Actividades profesionales confiables en cirugía mínimamente invasiva: iniciando el camino para lograr competencias en el futuro graduado de medicina / Entrustable professional activities in minimally invasive surgery: starting the way to achieve competencies in the future medical graduate

Objetivos: El desarrollo de actividades profesionales confiables (APROC) para el graduado de medicina en Cirugía Mínimamente Invasiva (CMI) es una necesidad en Colombia. La evidencia disponible es limitada. Este estudio describe la experiencia preliminar con una intervención orientada a esta necesidad, en el marco de un modelo de educación basada en competencias (EBC). Materiales y Métodos: Se diseñó una intervención orientada al desarrollo de actitudes, conocimientos y habilidades prácticas en CMI para estudiantes de medicina, mediante un enfoque de aula invertida extendida. Se realizaron evaluaciones pre y posintervención mediante el cuestionario Team-STEPPS (actitudes), exámenes de conocimiento y OSATS (habilidades prácticas). Se realizaron comparaciones pre y posintervención (t-test (p < 0,05) y mediciones del tamaño del efecto de la intervención (prueba d Cohen). Finalmente se evaluó la satisfacción estudiantil. Resultados: Un total de 99 estudiantes participaron en el estudio. Se encontraron diferencias estadísticamente significativas (p < 0,05) entre las mediciones pre y posintervención, y gran efecto en las actitudes, conocimientos y habilidades prácticas (d > 0,80). Se evidenció alta satisfacción estudiantil. Discusión: El diseño instruccional a través de metodologías interactivas permite desarrollar APROC en CMI, desde el pregrado. Estos resultados son similares a los reportados en otras intervenciones en el marco de la EBC. Conclusión: Nuestra intervención demostró efectos positivos sobre competencias estudiantiles orientadas al desarrollo de APROC en CMI para el futuro graduado. Aun es necesario medir estas competencias en la práctica real y al finalizar la carrera, para determinar si estas actividades pueden ser totalmente confiables a los participantes en su futura práctica profesional.

Aims: The development of entrustable professional activities (EPAs) in minimally invasive surgery (MIS) for undergraduates is a need in Colombia. The available evidence is limited. This study aims to describe the preliminary experience with an intervention oriented to this need, embedded in the framework of a competence-based education model (CBE). Materials and Methods: An intervention was designed for the development of EPAs in MIS oriented to the development of attitudes, knowledge and practical skills in medical students. Intervention was delivered through an extended inverted classroom approach. Pre- and postintervention measures were performed by using the Team-STEPPS questionnaire (attitudes), knowledge assessments and OSATS (practical skills). Comparisons were performed by t-test tests (p < 0.05) and the effect size of the intervention was calculated by the Cohen d test. Finally, the student's satisfaction was evaluated. Results: A total of 99 students participated in the study. The intervention showed statistically significant differences (p < 0.05), and great effect on attitudes, knowledge and practical skills (d > 0.80). Likewise, high student satisfaction was evidenced. Discussion: Interactive instructional design fosters development of EPAs in MIS for medical undergraduates. These results are similar to those reported in other interventions under the CBE model. Conclusion: Our intervention showed positive effects on competences oriented to the development of EPAs in MIS for the future graduate. Still is necessary to assess these competencies in real practice and at the end of medical career, in order to evaluate if these activities can be totally reliable to the participants in their future professional practice.

Volatile, isotope, and organic analysis of martian fines with the Mars Curiosity rover.

Samples from the Rocknest aeolian deposit were heated to ~835°C under helium flow and evolved gases analyzed by Curiosity's Sample Analysis at Mars instrument suite. H2O, SO2, CO2, and O2 were the major gases released. Water abundance (1.5 to 3 weight percent) and release temperature suggest that H2O is bound within an amorphous component of the sample. Decomposition of fine-grained Fe or Mg carbonate is the likely source of much of the evolved CO2. Evolved O2 is coincident with the release of Cl, suggesting that oxygen is produced from thermal decomposition of an oxychloride compound. Elevated δD values are consistent with recent atmospheric exchange. Carbon isotopes indicate multiple carbon sources in the fines. Several simple organic compounds were detected, but they are not definitively martian in origin.

Alternative identification test relying upon sexual reproductive abilities of Candida lusitaniae strains isolated from hospitalized patients.

The in vitro mating ability of Candida lusitaniae (teleomorph Clavispora lusitaniae) clinical isolates has been investigated. Studying the effects of culture conditions, we showed that ammonium ion depletion in the medium is a major trigger of the sexual cycle. Moreover, a solid support is required for mating, suggesting a role for adhesion factors in addition to the mating type gene recognition function. Monitoring of mating and meiosis efficiency with auxotrophic strains showed great variations in ascospore yields, which appeared to be strain and temperature dependent, with an optimal range of 18 to 28 degrees C. The morphogenetic events taking place from mating to ascospore release were studied by scanning and electron microscopy, and the ultrastructure of the conjugation canal, through which intercellular nuclear exchanges occur, was revealed. Labeling experiments with a lectin-fluorochrome system revealed that the nuclear transfer was predominantly polarized, thus allowing a distinction between the nucleus donor and the nucleus acceptor strains. The direction of the transfer depended on the strain combination used, rather than on the genotypes of the strains, and did not appear to be controlled by the mating type genes. Finally, we demonstrated that all of the 76 clinical isolates used in this study were able to reproduce sexually when mated with an opposite mating type strain, and we identified a 1:1 MATa/MATalpha ratio in the collection. These results support the idea that there is no anamorph state in C. lusitaniae. Accordingly, the mating type test, which is easy to use and can usually be completed within 48 h, is a reliable alternative identification system for C. lusitaniae.

PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea.

We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves.

Fungal auxin overproduction affects the anatomy of Hebeloma cylindrosporum-Pinus pinaster ectomycorrhizas.

We studied the effect of fungal auxin overproduction on the growth polarity of cortical cells in pine mycorrhizas by comparing the anatomy of Pinus pinaster (Ait.) Sol. mycorrhizas formed by an IAA-overproducing mutant of Hebeloma cylindrosporum Romagnesi or by the corresponding wild type with non-mycorrhizal short roots. Both wild- type and mutant strains induced an increase in root diameter that was mostly a result of the influence of the fungus on root cortical development. Both strains affected growth polarity of P. pinaster cortical cells and induced a change in their shape. The main modifications were a large reduction in axial diameter and an increase in the radial diameter of the cortical cells. The modifications were more marked with the mutant than with the wild type. The mutant induced a 43% reduction in cortical cell elongation and a 35% increase in radial diameter, whereas the corresponding changes induced by the wild type were 30 and 10%, respectively. The volume of cortical cells in mature mycorrhizas was generally lower than in uninoculated short roots indicating that wild-type and mutant strains induced a reorientation of cortical cell growth but did not induce an increase in turgor pressure of the cells. Immunolocalization allowed visualization of alpha-tubulin in root cortical cells, but no obvious modification in alpha-tubulin distribution was detected as a consequence of symbiosis establishment. Likewise, cytochemical localization of polysaccharides in cortical cell walls did not show significant modification following symbiosis establishment and Hartig net formation. The only noticeable modification was a reduction in cortical cell wall thickness in mycorrhizas compared with uninoculated short roots. The possible involvement of fungal auxin in the observed modifications is discussed.

Plant genome complexity may be a factor limiting in situ the transfer of transgenic plant genes to the phytopathogen Ralstonia solanacearum.

The development of natural competence by bacteria in situ is considered one of the main factors limiting transformation-mediated gene exchanges in the environment. Ralstonia solanacearum is a plant pathogen that is also a naturally transformable bacterium that can develop the competence state during infection of its host. We have attempted to determine whether this bacterium could become the recipient of plant genes. We initially demonstrated that plant DNA was released close to the infecting bacteria. We constructed and tested various combinations of transgenic plants and recipient bacteria to show that the effectiveness of such transfers was directly related to the ratio of the complexity of the plant genome to the number of copies of the transgene.

Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco.

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

Purification and properties of the chloroplastic form of biotin holocarboxylase synthetase from Arabidopsis thaliana overexpressed in Escherichia coli.

Holocarboxylase synthetases (HCSs) are key enzymes in biotin utilisation in both prokaryotes and eukaryotes. In a previous study, we demonstrated that, in plants, HCS activity is localised in cytosol, chloroplasts and mitochondria. We also described the cloning and sequencing of a full-length cDNA encoding an Arabidopsis thaliana HCS isoform with a putative organelle-transit peptide. In the study reported here, this cDNA was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated using an efficient three-step purification procedure. Polyclonal antibodies raised against pure HCS were produced to elucidate the subcellular localisation of this protein. Immunodetection carried out by Western blotting of isolated pea leaf subcellular compartments specifically revealed a single polypeptide that was ascribed to the chloroplast compartment. Immunocytochemistry of thin-cut sections from tobacco leaves, transformed by the complete coding sequence of A. thaliana HCS cDNA via Agrobacterium tumefaciens, confirmed that the enzyme encoded by this cDNA is the chloroplastic isoform. Moreover, physicochemical, biochemical and kinetic properties of the pure recombinant HCS were determined. The native recombinant enzyme is a 37-kDa monomer. In contrast to the major part of HCS activity measured in leaf extracts, the recombinant chloroplastic enzyme did not require addition of Mg2+ to be fully active, but was substantially inhibited by EDTA. This suggested that the chloroplastic HCS may contain a tightly-bound divalent cation required for enzyme activity. The recombinant enzyme was able to biotinylate efficiently apo-biotin carboxyl carrier protein (BCCP) from E. coli and apo-methylcrotonoyl-CoA carboxylase (MCCase) from A. thaliana. Apparent Km values for the enzyme substrates D-biotin, ATP and apo-MCCase were found to be 130 nM, 4.4 microM and 32 microM, respectively. Steady-state kinetic analyses of the HCS-catalysed reaction were investigated with respect to reaction mechanism and inhibition by AMP, one of the end-products of the enzyme-catalysed reaction. Substrate interaction and product inhibition patterns indicated that ATP and D-biotin bind sequentially, in an ordered manner, to the enzyme and that ATP or D-biotin and apo-BCCP bind in ping-pong fashion.

[Stress, seen from outside]. / Le stress, vu de l'extérieur.

This article draws from the vast literature on stress in nursing and discusses the preoccupation that researchers and specialists have with this area. It asks the reader to take a moment to reflect on the types of stressful situations that nurses face on a daily basis. The authors focus on the potential tensions that can ensue following exposure to stress and the resulting psychosomatic, psychological, behavioral and organizational consequences. The authors challenge legislators, governments, administrators, members of the multidisciplinary team and nursing organizations to tackle the issue of stress in nursing and make it a priority. Research conducted during the last 20 years provides overwhelming evidence that nurses work in a high-stress environment. The sources of stress vary and their consequences are significant. However, it is recognized that certain personal factors such as type "A" personality can influence the way nurses deal with stress. Ultimately, the authors conducted their literature review with the aim of proposing anti-stress strategies.

Detection of picogram amounts of nucleic acid by dot blot hybridization.

An increasing number of human proteins isolated from cell sources are being produced for pharmaceutical use. Consequently, federal agencies have required the quantitative determination of residual nucleic acids that copurify with the potential protein products. We have conducted these assays in connection with our application for licensure of Alferon Injection. We report a sensitive dot blot hybridization assay that was used to quantitate picogram (or less) amounts of nucleic acids which copurified with human proteins isolated from recombinant (S. cerevisiae) or natural (leukocytes) sources.

[Lea's secret. Confidentiality at any cost?]. / Le secret de Léa. "La confidentialité à tout prix?"

PIP: The case of 17-year old student seeking abortion information in a school clinic illustrates the ethical problems of nurses who must balance the patient's right to confidentiality with the conflicting claims of other interested parties, in this case the student's mother. The student desired to end the pregnancy because she wanted to pursue her studies and lacked the financial means to continue the pregnancy and assume responsibility for the child. She did not wish to confide in her mother, who had recently concluded a painful divorce. The student's mother subsequently approached the clinic nurse to inquire whether her daughter had sought services. Quebec's Code of Ethics for nurses states that the nursing professional should respect the confidentiality of all information obtained in the course of exercising the profession, and that confidential information should only be divulged with the authorization of the client or if the law so dictates. Quebec's law for protection of public health stipulates that medical majority is attained at age 14. The Code of Ethics for Nurses also states that nursing professionals should not reveal whether an individual has sought their professional services if the fact of having done so could cause a prejudice to the individual. In view of these professional obligations, the nurse concluded that her primary duty was to maintain the confidentiality of the young student. The profession of nursing depends to a large extent on human relations. Such tools as the Code of Ethics provide principles to orient the behavior of nurses.^ieng

Production of isolated somatic embryos from sunflower thin cell layers.

We describe here a two step procedure which allows the easy isolation of somatic embryos from Sunflower (Helianthus annuus L.) hypocotyl tissues. Thin cell layers composed of the epidermis plus 3 to 6 parenchyma cell layers were incubated for 5 days in a basal Murashige and Skoog medium using an auxin to cytokinin weight ratio of 1/1. The epidermis layers were then transferred to a Gamborg medium containing a high level of sucrose. After one week of incubation in this medium, many somatic embryos started to be released from the parental epidermal tissue. Even though the germination of these embryos is difficult, we have been able to induce secondary embryos and regenerate fertile plants.

Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.

Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency.

Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component human alpha-ketoacid dehydrogenase complexes.

cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (greater than 98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.

Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex.

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.

Cloning of rat brain succinyl-CoA:3-oxoacid CoA-transferase cDNA. Regulation of the mRNA in different rat tissues and during brain development.

3-Oxoacid CoA-transferase, which catalyses the first committed step in the oxidation of ketone bodies, is uniquely regulated in developing rat brain. Changes in 3-oxoacid CoA-transferase activity in rat brain during the postnatal period are due to changes in the relative rate of synthesis of the enzyme. To study the regulation of this enzyme, we identified, with a specific polyclonal rabbit anti-(rat 3-oxoacid CoA-transferase), two positive cDNA clones (approx. 800 bp) in a lambda gtll expression library, constructed from poly(A)+ RNA from brains of 12-day-old rats. One of these clones (lambda CoA3) was subcloned into M13mp18 and subjected to further characterization. Labelled single-stranded probes prepared by primer extension of the M13mp18 recombinant hybridized to a 3.6 kb mRNA. Rat brain mRNA enriched by polysome immunoadsorption for a single protein of size 60 kDa which corresponds to the precursor form of 3-oxoacid CoA-transferase was also found to be similarly enriched for the hybridizable 3.6 kb mRNA complementary to lambda CoA3. Affinity-selected antibody to the lambda CoA3 fusion protein inhibited 3-oxoacid CoA-transferase activity present in rat brain mitochondrial extracts. The 3.6 kb mRNA for 3-oxoacid CoA-transferase was present in relative abundance in rat kidney and heart, to a lesser extent in suckling brain and mammary gland and negligible in the liver. The specific mRNA was also found to be 3-fold more abundant in the brain from 12-day-old rats as compared with 18-day-old foetuses and adult rats, corresponding to the enzyme activity and relative rate of synthesis profile during development. These data suggest that 3-oxoacid CoA-transferase enzyme activity is regulated at a pretranslational level.

Isolation of a cDNA clone for the dihydrolipoamide acetyltransferase component of the human liver pyruvate dehydrogenase complex.

Dihydrolipoamide acetyltransferase (E2) forms the structural core of pyruvate dehydrogenase complex. A cDNA clone (lambda E2-1) for mammalian E2 was identified from a human liver lambda gt11 library using anti-E2 serum. Affinity-selected antibodies using the fusion protein from lambda E2-1 immuno-reacted specifically with E2 of purified pyruvate dehydrogenase complex on immuno-blot analysis. The cDNA insert was approximately 2.3 kb in length with an internal EcoR1 site generating 1.4 and 0.9 kb fragments. A synthetic 17-mer oligodeoxynucleotide mixture based on the amino acid sequence surrounding the lipoic acid-containing lysine residue in bovine kidney E2 hybridized with the 2.3 kb cDNA insert and the 1.4 kb fragment.