Testing locus coeruleus-norepinephrine accounts of working memory, attention control, and fluid intelligence.

The current set of studies examined the relationship among working memory capacity, attention control, fluid intelligence, and pupillary correlates of tonic arousal regulation and phasic responsiveness in a combined sample of more than 1,000 participants in two different age ranges (young adults and adolescents). Each study was designed to test predictions made by two recent theories regarding the role of the locus coeruleus-norepinephrine (LC-NE) system in determining individual differences in cognitive ability. The first theory, proposed by Unsworth and Robison (2017a), posits two important individual differences: the moment-to-moment regulation of tonic arousal, and the phasic responsiveness of the system to goal-relevant stimuli. The second theory, proposed by Tsukahara and Engle (2021a), argues that people with higher cognitive abilities have greater functional connectivity between the LC-NE system and cortical networks at rest. These two theories are not mutually exclusive, but they make different predictions. Overall, we found no evidence consistent with a resting-state theory. However, phasic responsiveness was consistently correlated with working memory capacity, attention control, and fluid intelligence, supporting a prediction made by Unsworth and Robison (2017a). Tonic arousal regulation was not correlated with working memory or fluid intelligence and was inconsistently correlated with attention control, which offers only partial support for Unsworth and Robison's (2017a) second prediction.

In vivo discovery of immunotherapy targets in the tumour microenvironment.

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments.

Synthesis and Chiroptical Properties of Hexa-, Octa-, and Deca-azaborahelicenes: Influence of Helicene Size and of the Number of Boron Atoms.

Four members of a new class of cycloborylated hexa-, octa-, and deca-helicenes (1 a-d) have been prepared in enantiopure form, along with two cycloplatinated deca-helicenes (1 d', 1 d1 ), further extending the family of cycloplatinated hexa- and octa-helicenes reported previously. The azabora[n]helicenes display intense electronic circular dichroism and large optical rotations; the dependence of the optical activity on the size of the helix (n=6, 8, 10) and the number of boron atoms (1 or 2) has been examined in detail both experimentally and theoretically. The photophysical properties (nonpolarized and circularly polarized luminescence) of these new fluorescent organic helicenes have been measured and compared with the corresponding organometallic phosphorescent cycloplatinated derivatives (1 a1 -d1 ).

Nanowell-based immunoassays for measuring single-cell secretion: characterization of transport and surface binding.

Arrays of subnanoliter wells (nanowells) provide a useful system to isolate single cells and analyze their secreted proteins. Two general approaches have emerged: one that uses open arrays and local capture of secreted proteins, and a second (called microengraving) that relies on closed arrays to capture secreted proteins on a solid substrate, which is subsequently removed from the array. However, the design and operating parameters for efficient capture from these two approaches to analyze single-cell secretion have not been extensively considered. Using numerical simulations, we analyzed the operational envelope for both open and closed formats, as a function of the spatial distribution of capture ligands, their affinities for the protein, and the rates of single-cell secretion. Based on these analyses, we present a modified approach to capture secreted proteins in-well for highly active secreting cells. This simple method for in-well detection should facilitate rapid identification of cell lines with high specific productivities.

Robust method for chromatogram shape analysis to improve early detection of performance drifts and adverse changes in process parameters during purification column operations.

The biopharmaceutical industry is under increased pressure to maximize efficiency, enhance quality compliance, and reduce the cost of drug substance manufacturing. Ways to reduce costs associated with manufacturing of complex biological molecules include maximizing efficiency of chromatography purification steps. For example, process analytical technology (PAT) tools can be employed to improve column resin life, prevent column operating failures, and decrease the time it takes to solve investigations of process deviations. We developed a robust method to probe the shape of the chromatogram for indications of column failure or detrimental changes in the process. The approach herein utilizes raw data obtained from manufacturing followed by a pre-processing routine to align chromatograms and patch together the different chromatogram phases in preparation for multivariate analysis. A principal component analysis (PCA) was performed on the standardized chromatograms to compare different batches, and resulted in the identification specific process change that affected the profile. In addition, changes in the chromatogram peaks were used to create predictive models for impurity clearance. This approach has the potential for early detection of column processing issues, improving timely resolution in large-scale chromatographic operations.

Noradrenaline tracks emotional modulation of attention in human amygdala.

The noradrenaline (NA) system is one of the brain's major neuromodulatory systems; it originates in a small midbrain nucleus, the locus coeruleus (LC), and projects widely throughout the brain.1,2 The LC-NA system is believed to regulate arousal and attention3,4 and is a pharmacological target in multiple clinical conditions.5,6,7 Yet our understanding of its role in health and disease has been impeded by a lack of direct recordings in humans. Here, we address this problem by showing that electrochemical estimates of sub-second NA dynamics can be obtained using clinical depth electrodes implanted for epilepsy monitoring. We made these recordings in the amygdala, an evolutionarily ancient structure that supports emotional processing8,9 and receives dense LC-NA projections,10 while patients (n = 3) performed a visual affective oddball task. The task was designed to induce different cognitive states, with the oddball stimuli involving emotionally evocative images,11 which varied in terms of arousal (low versus high) and valence (negative versus positive). Consistent with theory, the NA estimates tracked the emotional modulation of attention, with a stronger oddball response in a high-arousal state. Parallel estimates of pupil dilation, a common behavioral proxy for LC-NA activity,12 supported a hypothesis that pupil-NA coupling changes with cognitive state,13,14 with the pupil and NA estimates being positively correlated for oddball stimuli in a high-arousal but not a low-arousal state. Our study provides proof of concept that neuromodulator monitoring is now possible using depth electrodes in standard clinical use.

The impact of divided attention on automatic postural responses: A systematic review and meta-analysis.

Quick responses to a loss of balance or "automatic postural responses" (APRs) are critical for fall prevention. The addition of a distracting task- dual-tasking (DT), typically worsens performance on mobility tasks. However, the effect of DT on APRs is unclear. We conducted a systematic review and meta-analyses to examine the effects of DT on spatial, temporal, and neuromuscular components of APRs and the effect of DT on cognitive performance. A Meta-analysis of 19 cohorts (n = 329) showed significant worsening in spatial kinematic features of APRs under DT conditions (P = 0.01), and a meta-analysis of 9 cohorts (n = 123) demonstrated later muscle onset during DT (P = 0.003). No significant DT effect was observed for temporal kinematic outcomes in 18 cohorts (n = 328; P = 0.47). Finally, significant declines in cognitive performance were evident in 20 cohorts (n = 400; P = 0.002). These results indicate that, despite the somewhat reactive nature of APRs, the addition of a secondary task negatively impacts some aspects of the response. These findings underscore the importance of cortical structures in APR generation. Given the importance of APRs for falls, identifying aspects of APRs that are altered under DT may inform fall-prevention treatment approaches.

Ca2+ waves initiate antigen-stimulated Ca2+ responses in mast cells.

Ca(2+) mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca(2+) sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca(2+) waves that originate most frequently from the tips of extended cell protrusions, as well as Ca(2+) oscillations throughout the cell that usually follow the initiating Ca(2+) wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca(2+) puffs at the region of cell contact. Initiating Ca(2+) waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca(2+) release from endoplasmic reticulum stores and to extracellular Ca(2+), but they do not depend on store-operated Ca(2+) entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca(2+) signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca(2+) response.

Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays.

Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-Fc epsilonRI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-Fc epsilonRI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-Fc epsilonRI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-Fc epsilonRI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (alpha5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-Fc epsilonRI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-Fc epsilonRI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the Fc epsilonRI beta subunit but enhances stimulated Ca(2+) release from intracellular stores. The results suggest that paxillin associated with clustered IgE-Fc epsilonRI has a net positive effect on Fc epsilonRI signaling.

Insight into the Influence of the Chiral Molecular Symmetry on the Chiroptics of Fluorescent BINOL-Based Boron Chelates.

The prominent influence of the molecular symmetry, as defined by the symmetry point group, on the chiroptical behavior (electronic circular dichroism and, especially, circularly polarized luminescence) of simple fluorescent boron chelates (BODIPY and related BOPHY analogues) is studied and discussed. It is shown that increasing the dye symmetry by means of the D3 chiral symmetry group is a workable design option to enhance the level of differential emission of right- and left-circularly polarized light in BODIPY dyes and related emitters, and that the influence of the level of symmetry is stronger than the influence of the higher number of chiral moieties perturbing the acting achiral chromophore.

F-actin and myosin II accelerate catecholamine release from chromaffin granules.

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Caracterización de pacientes con enfermedad pulmonar obstructiva crónica en la unidad de cuidados intensivos

RESUMEN Introducción: La enfermedad pulmonar obstructiva crónica se caracteriza por la limitación del flujo aéreo, no completamente reversible, progresiva y asociada a respuesta inflamatoria anormal de los pulmones. Objetivos: Caracterizar a pacientes con antecedentes de enfermedad pulmonar obstructiva crónica, ingresados en cuidados intensivos. Métodos: Se realizó un estudio descriptivo, retrospectivo en una serie de 347 pacientes con enfermedad pulmonar obstructiva crónica ingresados en cuidados intensivos. Se estudiaron las variables edad, sexo, causa de ingreso, estado al egreso, valor de la escala APACHE II al ingreso, ventilación mecánica invasiva, traqueostomía, y causa directa de muerte según necropsias realizadas. Se calcularon distribuciones de frecuencia absoluta y relativa, medidas de tendencia central y para comparación, las pruebas de ji cuadrado y t de Student, con un nivel de significación del 5 %. Resultados: La edad media fue de 72,1 ± 8,2 años. Predominaron el sexo femenino (52,7 %) y el grupo de edad de 60-79 años (67,1 %). La proporción sexo femenino/ masculino fue de 1,1:1,0. Egresaron fallecidos 64,8 % de los pacientes. Hubo diferencias significativas entre la edad media de los fallecidos y los vivos (p = 0,001). La causa de ingreso clínico tuvo el mayor número de ingresos (86,7 %). La media del valor del APACHE II fue superior en los fallecidos (21,0 vs. 15,2). El 81,3 % de los pacientes recibió ventilación mecánica, y al 17,3 % se le realizó traqueostomía. La principal causa de muerte fue la bronconeumonía bacteriana (67,2 %). Conclusiones: Los pacientes con enfermedad pulmonar obstructiva crónica ingresados en la unidad de cuidados intensivos tienen alta mortalidad y está asociada a tres principales causas directas de muertes: bronconeumonía bacteriana, tromboembolismo pulmonar y el choque séptico.

ABSTRACT Introduction: COPD is characterized by limitation of the airflow, progressive, partially reversible and associated to an abnormal inflammatory responsive of the lungs. Objective: To characterize patients with COPD admitted at the Intensive Care Unit. Methods: A descriptive, retrospective study was carried out in a series of 347 patients with chronic obstructive pulmonary disease admitted to intensive care. The variables age, sex, cause of admission, discharge status, APACHE II scale value at admission, invasive mechanical ventilation, tracheostomy, and direct cause of death according to autopsies performed were studied. Absolute and relative frequency distributions, measures of central tendency, and for comparison, the chi-square and Student's t tests were calculated, with a significance level of 5%. Results: The mean age was of 72,1 ± 8,2 years. The female sex (52.7%) and the age group of 60-79 years (67.1%) predominated. The female / male sex ratio was 1.1: 1.0. 64.8% of the patients were discharged dead. There were significant differences between the mean age of the deceased and the living (p = 0.001). The cause of clinical admission had the highest number of admissions (86.7%). The mean APACHE II value was higher in the deceased (21.0 vs. 15.2). 81.3% of the patients received mechanical ventilation, and 17.3% underwent a tracheostomy. The main cause of death was bacterial bronchopneumonia (67.2%). Conclusions: Patients with chronic obstructive pulmonary disease admitted to the intensive care unit have high mortality and it is associated with three main direct causes of death: bacterial bronchopneumonia, pulmonary thromboembolism and septic shock.

A new toolbox for assessing single cells.

Unprecedented access to the biology of single cells is now feasible, enabled by recent technological advancements that allow us to manipulate and measure sparse samples and achieve a new level of resolution in space and time. This review focuses on advances in tools to study single cells for specific areas of biology. We examine both mature and nascent techniques to study single cells at the genomics, transcriptomics, and proteomics level. In addition, we provide an overview of tools that are well suited for following biological responses to defined perturbations with single-cell resolution. Techniques to analyze and manipulate single cells through soluble and chemical ligands, the microenvironment, and cell-cell interactions are provided. For each of these topics, we highlight the biological motivation, applications, methods, recent advances, and opportunities for improvement. The toolbox presented in this review can function as a starting point for the design of single-cell experiments.

Factores de riesgo de infección respiratoria aguda en menores de un año.Veguitas. Yara. Granma. 2018 / Risk factors for acute respiratory infection in children under one year old. Veguitas.Yara. Granma 2018 / Fatores de risco para infecção respiratória aguda em crianças menores de um ano. Veguitas. Yara Granma 2018

RESUMEN Las infecciones respiratorias agudas tienen alta incidencia en los infantes menores de un año. Se realizó un estudio descriptivo, corte transversal, para identificar factores de riesgo asociados a las infecciones respiratorias agudas en menores de un año, en la policlínica Ramón Heredia Umpierre, del municipio Yara, provincia Granma, en el periodo Enero - agosto 2018. El universo estuvo constituido por 86 infantes menores de un año con infección respiratoria aguda. Se confeccionó una planilla que recogió factores de riesgo, que constituyeron las variables: edad, sexo, lactancia mixta y artificial, bajo peso al nacer, hacinamiento, inmunización incompleta, padres fumadores, posesión de animales domésticos y enfermedades asociadas. Los factores de riesgo más significativo fueron: lactancia mixta y artificial (68,6 %), posesión de animales domésticos (60,4%) y el hábito de fumar de los padres (44,1 %). La modificación de factores de riesgo de las infecciones respiratorias agudas puede disminuir la incidencia de la enfermedad.

ABSTRACT Acute respiratory infections have a high incidence in infants under one year. A descriptive, cross-sectional study was conducted to identify risk factors associated with acute respiratory infections in children under one year of age, in the Ramón Heredia Umpierre polyclinic, in the Yara municipality, Granma province, in the period January - August 2018. The universe It consisted of 86 infants under one year of age with acute respiratory infection. A form was prepared that included risk factors, which constituted the variables: age, sex, mixed and artificial lactation, low birth weight, overcrowding, incomplete immunization, smoking parents, possession of domestic animals and associated diseases. The most significant risk factors were mixed and artificial lactation (68.6%), possession of domestic animals (60.4%) and parental smoking (44.1%). Modification of risk factors for acute respiratory infections may decrease the incidence of the disease.

RESUMO As infecções respiratórias agudas têm uma alta incidência em bebês com menos de um ano. Foi realizado um estudo transversal e descritivo para identificar fatores de risco associados a infecções respiratórias agudas em crianças menores de um ano, na policlínica Ramón Heredia Umpierre, no município de Yara, província de Granma, no período de janeiro a agosto de 2018. O universo Consistia em 86 crianças com menos de um ano de idade com infecção respiratória aguda. Foi elaborado um formulário que incluía fatores de risco, que constituíam as variáveis: idade, sexo, lactação mista e artificial, baixo peso ao nascer, superlotação, imunização incompleta, pais fumantes, posse de animais domésticos e doenças associadas. Os fatores de risco mais significativos foram lactação mista e artificial (68,6%), posse de animais domésticos (60,4%) e tabagismo dos pais (44,1%). A modificação dos fatores de risco para infecções respiratórias agudas pode diminuir a incidência da doença.

Functional single-cell analysis of T-cell activation by supported lipid bilayer-tethered ligands on arrays of nanowells.

Supported lipid bilayers are an important biomolecular tool for characterizing immunological synapses. Immobilized bilayers presenting tethered ligands on planar substrates have yielded both spatio-temporal and structural insights into how T cell receptors (TCRs) reorganize during the initial formation of synapses upon recognition of peptide antigens bound to major histocompatibility complex (MHC) molecules. The prototypical configuration of these assays, however, limits the extent to which the kinetics and structure of the supramolecular activation clusters of the synapse (that occur in seconds or minutes) can be related to subsequent complex cellular responses, such as cytokine secretion and proliferation, occurring over hours to days. Here we describe a new method that allows correlative measures of both attributes with single-cell resolution by using immobilized lipid bilayers and tethered ligands on the surface of dense arrays of subnanoliter wells. This modification allows each nanowell to function as an artificial antigen-presenting cell (APC), and the synapses formed upon contact can be imaged by fluorescence microscopy. We show that the lipid bilayers remain stable and mobile on the surface of the PDMS, and that modifying the ligands tethered to the bilayer alters the structure of the resulting synapses in expected ways. Finally, we demonstrate that this approach allows the subsequent characterization of secreted cytokines from the activated human T cell clones by microengraving in both antigen- and pan-specific manners. This new technique should allow detailed investigations on how biophysical and structural aspects of the synapse influence the activation of individual T cells and their complex functional responses.

Micropatterned ligand arrays to study spatial regulation in Fc receptor signaling.

Fc receptor signaling plays a fundamental role in immune responses. A plethora of Fc -receptors (e.g., Fc gamma, Fc-alpha, and Fc-epsilon) are expressed on different immune cells, including natural killer cells, macrophages, mast cells, and neutrophils. Receptor clustering and activation by multivalent ligands or opsonized particles induce a signaling cascade that leads to targeted secretion of chemical mediators (i.e., histamine, cytokines, and chemokines) and phagocytosis, among other responses. Spatial targeting and compartmentalization are common mechanisms of regulation in Fc receptor signaling. However, the tools for studying these dynamic interactions have been limited. To overcome these limitations in our model system, microfabricated surfaces containing spatially defined ligands are used to cluster- and activate IgE receptors (FcεRI), involved in allergic responses by mast cells. Micron-scale control of cell activation allows investigation of spatially regulated mechanisms for intracellular signaling with -fluorescence microscopy. This approach in conjunction with biochemical techniques has proven to be valuable for investigating immune receptor signaling.

Paclitaxel delivery to brain tumors from hydrogels: a computational study.

Malignant gliomas are aggressive forms of primary brain tumors characterized by a poor prognosis. The most successful treatment so far is the local implantation of polymer carriers (Gliadel® wafers) for the sustained release of carmustine. To improve the effectiveness of local drug treatment, new polymer carriers and pharmacological agents are currently being investigated. Of particular interest is a set of novel thermo-gelling polymers for the controlled release of hydrophobic drugs such as paclitaxel (e.g., OncoGel™). Herein, we use computational mass transport simulations to investigate the effectiveness of paclitaxel delivery from hydrogel-forming polymer carriers. We found similar (within 1-2 mm) therapeutic penetration distances of paclitaxel when released from these hydrogels as compared with carmustine released from Gliadel® wafers. Effective therapeutic concentrations were maintained for >30 days for paclitaxel when released from the hydrogel as compared with 4 days for carmustine released from Gliadel® wafers. Convection in brain tissue prevented the formation of a uniform drug concentration gradient around the implant. In addition, the surface area to volume ratio of the gel is an important factor that should be considered to maintain a controlled release of paclitaxel within the degradation lifetime of the polymer matrix.

Solving the two-center nuclear shell-model problem with arbitrarily oriented deformed potentials.

A general new technique to solve the two-center problem with arbitrarily oriented deformed realistic potentials is demonstrated, which is based on the powerful potential separable expansion method. As an example, molecular single-particle spectra for (12)C+(12)C-->(24)Mg are calculated using deformed Woods-Saxon potentials. These clearly show that nonaxial symmetric configurations play a crucial role in molecular resonances observed in reaction processes for this system at low energy.

Nanobiotechnology and cell biology: micro- and nanofabricated surfaces to investigate receptor-mediated signaling.

Advances in microfabrication and nanofabrication are opening new opportunities to investigate complicated questions of cell biology in ways not before possible. In particular, the spatial regulation of cellular processes can be examined by engineering the chemical and physical environment to which the cell responds. Lithographic methods and selective chemical modification schemes can provide biocompatible surfaces that control cellular interactions on the micron and submicron scales on which cells are organized. Combined with fluorescence microscopy and other approaches of cell biology, a widely expanded toolbox is becoming available. This review illustrates the potential of these integrated engineering tools, with an emphasis on patterned surfaces, for investigating fundamental mechanisms of receptor-mediated signaling in cells. We highlight progress made with immune cells and in particular with the IgE receptor system, which has been valuable for developing technology to gain new information about spatial regulation in signaling events.

Análisis técnico administrativo e instrumental Hospital General Dr. Domingo Luciani año 1989, 1er semestre 1990 / Instrumental and administrative technical analysis General Hospital Dr. Domingo Luciani year 1989. First semestre 1990

El estudio comprende un diagnóstico de la situación de salud del Distrito Sanitario 5 al cual pertenece el hospital, asimismo, son analizados e investigados los recursos que en el érea de salud posee el Distrito, su situación epidemiológica, aspectos geopolíticos, sociales y culturales que inciden sobre las condiciones sanitarias del Distrito, evidenciar el beneficio hospitalario hacia la comunidad a la que sirve y tratar de reflejar si se cumple el criterio de regionalización hospitalaria, se llego a condiciones que engloban el proceso salud-enfermedad y su vinculación con el ambiente, la organización de los servicios y su capacidad operativa, y se recomienda acciones a seguir según fallas encontradas