Self-perceived competencies on evidence-based medicine in medical students and physicians registered in a virtual course: a cross-sectional study.

BACKGROUND: Evidence-based medicine (EBM) is defined as the integration of the best available evidence from scientific studies with clinical experience (and context) and with patients' values and preferences. The objective of the present study was to describe self-perceived EBM competencies in physicians and medical students enrolled in a massive virtual EBM course. METHODS: Analytical cross-sectional study. People interested in a free virtual EBM course fulfilled their data in a virtual form for their registration in September 2020. In this form, 22 competencies related to four dimensions of EBM were evaluated: asking a clinical question, search, analysis, and application; using a 5-option Likert scale. The resulting database was analyzed, selecting people who claimed to be physicians or medical students of 18 years or more. RESULTS: 1793 participants were included: 1130 medical students and 663 physicians; more than 80% lived in Peru. The frequency of participants who agreed or strongly agreed with feeling qualified in each competence ranged: from 39.2% to 57.8% for the competencies of the 'Asking a clinical question' dimension, from 39.2% to 56.1% for 'Search,' from 19.9% to 32.0% for 'Analysis,' and from 19.6% to 29.9% for 'Application.' Both in physicians and students, the lowest frequencies were for the competencies of interpretation of impact measures, graphs, and results of systematic reviews; as well as shared decision making and calculation of expected benefit. Physicians who graduated more recently scored better on competencies from search and analysis dimensions. CONCLUSION: Among physicians and medical students enrolled in the course, self-perception of competencies was lower in the dimensions of analysis and application. More recently graduated physicians seem to have a greater self-perception of their research and analysis skills, probably due to curricular updates.List of abbreviations: EBM: Evidence-based medicine; CIMBE, for its acronym in Spanish: International Course on Evidence-Based Medicine; SOCIMEP, for its acronym in Spanish: Peruvian Medical Student Scientific Society.

Problematic Internet Use Associated with Symptomatic Dry Eye Disease in Medical Students from Peru.

PURPOSE: Dry eye is a multifactorial ocular surface disease (DED) characterized by a loss of tear film homeostasis, which is widely associated with alterations in mental health. Problematic internet use (PIU) is defined as the feeling of concern about using this tool irresistibly, for longer periods than usual, accompanied by anguish that results from not doing so without reaching mania or hypomania behaviors. Both PIU and DED present a theoretical link; however, there are no published studies that report its relationship with problematic internet use. Therefore, this study aims to determine the association between PIU and symptomatic DED in Peruvian medical students. PATIENTS AND METHODS: Analytical cross-sectional study that included human medical students from Peru. For the measurement of main variables, the Ocular Surface Disease Index (OSDI) and the Internet Addiction Test (IAT) were used. To study their relationship, the Poisson regression analysis was used, we consider a p-value <0.05 as significant. RESULTS: Data from 844 medical students were analyzed, 35.7% male and 64.3% female, with an average age of 21.8 ± 3.3 years. Likewise, the prevalence of symptomatic DED was 70.9%, and the internet's controlled use was 85.3%. In the analysis adjusted for symptomatic DED, the men showed significant differences in the controlled use of the internet (p <0.003), of which those who had PIU, 50% had severe symptomatic DED, as well as 80% of those addicted to the internet. The adjusted model showed that the PIU increases the prevalence of symptomatic DED in men (PR = 1.17, 95% CI: 1.06-1.29); however, in women, no association was found between both variables. CONCLUSION: PIU and symptomatic DED showed a significant association in male medical students from Peru.

Complications of percutaneous nephrolithotomy in the prone position according with modified Clavien-Dindo grading system.

INTRODUCTION: Clavien-Dindo classification system has been proposed to grade perioperative complications in percutaneous nephrolithotomy. The complications of this technique that have taken place in the last 2 years are reported in this paper according Clavien-Dindo classification. MATERIALS AND METHODS: Between 2011 and 2012 a total of 255 percutaneous nephrolithotomy were performed at our center for stones more than 2 cm in size. In order to determine the incidence of complications classified in the modified Clavien-Dindo system, statistical analysis of the data obtained was carried out. RESULTS: During the period analyzed, 255 percutaneous nephrolithotomy were performed in 249 patients, 41% of the right side, 57% of the left side and 2% bilateral and simultaneous. 137 and 112 patients were males and females, respectively. The most prevalent comorbidities were: hypertension (AHT) in 101 patients (40.6%), BMI>30 in 81 patients (32%), diabetes mellitus in 46 patients (18.5%) and coagulation abnormalities in 24 patients (9.6%). A total of 70 cases (27.4%) were distributed according to Clavien-Dindo classification: grade i, 8.4%, grade ii 8.4%, grade iiia 4.4%, grade iiib 6% grade iva .8%, grade ivb: 0% and grade v 0%. CONCLUSIONS: A graded classification scheme for reporting the complications of percutaneous nephrolithotomy is useful for monitoring and reporting outcomes. We propose a standardized use of this classification in order to make the results comparable among different centers performing the technique.

Expression of cytokines and their regulation during amoebic liver abscess development.

Amoebic liver abscess (ALA) is the most important extraintestinal complication of Entamoeba histolytica infection. Amoebic liver abscess development causes severe destruction of the liver tissue concomitant with a strong inflammatory reaction. We analyse the in situ expression of TNF-α, IFN-γ, IL-1ß, 1L-8 and IL-10 at different stages of ALA development in a susceptible animal model. Results showed that after inoculation, neutrophils (PMN) and some macrophages infiltrated the liver and were positive for TNF-α and IFN-γ at the acute phase of amoeba infection. The presence of these cytokines was transient and decreased as tissue damage progressed. In contrast, IL-1ß and IL-8 were detected mainly in neutrophils and macrophages from the periods of acute infection to subacute and chronic infection and decreased when granulomas were formed. The IL-10 was expressed in PMN and mononuclear cells and only during a short period at the onset of acute infection. The qRT-PCR of mRNA revealed a relationship with the expression of the cytokines in cells found in the ALA. Furthermore, our data suggest that IL-10 does not regulate local production of these cytokines. Our results indicate that an exacerbated inflammatory milieu is established and contributes to liver tissue damage and probably supports the survival of the parasites.

The neuronal cytoskeleton as a potential therapeutical target in neurodegenerative diseases and schizophrenia.

The cytoskeleton plays a key role in maintaining the highly asymmetrical shape and structural polarity of neurons that are essential for neuronal physiology. Cytoskeletal reorganization plays a key role in neuritogenesis. In neurodegenerative diseases, the cytoskeleton is abnormally assembled and impairment of neurotransmission occurs. In Alzheimer's disease, abundant amyloid plaques and neurofibrillary tangles constitute the two major neuropathologic alterations present in the brain. Neurofibrillary tangles are formed of paired helical filaments consisting nearly entirely of the microtubule-associated protein tau. Under normal conditions tau binds to microtubules, stabilizing neuron structure and integrity. Hyperphosphorylation of tau is assumed to be the cause of formation of paired helical filaments. Another example of cytoskeletal abnormalities present in neurodegenerative diseases are the Lewy bodies considered as cytopathologic markers of Parkinson's disease. Lewy bodies are constituted of tubulin, MAP1, and MAP2. Neuronal shape, loss of dendrites and spines, as well as irregular distribution of neuronal elongations occur in specific brain areas of schizophrenic patients. Increase in non-phosphorylated MAP2 and MAP1B at hippocampus has been suggested as responsible for somatodendritic and cytoarchitectural abnormalities found in schizophrenia. In addition, neurofibrillary tangles are more frequent among schizophrenic patients who received pharmacologic antipsychotic treatment. Cumulative evidence suggests that neurodegenerative diseases and psychiatric illnesses are associated with cytoskeletal alterations in neurons that, in turn, loose synaptic connectivity and the ability to transmit incoming axonal information to the somatodendritic domain. We will review evidence supporting that the neuronal cytoskeleton is disrupted in neurodegenerative and some psychiatric diseases, and therefore could be a target for drug therapy. In addition, current data indicating that melatonin, a hormone secreted by the pineal gland, promotes neuritogenesis through cytoskeletal rearrangements and in addition to the potential therapeutic use of melatonin in neurodegenerative diseases will be discussed.

Over-expression of betaI tubulin in MDCK cells and incorporation of exogenous betaI tubulin into microtubules interferes with adhesion and spreading.

Little is known about the presence and distribution of tubulin isotypes in MDCK cells although essential epithelial functions in these monolayers are regulated by dynamic changes in the microtubule architecture. Using specific antibodies, we show here that the betaI, betaII, and betaIV isotypes are differentially distributed in the microtubules of these cells. Microtubules in subconfluent cells radiating from the perinuclear region contain betaI and betaII tubulins, while those extending to the cell edges are enriched in betaII. Confluent cells contain similar proportions of betaI and betaII along the entire microtubule length. betaIV is the less abundant isotype and shows a similar distribution to betaII. The effect of modifying tubulin isotype ratios in the microtubules that could affect their dynamics and function was analyzed by stably expressing in MDCK cells betaI tubulin from CHO cells. Three recombinant clones expressing different levels of the exogenous betaI tubulin were selected and subcloned. Clone 17-2 showed the highest expression of CHO beta1 tubulin. Total betaI tubulin levels (MDCK+CHO) in the clones were approximately 1.8 to 1.1-fold higher than in mock-transfected cells only expressing MDCK beta1 tubulin. In all the cells, betaII tubulin levels remained unchanged. The cells expressing CHO beta1 tubulin showed defective attachment, spreading, and delayed formation of adhesion sites at short times after plating, whereas mock-transfected cells attached and spread normally. Analysis of cytoskeletal fractions from clone 17-2 showed a MDCK betaI/CHO betaI ratio of 1.89 at 2 h that gradually decreased to 1.0 by 24 h. The ratio of the two isotypes in the soluble fraction remained unchanged, although with higher values than those found for the polymerized betaI tubulin. By 24 h, the transfected cells had regained normal spreading and formed a confluent monolayer. Our results show that excess levels of total betaI tubulin, resulting from the expression of the exogenous beta1 isotype, and incorporation of it into microtubules affect their stability and some cellular functions. As the levels return to normal, the cells recover their normal phenotype. Regulation of betaI tubulin levels implies the release of the MDCK betaI isotype from the microtubules into the soluble fraction where it would be degraded.

Extracellular matrix-induced signaling in Entamoeba histolytica: its role in invasiveness.

Entamoeba histolytica trophozoites can invade the mucosa of the host large intestine to cause disease. Here, Isaura Meza describes recent work indicating that the trophozoites respond to the binding of extracellular matrix proteins by activating receptor-mediated signal transduction pathways. Activation of phosphokinase C or adenylyl cyclase induces protein phosphorylation, actin gene expression and reorganization of the actin cytoskeleton to form adhesive structures that promote interaction with the substrate and the release of proteases. The release of proteases at the sites of contact, with the subsequent degradation of the substrate and generation of chemotactic peptides, facilitates locomotion and dissemination of the invading trophozoites.

Actin induction during PMA and cAMP-dependent signal pathway activation in Entamoeba histolytica trophozoites.

Activation of PKC or cAMP-dependent signalling pathways in Entamoeba histolytica triggers the phosphorylation of proteins involved in actin rearrangements necessary for adhesion and locomotion. Analogous motifs to SRE and CRE sequences--known to respond to PMA and cAMP--were identified within the 5' regulatory region (5'RR) of one of the parasite actin genes. These sequences could be involved in the actin transcriptional upregulation reported during signalling. To test this hypothesis, a plasmid containing the 5'RR of the actin gene fused to the bacterial neomycin gene (neo) was used for stable transfection. Expression of neo and endogenous actin was measured after stimulation of transfected amoebae by PMA and dcAMP. It was found that both compounds induced neo and actin expression and showed a co-operative effect in the induction of neo. Induction by PMA or dcAMP failed if the directing amoebic 5'RR lacked SRE and CRE motifs. Transfection of amoebae with plasmid constructs, containing either progressive deletions of the actin 5'RR or site-directed mutations of the SRE and CRE-like motifs, corroborated that these sequences and a co-ordinated participation of PKC- and PKA-activated transcription factors are responsible for the increments in neo and actin mRNAs. In vivo, these PMA and cAMP-response elements could play an important role in regulating actin expression and organization in signalling processes activated during tissue invasion.

Specific and reversible inhibition of Entamoeba histolytica cysteine-proteinase activities by Zn2+: implications for adhesion and cell damage.

BACKGROUND: Cysteine-proteinases are thought to play an important role in E. histolytica pathogenicity. Although effective blockage of proteolytic activities can be obtained with several known inhibitors, the high cellular toxicity of most of the inhibitors precludes experimentation with live cells or animal models. Specific cysteine-proteinase inhibitors that could be utilized in studies of virulence are of great need in the field of amebiasis. METHODS: Cysteine-proteinase activities were determined in trophozoite lysates by azocasein degradation and after PAGE and gelatin zymograms. Inhibition of the activities was assessed in the presence of 0.01-2.5 mM concentrations of divalent cations of the IIB and VIII series such as Zn, Cd, Hg, Ni, and Co. Reversibility was induced with 25 mM L-cysteine or 50 mM L-histidine and by metal chelation with 5 mM phenantroline. The inhibitory effect of ZnCl2 was tested with live cells in fibronectin-binding and cytotoxicity assays. RESULTS: ZnCl2 specifically inhibited cysteine-proteinase activities in trophozoite lysates in a concentration-dependent manner. Additionally, 1.0-2.5 mM ZnCl2 blocked proteolysis in more than 70%. This inhibition was completely reverted by L-cysteine, L-histidine, or phenantroline. Similar results were obtained by analyzing individual cysteine-proteinase activities separated in gelatin zymograms. At these concentrations, ZnCl2 significantly interfered with trophozoite adhesion, thus making amebas deficient in substrate degradation and cell damage. CONCLUSIONS: ZnCl2 is an effective inhibitor of amebic cysteine-proteinases. Its low toxicity at relatively high concentrations, high solubility, and low cost make it adequate for live cell experimentation and animal models of amebic virulence.

Entamoeba histolytica: identification of functional Gs and Gi proteins as possible signal transduction elements in the interaction of trophozoites with fibronectin.

Trophozoites of Entamoeba histolytica adhere to several components of the extracellular matrix. Binding is mediated by specific receptors identified in the parasite surface. Interaction of trophozoites with FN induces the formation of special adhesion structures that are dynamic cytoskeleton membrane complexes and facilitate both adhesion and substrate degradation. The process requires activation of signaling pathways in which PLC, IP3, Ca2-, and PKC participate. These observations, and recent experiments showing increments in cAMP in the trophozoites during the interaction with FN, suggest that FN receptors in the amebic surface could be coupled to G-proteins. We report here that trophozoite plasma membrane peptides of 92, 49, 42, 37, and 21 kDa are ADP-ribosylated by Vibrio cholerae and Bordetella pertussis toxins. Three of them are also recognized by antibodies prepared against the alpha-subunit of Gs-and Gi-proteins. Adenylyl cyclase activity detected in isolated membranes was strongly stimulated by treatment with the toxins. Forskolin (an agonist of the enzyme) and FN also induced increments in the enzymatic activity. Live amebas incubated with the toxins showed enhanced adhesion to FN substrates and a striking reorganization of polymerized actin. The actin rearrangement is reminiscent of the one induced by either forskolin or dibutyril cyclic AMP treatment. Our present data show the presence and the functionality of Gs- and Gi-like proteins and their apparent activation during in vitro interaction of amebas with FN and complement previous observations indicating the operation of signal transduction mechanisms in E. histolytica.

Myosin II-actin interaction in MDCK cells: role in cell shape changes in response to Ca2+ variations.

Cultured MDCK cell monolayers respond to a low level of extracellular calcium ([Ca2+]e < or = 5 microM) with a loss of transepithelial electrical resistance and transport function, and changes in position of a circumferential ring of actin filaments tethered to the plasma membrane at the zonula adhaerens. Keeping this cytoskeletal structure in place seems necessary to preserve the architecture of the tight junctions and therefore their sealing capacity. All three effects are reversible upon restituting normal [Ca2+]e. Recent work provided evidence of actin-myosin interactions at the filament ring, thus suggesting a contraction process involved in the alteration of the actin cytoskeleton. We now report that active contraction does occur and causes an extensive morphological transformation of MDCK cells. A marked increase in cell height simultaneous with a decrease in width and area of contact to the substratum was seen within 10 min of removal of [Ca2+]e; recovery began immediately after replacing calcium, although it took longer for completion. Conventional and confocal epifluorescence studies showed actin colocalized with myosin II at various planes of resting or contracted cells, in particular at the ring level. Electron-micrographs revealed the circumferential actin ring associated with the plasma membrane in a waist-like constriction where Ca2+ was removed from the cultures. Contraction, as well as relaxation, in response to [Ca2+]e variations were inhibited by cytochalasin-D (an actin-filament disrupting drug), by okadaic acid( an inhibitor of myosin light-chain dephosphorylation), and by 2,3-butanedione monoxime (a blocker of myosin II ATPase activity). Similarly, no response was observed in cells previously depleted of metabolic energy by 2,4-dinitrophenol and 2-deoxy-D-glucose preincubation. The actin-myosin mediated reversible structural transformation of MDCK cells in response to [Ca2+]3 poses new questions for the interpretation of in vitro experiments, as well as for the understanding of epithelial function.

Fibronectin-derived fragments as inducers of adhesion and chemotaxis of Entamoeba histolytica trophozoites.

Active migration of Entamoeba histolytica trophozoites through extracellular matrixes might play a role in host tissue destruction. Trophozoites degrade soluble fibronectin (FN) bound to their surface and adhere to substrate-bound FN, producing local degradation. FN proteolytic fragments were used to determine the nature of adhesion and motility-promoting domains within the protein. The 70-kDa fragment (amino-terminal end) promoted the highest adhesion, followed by the 120-kDa fragment, which contains the cell-binding domain. The 25-kDa fragment (carboxy-terminal end of the A chain) promoted half the adhesion, while two Hep II-binding fragments had no effect. The 70- and 120-kDa fragments also stimulated directed migration and chemokinesis. Intact FN and the 25-kDa fragment showed lower stimulation. The Hep II-binding fragments had no activity. Results support previous evidence for distinct cell-surface components as mediators of adhesion to FN and trophozoite motility and the potential importance of cell matrix recognition and degradation in their invasive behavior.

Myosin I interactions with actin filaments and trans-Golgi-derived vesicles in MDCK cell monolayers.

In MDCK cell cultured monolayers, as well as in natural and other cultured epithelia, the proper organization of the actin filament ring, tethered to the plasma membrane at the zonula adhaerens, is apparently necessary for their functioning as a transporting epithelium. It has been proposed that actin filaments, in conjunction with motor proteins, could provide the structural basis that regulates the tight junction (TJ) sealing capacity as well as the transport of membrane-tagged proteins required for cell polarization. To test this hypothesis, the authors analyzed the localization and possible association of the actin-binding motor protein myosin I with actin filaments during changes in the actin ring position and organization, and also with trans-Golgi-derived vesicles. Modifications of the ring were induced subjecting the cells to external Ca2+ depletion and restoration (Ca2+ switch), or by treatment with drugs known to depolymerize actin filaments (cytochalasin D, CD). The distribution of myosin I and actin, both in intact cells and in cellular fractions, was monitored using heterologous cross-reacting antibodies and phalloidin. The authors identified an isoform of myosin I of approximately 110-125 KDa, homologous to myosin IB of Acanthamoeba, a fraction of which colocalized with the peripheral actin ring. The association seems transient as, once the ring retracted as result of Ca2+ depletion, or became disorganized by CD, myosin not longer colocalized with the actin fibers but appeared dispersed in the cytoplasm. Furthermore, a significant fraction of the total myosin I in the cell was associated to Golgi-derived vesicles which could also associate in vitro with actin filaments. The authors' data support, then, the participation of myosin I, in association with actin filaments, in vesicle translocation to and from the cell membrane as proposed for natural epithelia, and provide a further insight into the structural organization that maintains epithelial cell polarity in cultured monolayers.

Up-regulation of action mRNA and reorganization of the cytoskeleton in Entamoeba histolytica trophozoites.

Actin mRNA levels were measured in Entamoeba histolytica trophozoites after experimentally inducing changes in the organization of the cytoskeleton. The treatment of trophozoites with forskolin, N6,2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate, and phorbol myristate acetate induced the organization of actin into multiple dots and defined structures with a concomitant increase in F-actin content. Cytochalasin D elicited polarization of the structured actin and formation of aggregates, as well as an increment in F-actin. Simultaneously, up-regulation of actin mRNA levels was produced by all the drugs. De novo synthesis of actin mRNA, as measured by nuclear run-ons, showed increased transcription of actin mRNA. On the other hand, treatment of cells with actinomycin D blocked the elevation of actin mRNA synthesis induced by forskolin, dibutyryl cyclic AMP, or cytochalasin D whereas, the increment induced by PMA was not affected. These data indicate a regulatory control of actin mRNA synthesis at the transcriptional level by forskolin, dibutyryl cyclic AMP and cytochalasin D, and transcriptional as well as post-transcriptional controls by phorbol myristate acetate. The experiments presented here suggest the possibility that, regulation of actin mRNA transcription in E. histolytica trophozoites is linked to growth conditions, that are accompanied by reorganization of the actin cytoskeleton and thus, related to the motility and invasiveness of the parasite.