Optimising adjacent membrane segmentation and parameterisation in multicellular aggregates by piecewise active contours.

In fluorescence microscopy imaging, the segmentation of adjacent cell membranes within cell aggregates, multicellular samples, tissue, organs, or whole organisms remains a challenging task. The lipid bilayer is a very thin membrane when compared to the wavelength of photons in the visual spectra. Fluorescent molecules or proteins used for labelling membranes provide a limited signal intensity, and light scattering in combination with sample dynamics during in vivo imaging lead to poor or ambivalent signal patterns that hinder precise localisation of the membrane sheets. In the proximity of cells, membranes approach and distance each other. Here, the presence of membrane protrusions such as blebs; filopodia and lamellipodia; microvilli; or membrane vesicle trafficking, lead to a plurality of signal patterns, and the accurate localisation of two adjacent membranes becomes difficult. Several computational methods for membrane segmentation have been introduced. However, few of them specifically consider the accurate detection of adjacent membranes. In this article we present ALPACA (ALgorithm for Piecewise Adjacent Contour Adjustment), a novel method based on 2D piecewise parametric active contours that allows: (i) a definition of proximity for adjacent contours, (ii) a precise detection of adjacent, nonadjacent, and overlapping contour sections, (iii) the definition of a polyline for an optimised shared contour within adjacent sections and (iv) a solution for connecting adjacent and nonadjacent sections under the constraint of preserving the inherent cell morphology. We show that ALPACA leads to a precise quantification of adjacent and nonadjacent membrane zones in regular hexagons and live image sequences of cells of the parapineal organ during zebrafish embryo development. The algorithm detects and corrects adjacent, nonadjacent, and overlapping contour sections within a selected adjacency distance d, calculates shared contour sections for neighbouring cells with minimum alterations of the contour characteristics, and presents piecewise active contour solutions, preserving the contour shape and the overall cell morphology. ALPACA quantifies adjacent contours and can improve the meshing of 3D surfaces, the determination of forces, or tracking of contours in combination with previously published algorithms. We discuss pitfalls, strengths, and limits of our approach, and present a guideline to take the best decision for varying experimental conditions for in vivo microscopy.

Characterization of the Proteome of Theobroma cacao Beans by Nano-UHPLC-ESI MS/MS.

Cocoa seed storage proteins play an important role in flavour development as aroma precursors are formed from their degradation during fermentation. Major proteins in the beans of Theobroma cacao are the storage proteins belonging to the vicilin and albumin classes. Although both these classes of proteins have been extensively characterized, there is still limited information on the expression and abundance of other proteins present in cocoa beans. This work is the first attempt to characterize the whole cocoa bean proteome by nano-UHPLC-ESI MS/MS analysis using tryptic digests of cocoa bean protein extracts. The results of this analysis show that >1000 proteins could be identified using a species-specific Theobroma cacao database. The majority of the identified proteins were involved with metabolism and energy. Additionally, a significant number of the identified proteins were linked to protein synthesis and processing. Several proteins were also involved with plant response to stress conditions and defence. Albumin and vicilin storage proteins showed the highest intensity values among all detected proteins, although only seven entries were identified as storage proteins. A comparison of MS/MS data searches carried out against larger non-specific databases confirmed that using a species-specific database can increase the number of identified proteins, and at the same time reduce the number of false positives. The results of this work will be useful in developing tools that can allow the comparison of the proteomic profile of cocoa beans from different genotypes and geographic origins. Data are available via ProteomeXchange with identifier PXD005586.

[Foundations for the classification of medical specialist programs in a national qualifications framework]. / Fundamentos para la clasificación de los programas de médico especialista en un marco de cualificaciones nacional.

Qualification of learning outcomes in terms of knowledge, skills, responsibility and autonomy provided by medical specialist programs is of interest to State authorities, educational service providers, employers, and specialists. Countries that are signatories to the Bologna Process and others outside Europe, established that the guaranteed primary degree for medical studies is the Master in Medicine (second cycle). There is agreement that medical specialist programs are more advanced than a Master of Medicine but are different from the Doctor of Medicine (third cycle) in their clinical orientation. These programs usually do not have research components and occasionally they are not carried out in the higher education system. However, the level of qualification of medical specialist programs has not been established due to lack of consensus. In Chile, this decision becomes even more complicated due to the certification of "licenciatura" (first cycle) that medical schools provide. There are also gaps in the educational classification procedure employed by the country. However, the review of national qualification frameworks and government acts shows that some countries have validated these certifications as third cycle. Medical specialties certainly do not correspond to PhD programs and the eligibility of the qualification level must be guaranteed to all stakeholders, who require an agreement on the specific national framework of learning outcomes and competencies.

[Time to change the graduate-postgraduate liaison in Chilean medical education? Reflections about a double certification as Physician and Doctor in medical sciences]. / ¿Es tiempo de modificar la vinculación pregrado postgrado en la educación médica chilena? Reflexiones sobre una vía de doble titulación Médico cirujano/Doctor en ciencias médicas.

In the last 150 years, scientific research has produced extraordinary discoveries in Medicine and there is no doubt that research will continue contributing substantially in the future but there is no the same conviction regarding how to provision such capacities in medical graduates. In Chile, the Faculties of Medicine created several doctorate programs in Medical Sciences (Ph.D.) to strengthen medical research. However, the low number of physicians who apply to these programs is a caveat. These programs provide the advanced third-cycle competencies expected by students aspiring for an excellence research certification and their incorporation into academia. Universities stand out in their capacity to adapt themselves to educational needs, developing programs designed to fill specific gaps. Ph.D. programs intercalated to the medical career show that this is the correct approach. The development of specific doctorate programs for the small number of physicians interested in research and for medical students with a strong interest in research could be an innovative solution to motivate and encourage them to develop a career in clinical investigation. Using this approach, Medical Schools and Doctorate in Medical Sciences programs would jointly stimulate the training of medical scientists.

Sweet corn cob as a functional ingredient in bakery products.

Gluten free (GF) products are often inferior in quality attributes, nutritional content and consumer acceptability. The use of GF by-products is a novel strategy to improve the structure and nutritional profile of these products. Sweet corn cob (SCC) is a by-product of sweet corn processing containing a considerable amount of fibre and ferulic acid. The effect of baking on ferulic acid content, colour, texture and physical characteristics on muffins incorporated with SCC flour (SCCF) as a value-added food ingredient was investigated using a GF model system. The freeze-dried SCCF, containing ferulic acid (6.02 mg g-1) was used to replace the rice flour at varying levels of 10, 20, and 30%. In general, SCCF increased dietary fibre and free ferulic acid content of muffins. Inclusion of 20% SCCF showed an increase in terms of the height of the muffin and number of air cells in the crumb, along with a decrease in the hardness of muffins. Muffins with SCCF showed higher mean overall liking scores than rice flour muffin.

[Diagnostic methods in candidemia: a systematic review of literature with meta-analysis]. / Métodos diagnósticos en candidemia: una revisión sistemática de la literatura con meta-análisis.

UNLABELLED: Candidemia is a disease with high morbidity and mortality especially in critical care patients. Early diagnosis enables early treatment. OBJECTIVES: To conduct a systematic review of the literature in order to establish the best laboratory tests for the diagnosis of candidemia in critical patients. MATERIALS AND METHODS: We conducted a systematic review of available literature in PubMed. Serological studies were subjected to meta-analysis in metadisk-Beta 1.1.1. RESULTS: 4 studies of 1286 reviewed were included. Three were about serological tests and one about molecular testing (RT-PCR). The sensitivity and specificity for RT-PCR, antibody testing and antigen and antibody tests were 87% and 100%, 47.5% and 82.6%, 96% and 81%, respectively. Diagnostic Odds Ratio of antigenemia was 1.51 (95% CI = 0,032-70,964, p = 0.001). CONCLUSIONS: RT-PCR has better diagnostic performance, measuring antigenemia plus antibodies improves sensitivity, specificity, LR + and LR-- . There is insufficient evidence to support this.

UHPLC-MS/MS analysis of cocoa bean proteomes from four different genotypes.

In this study the proteomic profiles of cocoa beans from four genotypes with different flavour profiles were analysed by bottom-up label-free UHPLC-MS/MS. From a total of 430 identified proteins, 61 proteins were found significantly differentially expressed among the four cocoa genotypes analysed with a fold change of ≥2. PCA analysis allowed clear separation of the genotypes based on their proteomic profiles. Genotype-specific abundances were recorded for proteases involved in the degradation of storage proteins and release of flavour precursors. Different genotype-specific levels of other enzymes, which generate volatiles compounds that could potentially lead to flavour-inducing compounds, were also detected. Overall, this study shows that UHPLC-MS/MS data can differentiate cocoa bean varieties.

Proteomic and peptidomic UHPLC-ESI MS/MS analysis of cocoa beans fermented using the Styrofoam-box method.

This work characterises the peptide and protein profiles of Theobroma cacao beans of the genotype IMC 67 at different fermentation stages, using the Styrofoam-box fermentation method and employing UHPLC-ESI MS/MS for the analysis of peptides and proteins extracted from the beans. A total of 1058 endogenous peptides were identified and quantified over four fermentation time points. The majority of these peptides were formed after 2 and 4 days of fermentation, and originated predominantly from the proteolysis of two storage proteins - vicilin and a 21 kDa albumin. The changes in the peptide profile over fermentation were subsequently evaluated, and potential markers for assessing the degree of fermentation were identified. In particular, changes of the relative abundance of the major cocoa proteins detected can be proposed as potential markers for the fermentation stage. Furthermore, PCA of both the peptidomic and proteomic data has allowed differentiation of beans at different fermentation stages.

A nodal signaling pathway regulates the laterality of neuroanatomical asymmetries in the zebrafish forebrain.

Animals show behavioral asymmetries that are mediated by differences between the left and right sides of the brain. We report that the laterality of asymmetric development of the diencephalic habenular nuclei and the photoreceptive pineal complex is regulated by the Nodal signaling pathway and by midline tissue. Analysis of zebrafish embryos with compromised Nodal signaling reveals an early role for this pathway in the repression of asymmetrically expressed genes in the diencephalon. Later signaling mediated by the EGF-CFC protein One-eyed pinhead and the forkhead transcription factor Schmalspur is required to overcome this repression. When expression of Nodal pathway genes is either absent or symmetrical, neuroanatomical asymmetries are still established but are randomized. This indicates that Nodal signaling is not required for asymmetric development per se but is essential to determine the laterality of the asymmetry.

Activation of kinin B receptor triggers differentiation of cultured human keratinocytes.

BACKGROUND: Keratinocyte life span is modulated by receptors that control proliferation and differentiation, key processes during cutaneous tissue repair. The kinin B(1) receptor (B(1)R) has been reported in normal and pathological human skin, but so far there is no information about its role in keratinocyte biology. OBJECTIVES: To determine the consequence of kinin B(1)R stimulation on tyrosine phosphorylation, a key signalling mechanism involved in keratinocyte proliferation and differentiation. METHODS: Subconfluent primary cultures of human keratinocytes were used to investigate tyrosine phosphorylation, epidermal growth factor receptor (EGFR) transactivation, cell proliferation and keratinocyte differentiation. Cell proliferation was assessed by measuring bromodeoxyuridine incorporation whereas assessment of cell differentiation was based on the expression of filaggrin, cytokeratin 10 (CK10) and involucrin. RESULTS: The major proteins phosphorylated, after B(1)R stimulation, were of molecular mass 170, 125, 89 and 70 kDa. The 170- and 125-kDa proteins were identified as EGFR and p125(FAK), respectively. Phosphorylation was greatly reduced by GF109203X and by overexposure of keratinocytes to phorbol 12-myristate 13-acetate, indicating the participation of protein kinase C. B(1)R stimulation did not increase [Ca(2+)]i, but triggered EGFR transactivation, an event that involved phosphorylation of Tyr(845), Tyr(992) and Tyr(1068) of EGFR. B(1)R stimulation did not elicit keratinocyte proliferation, but triggered cell differentiation, visualized as an increase of filaggrin, CK10 and involucrin. Blockade of EGFR tyrosine kinase by AG1478, before B(1)R stimulation, produced an additional increase in filaggrin expression. CONCLUSIONS: The kinin B(1)R may contribute to keratinocyte differentiation and migration by triggering specific tyrosine signalling pathways or by interacting with the ErbB receptor family.

Water-soluble precursors of beef flavour: I. Effect of diet and breed.

The effects of diet and breed on the concentration of water-soluble flavour precursors, namely sugars, free amino acids, ribonucleotides, creatinine, carnosine and creatine, were studied in beef longissimus lumborum muscle. Diet had a significant effect on the concentration of free amino acids, with animals fed on grass silage having higher free amino acid levels than animals fed on a concentrate diet, whereas animals fed concentrates had a higher total reducing sugar content. Differences between a beef breed (Aberdeen Angus×Holstein-Friesian) and a dairy breed (Holstein-Friesian) were generally small.

Water-soluble precursors of beef flavour. Part II: Effect of post-mortem conditioning.

Changes in glycolytic metabolites, nucleotide degradation products, free amino acids and other amino compounds were monitored in beef muscle (M. longissimus lumborum), stored for 21days at 4°C, in order to evaluate how post-mortem conditioning may affect flavour formation in beef. The major effects observed in sugar-related substances were the dephosphorylation of the phosphates of glucose, fructose and mannose, to yield their free sugars, as well as the breakdown of inosine 5'-monophosphate, to give a sixfold increase in ribose. Total reducing sugars increased by only 15% during conditioning, while glycogen levels remained unchanged from 2days post-slaughter. Free amino acids increased during conditioning, particularly between days 7 and 14. Phenylalanine, methionine, lysine, leucine and isoleucine were the amino acids showing the greatest increase with conditioning time, with methionine, in particular, showing a sevenfold increase during the conditioning period. The effects of these precursor changes on cooked beef flavour are discussed.

Vertebrate gastrulation: calcium waves orchestrate cell movements.

A recent study reveals that the propagation of intercellular calcium signals is closely associated with the generation of convergent extension movements during Xenopus gastrulation. Such signals provide a mechanism whereby large populations of cells can communicate to generate orchestrated cell movements.

Prion Function and Pathophysiology in Non-Mammalian Models.

More than thirty years have passed since the discovery of the prion protein (PrP) and its causative role in transmissible spongiform encephalopathy. Since a combination of both gain- and loss-of-function mechanisms may underlay prion pathogenesis, understanding the physiological role of PrP may give important clues about disease mechanisms. Historically, the primary strategy for prion research has involved the use of human tissue, cell cultures and mammalian animal models. Nevertheless, experimental difficulties of in vivo studies and controversial observations obtained in these systems have stimulated the search for alternative animal models. PrPC is highly conserved in mammals, and PrPC-related orthologs are expressed in zebrafish, a vertebrate model organism suitable to study the mechanisms associated with human diseases. Invertebrate models, as they do not express PrPC have served to investigate the neurotoxic mechanisms of mammalian PrP. Here we overview most recent advances in the study of PrP function in normal and pathogenic conditions based on non-mammalian studies, highlighting the contribution of zebrafish, fly and worms to our current understanding of PrP biology.

Diseño y fabricación de modelos impresos en 3D como complemento para las clases prácticas de histología médica / Design and manufacturing of 3D printed models as a complement for medical histology practical classes

RESUMEN: Tradicionalmente, la Histología se ha apoyado del análisis de preparaciones histológicas a través del microscopio para su enseñanza. En este sentido, uno de los principales obstáculos que enfrentan los estudiantes al analizar los tejidos, es extrapolar una imagen bidimensional a una estructura tridimensional (3D). La impresión 3D permite subsanar esta limitación, haciendo posible fabricar material docente, con las características requeridas con un alto grado de detalle y bajo costo. El objetivo de este trabajo fue diseñar y fabricar modelos impresos en 3D como complemento para las clases prácticas de Histología Médica. Se fabricaron modelos impresos en 3D de la ultraestructura de la barrera de filtración glomerular (BFG) en su estado normal y síndrome nefrótico. Además, se fabricó un modelo de la capa muscular del esófago humano dando énfasis a la disposición helicoidal de sus fibras musculares. Los modelos de epidermis permitieron identificar sus distintos estratos: estrato córneo, estrato granuloso, estrato espinoso, y estrato basal. Dentro los beneficios derivados de la impresión de modelos en 3D podemos destacar el bajo costo económico de su fabricación, alta reproducibilidad, bioseguridad, y potencial para favorecer el aprendizaje y la enseñanza de la Histología. No obstante, es necesario analizar la percepción y beneficio sobre el aprendizaje de los estudiantes derivados de la aplicación de los modelos mediante técnicas de evaluación cuantitativas y cualitativas.

SUMMARY: Traditionally, Histology has relied on the analysis of histological slides through the microscope for its teaching. In this sense, one of the main obstacles faced by students when analyzing tissues is to extrapolate a two-dimensional image to a three-dimensional (3D) structure. 3D printing makes it possible to overcome this limitation, making it possible to manufacture teaching material with the required characteristics with a high degree of detail and low cost. The objective of this work was to design and manufacture 3D printed models as a complement for the practical classes of Medical Histology. 3D printed models of the ultrastructure of the glomerular filtration barrier (GFB) in its normal state and nephrotic syndrome were fabricated. In addition, a model of the muscular layer of the human esophagus was fabricated emphasizing the helical arrangement of its muscle fibers. The epidermis models allowed the identification of its different layers: stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. Among the benefits derived from 3D printing of models, we can highlight the low economic cost of manufacturing, biosafety and potential to favor the learning and teaching of Histology. However, it is necessary to analyze the perception and benefit on student learning derived from the application of the models by means of quantitative and qualitative evaluation techniques.

Catalytic hydrodeoxygenation of methyl-substituted phenols: correlations of kinetic parameters with molecular properties.

The hydrodeoxygenation of methyl-substituted phenols was carried out in a flow microreactor at 300 degrees C and 2.85 MPa hydrogen pressure over a sulfided CoMo/Al(2)O(3) catalyst. The primary reaction products were methyl-substituted benzene, cyclohexene, cyclohexane, and H(2)O. Analysis of the results suggests that two independent reaction paths are operative, one leading to aromatics and the other to partially or completely hydrogenated cyclohexanes. The reaction data were analyzed using Langmuir-Hinshelwood kinetics to extract the values of the reactant-to-catalyst adsorption constant and of the rate constants characterizing the two reaction paths. The adsorption constant was found to be the same for both reactions, suggesting that a single catalytic site center is operative in both reactions. Ab initio electronic structure calculations were used to evaluate the electrostatic potentials and valence orbital ionization potentials for all of the substituted phenol reactants. Correlations were observed between (a) the adsorption constant and the two reaction rate constants measured for various methyl-substitutions and (b) certain moments of the electrostatic potentials and certain orbitals' ionization potentials of the isolated phenol molecules. On the basis of these correlations to intrinsic reactant-molecule properties, a reaction mechanism is proposed for each pathway, and it is suggested that the dependencies of adsorption and reaction rates upon methyl-group substitution are a result of the substituents' effects on the electrostatic potential and orbitals rather than geometric (steric) effects.

Novel biomechanics demonstrate gait dysfunction due to hamstring tightness.

BACKGROUND: Children with cerebral palsy and hamstring tightness often demonstrate limited terminal swing knee extension. The conventional clinical measure of popliteal angle describes static hamstring tightness, but is not consistent with dynamic limitation. We hypothesize hamstring tightness, determined via modification of the conventional popliteal angle measure, is directly related to decreased terminal swing knee extension in children with cerebral palsy and normal magnitude knee flexion moments. METHODS: Six patients with cerebral palsy and six normal subjects were evaluated via physical examination and instrumented gait analysis. Physical examination included popliteal angle measures at first hamstring resistance to passive extension (R(1)), and end-range extension (R(2)) with the hip in varying degrees of flexion. Passive R(1) data were used to calculate regression equations to predict R(1) during gait, resulting in a novel measure of Available Knee Extension. Hamstring EMG was also compared. FINDINGS: R(1) during physical examination was significantly correlated with Available Knee Extension at terminal swing (Pearson r = -0.7251, P < 0.0001). Patients walked with significantly decreased velocity (0.959 vs. 1.27 m/s, P = 0.0002) and decreased knee extension at terminal swing (25.6 vs. 2.05 degrees, P < 0.0001), in the presence of normal knee flexion moments (-0.289 vs. -0.306 Nm/kg, P = 0.5009), and significantly decreased power absorption (-0.821 vs. -1.43 W/kg, P < 0.0001). Eleven of 12 patient knees demonstrated negative Available Knee Extension at terminal swing, with markedly limited knee extension. Five of 12 normal knees demonstrated negative Available Knee Extension, but this was near full extension. Hamstring EMG onset times were not significantly different. INTERPRETATION: We believe Available Knee Extension, defined on the basis of clinical measures of first resistance to hamstring stretch, provides a biomechanical link between physical examination findings and dynamic limitations in terminal swing knee extension.

Performance of an inverted pendulum model directly applied to normal human gait.

BACKGROUND: In clinical gait analysis, we strive to understand contributions to body support and propulsion as this forms a basis for treatment selection, yet the relative importance of gravitational forces and joint powers can be controversial even for normal gait. We hypothesized that an inverted pendulum model, propelled only by gravity, would be inadequate to predict velocities and ground reaction forces during gait. METHODS: Unlike previous ballistic and passive dynamic walking studies, we directly compared model predictions to gait data for 24 normal children. We defined an inverted pendulum from the average center-of-pressure to the instantaneous center-of-mass, and derived equations of motion during single support that allowed a telescoping action. Forward and inverse dynamics predicted pendulum velocities and ground reaction forces, and these were statistically and graphically compared to actual gait data for identical strides. FINDINGS: Results of forward dynamics replicated those in the literature, with reasonable predictions for velocities and anterior ground reaction forces, but poor predictions for vertical ground reaction forces. Deviations from actual values were explained by joint powers calculated for these subjects. With a telescoping action during inverse dynamics, predicted vertical forces improved dramatically and gained a dual-peak pattern previously missing in the literature, yet expected for normal gait. These improvements vanished when telescoping terms were set to zero. INTERPRETATION: Because this telescoping action is difficult to explain without muscle activity, we believe these results support the need for both gravitational forces and joint powers in normal gait. Our approach also begins to quantify the relative contributions of each.

Expression of nk2.1a during early development of the thyroid gland in zebrafish.

We show here that a zebrafish orthologue of the Thyroid Transcription Factor-1 (TTF-1), nk2.1a, is expressed in the developing thyroid gland. Using a fate mapping approach we found that an early nk2.1a expression domain in the endoderm adjacent to the heart follows morphogenetic movements of the lower jaw, ending up in the region in which the mature thyroid gland is located. We therefore suggest that nk2.1a labels the thyroid precursor cells from somitogenesis stages onwards.