End-to-end image analysis pipeline for liquid-phase electron microscopy.

Liquid phase transmission electron microscopy allows the imaging of materials in liquid environments. The sample is encapsulated within electron-beam transparent windows and hence protected by the ultrahigh vacuum necessary within the electron gun. Such an approach allows to study biological and soft materials in their natural environment and offers the possibility of accessing their dynamic nature. Yet, the electron beam scattering from the windows and solvent increases the image noise and blur. Herein, we propose a pipeline to both de-noise and sharpen images obtained by liquid transmission electron microscopy. We develop the workflow in a way that it does not require any human interference, nor introduce artefacts, but actually unveils features of the imaged samples covered by the noise and the blur. LAY DESCRIPTION: Transmission Electron Microscopy TEM is one of the most powerful techniques for structural determination at the nanoscale, with the ability to image matter down to the atomic level. TEM is only possible by keeping the electron beam under high vacuum in order to avoid undesired scattering events in the beam path. High vacuum means that the TEM samples must conventionally be in solid-state. Thus, samples in liquid form or containing liquids, like water, need special preparation techniques which tend to alter the structure and chemical nature of the sample. Such alterations are particularly critical for biological and soft organic materials where the structures are controlled by the presence of water and/or other liquids. The development of new cameras, materials and sample holders have made possible for TEM to be performed on liquid samples. Liquid Phase Transmission Electron Microscopy (LTEM) offers the possibility to investigate nanoscopic structures in liquid state and monitor dynamic processes. However important limitations come from the liquid nature of samples in the imaging process such as the low contrast afforded by organic and biological materials and additional noise and blur introduced by the liquid sample and its thickness. Existing image analysis algorithms for TEM result inadequate for LTEM. The end-to-end image analysis method herein has the ability to recover the original images together with their sharpness, without introducing any artefacts. The proposed algorithms offer the great advantage of unveiling image details which are not usually seen during imaging, thus allowing a better understanding of the nature, structure and ultimately the function of the investigated structures. The fully automatised analysis method allows to efficiently process dozens of images in few hours, improving dramatically the performance of LTEM imaging.

Goals scored and received in 5vs4 GK game strategy are constrained by critical moment and situational variables in elite futsal.

The aim of this study was to identify the importance and meaning of goals using the goalkeeper as an outfield player in elite futsal according to critical and situational variables. The sample consisted of 11,446 actions corresponding to 1,325 matches from the 1st division Spanish Futsal League during the seasons from 2010 to 2015. Multinomial logistic regression and classification tree multivariate models were used to identify the best predictor variables related to the likelihood of scoring goals, receiving goals, or no goals. Results from Multinomial logistic regression emphasised goals scored in balanced matches and playing with the goalkeeper as an outfield player before the last eight minutes. When the teams were drawing or losing, finished with goals received or without goals. The classification tree results identified a greater likelihood of scoring goals when the teams were winning, in balanced matches, and within the last eight minutes. Conversely, a greater likelihood of suffering goals was observed using the goalkeeper as an outfield player when the teams were losing, in unbalanced matches and in the last eight minutes. The identified trends will allow futsal coaches to recognise the most suitable situations for achieving efficacy when using the goalkeeper as an outfield player strategy.

¿Debemos preocuparnos por la coordinación corporal de los escolares de la Educación Secundaria Obligatoria? / Why should we care about motor coordination of Secondary School children?

Existe una preocupación sobre el nivel de competencia coordinativa de los escolares en la Educación Secundaria Secundaria (ESO) española y su impacto en sus actividades cotidianas y en su salud. El objetivo de este estudio fue evaluar la coordinación de una muestra de escolares de primero de la ESO. Participaron 120 escolares, 65 chicos y 55 chicas, de cuatro institutos de Educación Secundaria de la Comunidad de Madrid, de edades comprendidas entre 12 y 14 años. Todos los participantes llevaron a cabo el test de coordinación corporal (KKTK) diseñado para detectar los problemas de coordinación motriz en las edades escolares. Los resultados permitieron el establecimiento de tres grupos de coordinación: normal (38,3%), sintomático (31,35%) y problemático (30%). Estos resultados indicaron que un 61,7% de los escolares participantes en este estudio presentaban algún tipo de dificultad de coordinación, al encontrarse en los niveles sintomático y problemático. La presencia de las chicas en el nivel sintomático y problemático fue predominante. Los resultados confirman que los problemas de coordinación motriz existen en la población escolar y que no solo suponen un problema educativo sino también de salud, ya que impiden estilos de vida más activos entre los adolescentes, especialmente entre las chicas (AU)

There is concern about the level of motor coordination among Spanish secondary students and its impact on their daily activities and health. The aim of this study was to evaluate the coordination of a sample of secondary school children. The study included 120 schoolchildren, 65 boys and 55 girls of four Secondary Schools of the Community of Madrid, aged between 12 and 14 years. All participants performed the Body Coordination Test (KKTK), designed to detect motor coordination problems in schoolchildren aged 12 to 14 years. The results allowed to establish three coordinating groups: normal (38.3%) symptomatic (31.35%) and problematic (30%). These results indicated that 61.7% of the students participating in this study formed the Symptomatic and Problematic groups. Girls were predominant in the symptomatic and problematic levels. This study confirms that motor coordination problems exist among Spanish secondary schoolchildren. This is not only an education but also a health problem because it prevents more active life styles among adolescents, especially among girls (AU)

High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human African trypanosomiasis, leishmaniasis, and Chagas disease.

African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compounds against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chemical libraries using phenotypic and target-based approaches. Combinatorial chemistry libraries contain hundreds of thousands of compounds; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chemical diversity that can serve as excellent templates for the synthesis of novel, biologically active molecules. We report here a validated HTS platform for the screening of microbial extracts against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial extracts from the MEDINA Natural Products library. Tandem liquid chromatography-mass spectrometry showed that 48 extracts contain potentially new compounds that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product extracts against the above-mentioned kinetoplastid parasites.

[Iodine nutritional status in the school-aged population in Alicante (Spain)]. / Situación nutricional de yodo en la población escolar de Alicante.

BACKGROUND: According to the World Health Organization (WHO), goiter is endemic in Spain. The main cause of endemic goiter is iodine deficiency, which is also the principal cause of mental retardation and avoidable cerebral palsy throughout the world. MATERIAL AND METHODS: We conducted an observational study to determine the prevalence of endemic goiter and nutritional iodine status in the province of Alicante. Urinary iodine excretion was measured in a morning urine sample, and thyroid volume was measured by means of a thyroid ultrasound scan. A case of goiter was diagnosed if thyroid volume was above the 97th percentile adjusted by age, as published by the WHO. RESULTS: No cases of goiter were found. In addition, the median urinary iodine excretion levels adjusted by age were within the normal range, as defined by the WHO's criteria. CONCLUSIONS: Endemic goiter was not found in the province of Alicante and urinary iodine excretion values demonstrated adequate iodine intake. Further ultrasound studies are needed to establish reference thyroid volumes for our population.

Situación nutricional de yodo en la población escolar de Alicante / Iodine nutritional status in the school-aged population in Alicante (Spain)

Antecedentes Según la Organización Mundial de la salud (OMS), España se consideró un país afectado de endemia bociosa. El déficit de yodo es el responsable de dicha endemia además de ser la principal causa de retraso mental y parálisis cerebral evitable en el mundo. Material y métodos Estudio observacional para determinar la prevalencia de bocio endémico y el estado nutricional de yodo en la provincia de Alicante. Para ello se midió la yoduria en una muestra aislada y el volumen tiroideo mediante ecografía. Se consideró bocio todo volumen tiroideo superior al percentil 97 por edad publicado por la OMS. Resultados No se ha encontrado ningún caso de bocio. Así mismo las cifras de yoduria obtenidas también pueden ser consideradas dentro de la normalidad bajo los criterios de la OMS. Conclusiones Se puede decir que la provincia de Alicante no padece endemia bociosa y que además las cifras de yoduria demuestran una adecuada ingesta de yodo. Se evidencia la necesidad de realizar más estudios ecográficos de tiroides en otras zonas para establecer volúmenes tiroideos de referencia para nuestra población

Background According to the World Health Organization (WHO), goiter is endemic in Spain. The main cause of endemic goiter is iodine deficiency, which is also the principal cause of mental retardation and avoidable cerebral palsy throughout the world. Material and methods We conducted an observational study to determine the prevalence of endemic goiter and nutritional iodine status in the province of Alicante. Urinary iodine excretion was measured in a morning urine sample, and thyroid volume was measured by means of a thyroid ultrasound scan. A case of goiter was diagnosed if thyroid volume was above the 97th percentile adjusted by age, as published by the WHO. Results No cases of goiter were found. In addition, the median urinary iodine excretion levels adjusted by age were within the normal range, as defined by the WHO's criteria. Conclusions Endemic goiter was not found in the province of Alicante and urinary iodine excretion values demonstrated adequate iodine intake. Further ultrasound studies are needed to establish reference thyroid volumes for our population

[An unusual cause of failure of weaning from mechanical ventilation in an infant]. / Una causa inusual de fracaso del destete de la ventilación mecánica en un lactante.

The weaning process is a critical phase in patients undergoing mechanical ventilation. This process can be hampered by numerous causes, such as neuromuscular diseases and spinal muscular atrophy (SMA). We present a 6-month-old boy with respiratory distress, fever, marked hypotonia without motor developmental milestones, and areflexia. The patient showed progressive respiratory distress requiring mechanical ventilation. Definitive weaning was not achieved and the boy died from respiratory failure. Partial autopsy was performed with a diagnosis of SMA and genetic study of the parents. Neuromuscular diseases are an infrequent cause of respiratory insufficiency in suckling infants. The differential diagnosis is made between axonal and motor neuron diseases. The diagnosis was confirmed by muscular biopsy and genetic study.

Una causa inusual de fracaso del destete de la ventilación mecánica en un lactante / An unusual cause of failure of weaning from mechanical ventilation in an infant

El proceso de destete es una fase crítica del paciente tratado con soporte ventilatorio que puede verse dificultada por numerosas causas entre las que se encuentran las enfermedades neuromusculares, como la atrofia muscular espinal (AME). Lactante de 6 meses que presenta dificultad respiratoria y fiebre llamando la atención una marcada hipotonía generalizada sin logros motrices y arreflexia rotuliana. El niño sufre deterioro respiratorio con necesidad de asistencia ventilatoria mecánica sin lograrse un destete definitivo. Fallece por fallo respiratorio, realizándose necropsia parcial con diagnóstico de AME y estudio genético de progenitores. Las enfermedades neuromusculares son una causa poco frecuente de insuficiencia respiratoria en lactantes. Se realiza un diagnóstico diferencial entre las enfermedades de motoneurona, enfermedades axonales y enfermedades de placa motora. El diagnóstico fue confirmado por la biopsia muscular y el estudio genético

The weaning process is a critical phase in patients undergoing mechanical ventilation. This process can be hampered by numerous causes, such as neuromuscular diseases and spinal muscular atrophy (SMA). We present a 6-month-old boy with respiratory distress, fever, marked hypotonia without motor developmental milestones, and areflexia. The patient showed progressive respiratory distress requiring mechanical ventilation. Definitive weaning was not achieved and the boy died from respiratory failure. Partial autopsy was performed with a diagnosis of SMA and genetic study of the parents. Neuromuscular diseases are an infrequent cause of respiratory insufficiency in suckling infants. The differential diagnosis is made between axonal and motor neuron diseases. The diagnosis was confirmed by muscular biopsy and genetic study

Fibrosis endomiocárdica o enfermedad de Davies / Endomyocardial fibrosis or Davies’ disease

No disponible

Responsive brushes and gels as components of soft nanotechnology.

Progress in the development of generic molecular devices based on responsive polymers is discussed. Characterisation of specially synthesised polyelectrolyte gels, "grafted from" brushes and triblock copolymers is reported. A Landolt pH-oscillator, based on bromate/ sulfite/ferrocyanide, with a room temperature period of 20 min and a range of 3.1 < pH < 7.0, has been used to drive periodic oscillations in volume in a pH responsive hydrogel. The gel is coupled to the reaction and changes volume by a factor of at least 6. A continuously stirred, constant volume, tank reactor was set-up on an optical microscope and the reaction pH and gel size monitored. The cyclic force generation of this system has been measured directly in a modified JKR experiment. The responsive nature of polyelectrolyte brushes, grown by surface initiated ATRP, have been characterised by scanning force microscopy, neutron reflectometry and single molecule force measurements. Triblock copolymers, based on hydrophobic end-blocks and either polyacid or polybase mid-block, have been used to produce polymer gels where the deformation of the molecules can be followed directly by SAXS and a correlation between molecular shape change and macroscopic deformation has been established. The three systems studied allow both the macroscopic and a molecular response to be investigated independently for the crosslinked gels and the brushes. The triblock copolymers demonstrate that the individual response of the polyelectrolyte molecules scale-up to give the macroscopic response of the system in an oscillating chemical reaction.

[Cerebrovascular stroke as a sign of atrial myxoma in childhood]. / Accidente cerebrovascular como manifestación de mixoma auricular.

Primary cardiac tumors are uncommon with an estimated incidence of between 0.0017 % and 0.19 %. Most are benign. Whereas myxomas are the most common primary tumor in adults, in children they are exceptionally rare. Cardiac myxomas usually develop in the left atrium, 20 % occur in the right atrium and the remainder develops in the ventricles and rarely in the heart valves. Cerebrovascular strokes secondary to myxoma are rare in childhood. The diagnostic test of choice is transesophageal echocardiogram and early excision is the most effective treatment in preventing serious complications. We report a case of cerebral stroke as the only manifestation of an atrial myxoma in an 11-year-old-girl. The patient presented vertigo, right hemiparesis of the body and dysarthria without loss of consciousness. After diagnostic tests (computerized tomography, magnetic resonance imaging and cerebral angioresonance) she was diagnosed with an ischemic lesion in the left middle cerebral artery. Various investigations were performed to find the cause of the stroke, among them cardiologic study, and a mass in the left atrium suggestive of myxoma was found. The tumor was removed and the diagnosis of myxoma was confirmed by histopathological examination. Outcome was satisfactory. The presence of a cerebral ischemic episode, with or without concomitant heart disease, suggests the need to look for cardiac etiology.

Accidente cerebrovascular como manifestación de mixoma auricular / Cerebrovascular stroke as a sign of atrial myxoma in childhood

Los tumores cardíacos primarios son extremadamente raros, con una incidencia estimada entre el 0,0017 y el 0,19 por ciento. En su mayoría son benignos. Mientras los mixomas son los más frecuentes en adultos, en niños es un proceso infrecuente. Los mixomas se encuentran principalmente en la aurícula izquierda, el 20 por ciento en la aurícula derecha, el resto en los ventrículos y, muy rara vez, en las válvulas auriculoventriculares. Los accidentes cerebrovasculares secundarios a un mixoma son bastante raros, sobre todo en la edad pediátrica. Se considera que el ecocardiograma transesofágico es el estudio diagnóstico de elección y la resección temprana es el tratamiento más adecuado para evitar las complicaciones graves. Se presenta el caso de un mixoma auricular como causa de accidente cerebrovascular en una niña de 11 años, que ingresó tras presentar vértigo, pérdida de fuerza en hemicuerpo derecho y dificultad para el habla, sin pérdida de conocimiento para estudio. Tras la realización de estudios complementarios (tomografía computarizada, resonancia magnética y angiorresonancia cerebral) se diagnosticó infarto de la arteria cerebral media izquierda. Se realizaron diversos estudios para clasificar el origen del accidente cardiovascular, entre ellos, el estudio cardiológico, encontrándose una masa en aurícula izquierda indicativa de mixoma. Se realizó resección del tumor y se confirmó el diagnóstico de mixoma mediante el estudio anatomopatológico. La evolución clínica fue satisfactoria. Ante la presencia de un suceso isquémico en territorio cerebral, con o sin evidencia de enfermedad cardíaca, debe buscarse intencionadamente una causa cardíaca (AU)

Novel inhibitors of Trypanosoma cruzi dihydrofolate reductase.

There is an urgent need for the development of new drugs to treat Chagas' disease, which is caused by the protozoan parasite Trypanosoma cruzi. The enzyme dihydrofolate reductase (DHFR) has been a very successful drug target in a number of diseases and we decided to investigate it as a potential drug target for Chagas' disease. A homology model of the enzyme was used to search the Cambridge Structural Database using the program DOCK 3.5. Compounds were then tested against the enzyme and the whole parasite. Compounds were also screened against the related parasite, Trypanosoma brucei.

Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Leishmania major.

Kinetic properties of the dimeric enzyme dUTPase from Leishmania major were studied using a continuous spectrophotometric method. dUTP was the natural substrate and dUMP and PPi the products of the hydrolysis. The trypanosomatid enzyme exhibited a low K(m) value for dUTP (2.11 microM), a k(cat) of 49 s(-1), strict Michaelis-Menten kinetics and is a potent catalyst of dUDP hydrolysis, whereas in other dUTPases described, this compound acts as a competitive inhibitor. Discrimination is achieved for the base and sugar moiety showing specificity constants for different dNTPs similar to those of bacterial, viral, and human enzymes. In the alkaline range, the K(m) for dUTP increases with the dissociation of ionizable groups showing pK(a) values of 8.8, identified as the uracil moiety of dUTP and 10, whereas in the acidic range, K(m) is regulated by an enzyme residue exhibiting a pK(a) of 7.1. Activity is strongly inhibited by the nucleoside triphosphate analog alpha-beta-imido-dUTP, indicating that the enzyme can bind triphosphate analogs. The existence of specific inhibition and the apparent structural and kinetic differences (reflected in different binding strength of dNTPs) with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against leishmaniasis.

Novel inhibitors of Leishmanial dihydrofolate reductase.

The program DOCK3.5 was used to search the Cambridge Structural Database for novel inhibitors of Leishmanial dihydrofolate reductase. A number of compounds were obtained and screened against the enzyme and against the intact parasite Leishmania donovani and the related organisms Trypanosoma brucei and Trypanosoma cruzi. The compounds screened showed weak activity in both the enzyme assays and the in vitro assays.

Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease.

The intracellular pathogen Trypanosoma cruzi is the etiological agent of Chagas' disease. We have isolated a full-length cDNA encoding uracil-DNA glycosylase (UDGase), a key enzyme involved in DNA repair, from this organism. The deduced protein sequence is highly conserved at the C-terminus of the molecule and shares key residues involved in binding or catalysis with most of the UDGases described so far, while the N-terminal part is highly variable. The gene is single copy and is located on a chromosome of approximately 1.9 Mb. A His-tagged recombinant protein was overexpressed, purified and used to raise polyclonal antibodies. Western blot analysis revealed the existence of a single UDGase species in parasite extracts. Using a specific ethidium bromide fluorescence assay, recombinant T.cruzi UDGase was shown to specifically excise uracil from DNA. The addition of both Leishmania major AP endonuclease and exonuclease III, the major AP endonuclease from Escherichia coli, produces stimulation of UDGase activity. This activation is specific for AP endonuclease and suggests functional communication between the two enzymes.

Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.

Properties of Leishmania major dUTP nucleotidohydrolase, a distinct nucleotide-hydrolysing enzyme in kinetoplastids.

We have previously reported the presence, in the parasitic protozoan Leishmania major, of an enzyme involved in controlling intracellular dUTP levels. The gene encoding this enzyme has now been overexpressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical and enzymic analyses of the Leishmania enzyme show that it is a novel nucleotidohydrolase highly specific for deoxyuridine 5'-triphosphate. The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates. The reaction products are dUMP and PP(i) when dUTP is the substrate and dUMP and P(i) in the case of dUDP. The enzyme is sensitive to inhibition by the reaction product dUMP but not by PP(i). dUTPase activity is highly dependent on Mg(2+) concentrations and markedly sensitive to the phosphatase inhibitor, NaF. In summary, Leishmania dUTPase appears to be markedly different to other proteins characterized previously that accomplish the same function.