically tested further before it can be implemented in routine clinical practice.

Cardiovascular responses to graded degrees of hypoxaemia in the llama fetus.

The fetal llama exposed to an intense degree of hypoxaemia did not increase cerebral blood flow, but showed a marked peripheral vasoconstriction. The same cardiovascular response is observed in fetal sheep submitted to a extremely severe hypoxaemia, when the initial compensatory vasodilatory mechanisms in brain and heart fail. To investigate whether the fetal llama responses to acute hypoxaemia are adaptive, or whether they are the result of a breakdown of mechanisms of blood flow redistribution that favours the central nervous system, we studied seven fetal llamas (0.6-0.7 of gestation) chronically-catheterized during 1 h of graded and progressive hypoxaemia. Fetal ascending aorta blood gases and fetal cardiac output and its distribution (radiolabelled-microspheres) were measured after 60 min of normoxaemia (B) and at the end of 20 min (H20), 40 min (H40) and 60 min (H60) of hypoxaemia. Data were analysed by ANOVA and Newman-Keuls tests. Each treatment resulted in a lower (P < 0.05) percentage of haemoglobin saturation than hypoxaemia; H40 was lower than H20, and H60 was lower than H20 and H40. No statistical difference was observed among treatments for cardiac output or cerebral blood flow. These results demonstrate that fetal cardiac output and brain blood flow are maintained at all degrees of hypoxaemia, indicating that these cardiovascular responses are an adaptive response in the llama fetus, rather than an index of cardiorespiratory decompensation.

Renal function in the chronically cannulated fetal llama: comparison with studies in the ovine fetus.

Samples of maternal and fetal plasma, fetal urine, and amniotic fluid were collected from 8 chronically cannulated pregnant llamas, in the last third of gestation. The samples were obtained for up to 18 days post-surgery. Osmolality, sodium (Na), potassium (K), chloride (Cl), and urea were measured on 40 samples collected on days 1, 2, 3, 4-5, 6-7, 8-9, and 10-19. The osmolalities of maternal and fetal plasma, fetal urine and amniotic fluid, averaged over these 7 time periods, were, respectively, 312 +/- 2, 311 +/- 1, 484 +/- 14, and 317 +/- 1 mosmol kg-1. Values are given as mean +/- s.e. The major differences from fetal fluid values in the ovine fetus (from previously published values) were the higher osmolality and urea concentration of llama fetal urine. Urine flow rate measured in 6 fetuses, 4.5-6.5 kg body weight, was 5.8 +/- 0.4 mliter h-1; urea clearance rate was 55.5 +/- 11.8 mliter h-1. Glomerular filtration rate (GFR), measured with 51Cr-EDTA in 5 fetuses on 1-4 occasions, was 111.4 +/- 23.3 mliter h-1. Fractional reabsorptions (FR) of Na, K and Cl were 97.9 +/- 1, 75.9 +/- 13.5 and 97.7 +/- 0.4% respectively. The GFR (25 mliter kg-1 h-1) and urine flow rate (1 mL kg-1 h-1) were less than half and about one-tenth the respective values in ovine fetuses. As Na reabsorption is the major oxygen-consuming activity of the kidney, the llama fetal kidney requires only half the oxygen needed by the ovine fetal kidney to reabsorb the filtered sodium load. The reason for the formation of hypertonic, rather than hypotonic, urine in the fetal llama may be due to both greater morphological maturity of the kidney and the excretion of as yet unidentified osmotically active organic substances.

Cardiovascular responses to arginine vasopressin blockade during acute hypoxemia in the llama fetus.

The fetal llama has a marked increase in the peripheral vascular resistance and no augmentation of brain blood flow during hypoxemia. In spite of the substantial plasma arginine-vasopressin (AVP) increase during hypoxemia, up to 8 times greater than in fetal sheep, there are no changes of carotid and femoral blood flows during hypoxemia with a V1 receptor blockade, as is seen in the fetal sheep. The aim of this study was to assess the role of AVP function in mediating the combined ventricular output and organ blood flow in the hypoxemic llama fetus. Six fetal llamas at 0.65 of gestation were instrumented under general anesthesia, and cardiorespiratory responses and blood flows determined under normoxemic and hypoxemic conditions. The AVP effect was determined using a V1 antagonist during normoxemic and hypoxemic conditions. Organ blood flows were measured with the radioactive microsphere technique. No significant differences in organ blood flow or in their vascular resistances were seen between the control and treated fetuses during hypoxemia. We conclude that V1 blockade did not have any important role in the cardiovascular response to acute hypoxemia in the llama fetus, in contrast with lowland fetuses. AVP may be playing a role in other regions, possibly in kidney or lung, during hypoxemia.

Low-dose inhaled carbon monoxide reduces pulmonary vascular resistance during acute hypoxemia in adult sheep.

Carbon monoxide (CO) is produced by the action of the heme oxygenase (HO) complex through the oxidation of heme. CO, like nitric oxide (NO), is a molecular gas that among other actions stimulates guanylyl cyclase and increases cGMP levels in smooth muscle cells, regulating the vascular tone. Acute hypoxia generates pulmonary hypertension and increases the expression of inducible HO isoform (HO-1) in the vascular endothelium. Inhaled NO causes a potent pulmonary vasodilation. We hypothesized that inhaled CO might produce similar actions as NO on pulmonary vascular resistance (PVR). To test our contention, we studied the effects of inhaled CO (40 ppm) in the augmented PVR observed during hypoxemia. Five chronically instrumented German Merino sheep were submitted to a protocol consisting of 20 min of normoxemia (N), 20 min of isocapnic hypoxemia (H20), 20 min of isocapnic hypoxemia plus CO 40 ppm (H40), and 20 min of recovery (R). In the control protocol, we did not administer inhaled CO. Arterial gases and pH, percentage of carboxyhemoglobin (COHb), systemic and pulmonary arterial pressure, systemic and pulmonary vascular resistance, and cardiac output were measured during each period. During H20 period, there was a significant increase in cardiac output and PVR in sheep submitted to both protocols. The sheep treated with inhaled CO (H40 + CO) showed a modest but significant decrease (16%) in the elevated PVR. Our data indicate that inhaled CO decreases pulmonary vascular resistance associated with acute hypoxemia in adult sheep.

[Quantitative cultures from catheters, using the Brun Buisson technique]. / Cultivos cuantitativos de catéteres por la técnica de Brun Buisson.

The objective of this collaborative work carried out in the Fundación Favaloro and the Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia, was to determine optimal conditions for incubation (time and atmosphere) of quantitative cultures of catheters processed according to the technique of vortex agitation (Brun Buisson method). From 689 processed catheters, 551 yielded negative cultures. From the 138 positive cultures, 125 yielded monomicrobial cultures and 13 polimicrobial cultures (total number of microorganisms was 151). In the last situation each micoorganism was considered on an individual basis. A total of 58 episodes of catheter related bacteremias occurred, being 52 monomicrobial and 6 polimicrobial (total number of microorganisms was 64). When colony counts were compared in aerobic and in 5-10% CO2 atmospheres, a very good correlation was obtained (p = 0.27; r2 = 0.9268). No advantage was observed by incubating plates for more than 48 hours. Colony counts performed at the second versus the third day, and at the second day versus the seventh, gave very good correlation (p = 0.10 and r2 = 0.9996; p = 0.31 and r2 = 0.9995, respectively).

[The detection of the DNA of the hepatitis B virus in patients with probable non-A, non-B hepatitis]. / Detección del DNA del virus de la hepatitis B en pacientes con probable hepatitis noA-noB.

We found the presence of hepatitis B virus in 17 cases of non-A-non-B hepatitis using the DNA detection technique in serum of patients with a type of chronic hepatopathy. This finding supports the needs to determine this seric marker in all patients afflicted with chronic hepatopathy before the diagnosis of hepatitis B is excluded.