Evaluation of a lateral flow immunoassay to detectCTX-M extended-spectrum B-lactamases (ESBL)directly from positive blood cultures for its potentialuse in Antimicrobial Stewardship programs / Evaluación de una inmunocromatografía paradetectar beta-lactamasas de espectro extendidoCTX-M directamente de hemocultivos positivospara su potencial uso en programas deoptimización de antibioterapia

Background. Bloodstream infections (BSI) caused by extended-spectrum beta-lactamases Enterobacteriaceae (ESBL-E) are associated with high rates of treatment failure andincreased mortality, especially when appropriate antimicrobialtherapy is delayed. Our aim was to evaluate the anticipationof ESBLs detection and the potential improvement of the timeresponse of the Vitek 2 System (BioMérieux; France).Methods. We compared this lateral flow immunoassaywhen used directly on fluid from positive blood cultures to theVITEK2 AST system. We evaluated 80 isolates, 61 tested directlyon fluid from positive blood cultures and 19 tested on fluidfrom blood cultures spiked with known ESBL positive and negative organisms.Results. The concordance between the CTX-LFIA and thereference method (Vitek 2) had a Cohen´s Kappa coefficient of0.97, indicating a particularly good correlation between bothcompared methods.Conclusion. This lateral flow immunoassay can be morerapid than the Vitek 2 for earlier presumptive identification ofCTX- M ESBLs and can be useful to anticipate results and theadjustment of antimicrobial therapy. (AU)

Antecedentes. Las bacteriemias causadas por Enterobacteriaceae productoras beta-lactamasas de espectro extendido(BLEE) están asociadas con altas tasas de fallo de tratamientoy mortalidad, especialmente cuando se retrasa el tratamientoapropiado. Nuestro objetivo ha sido evaluar la anticipación dela detección de estas BLEE y la potencial mejora en el tiempode respuesta respecto al VITEK2 System (Biomerieux; Francia).Métodos. Se comparó una inmunocromatografía para sudetección con el VITEK2 AST system directamente del hemocultivo. Se evaluaton 80 aislados, 61 evaluados directamentede hemocultivos positivos y 19 de la misma manera pero inoculados con microorganismos productores y no productores deBLEE.Resultados. La concordancia entre la inmunocromatografía y el VITEK2 AST mostró un coeficiente Kappa de 0,97,indicando una buena correlación entre ambas técnicas.Conclusión. Esta inmunocromatografía puede ser másrápida que el VITEK2 para una identificación de BLEE tipo CTXM y puede ser útil para anticipar resultados y ajustar la terapiaantimicrobiana. (AU)

New STAG3 gene variant as a cause of premature ovarian insufficiency / Nueva variante del gen STAG3 causante de insuficiencia ovárica prematura

Objectives: To describe a case of ovarian failure secondary to a homozygous pathogenic variant in the STAG3 gene not previously reported. Materials and Methods: A 16-year-old patient with primary amenorrhea and absence of secondary sexual characteristics, with documented autoimmune hypothyroidism, poor genital and gonadal streak development which prompted the performance of clinical exome sequencing. A homozygous pathogenic variant not previously reported in the STAG3 gene, which has been associated with premature ovarian insufficiency (POI), was identified. Conclusions: In this case, clinical exome sequencing was key for identifying a STAG gene abnormality, probably associated with POI and long term prognosis for the patient. A new pathogenic variant c.2773delT; p.Ser925Profs*6 of the STAG3 gene associated with POI was established.

Objetivos: describir un caso de falla ovárica secundaria a una variante patogénica homocigota en el gen STAG3 no reportada previamente. Materiales y métodos: paciente de 16 años con amenorrea primaria y ausencia de características sexuales secundarias, en quien se documentó hipotiroidismo autoinmune, pobre desarrollo genital y cintilla gonadal, por lo cual se realizó secuenciación de exoma clínico. Se identificó una variante homocigota patogénica previamente no reportada en el gen STAG3, el cual ha sido relacionado con insuficiencia ovárica prematura (IOP). Conclusiones: en este caso, la realización de exoma clínico fue determinante para identificar una alteración del gen STAG, probablemente asociada a la IOP y el pronóstico a largo plazo de la paciente. Se establece una nueva variante patogénica c.2773delT; p.Ser925Profs*6 del gen STAG3 asociada a la IOP.

Modulation of Gq/PLC-Mediated Signaling by Acute Lithium Exposure.

Although lithium has long been one of the most widely used pharmacological agents in psychiatry, its mechanisms of action at the cellular and molecular levels remain poorly understood. One of the targets of Li+ is the phosphoinositide pathway, but whereas the impact of Li+ on inositol lipid metabolism is well documented, information on physiological effects at the cellular level is lacking. We examined in two mammalian cell lines the effect of acute Li+ exposure on the mobilization of internal Ca2+ and phospholipase C (PLC)-dependent membrane conductances. We first corroborated by Western blots and immunofluorescence in HEK293 cells the presence of key signaling elements of a muscarinic PLC pathway (M1AchR, Gq, PLC-ß1, and IP3Rs). Stimulation with carbachol evoked a dose-dependent mobilization of Ca, as determined with fluorescent indicators. This was due to release from internal stores and proved susceptible to the PLC antagonist U73122. Li+ exposure reproducibly potentiated the Ca response in a concentration-dependent manner extending to the low millimolar range. To broaden those observations to a neuronal context and probe potential Li modulation of electrical signaling, we next examined the cell line SHsy5y. We replicated the potentiating effects of Li on the mobilization of internal Ca, and, after characterizing the basic properties of the electrical response to cholinergic stimulation, we also demonstrated an equally robust upregulation of muscarinic membrane currents. Finally, by directly stimulating the signaling pathway at different links downstream of the receptor, the site of action of the observed Li effects could be narrowed down to the G protein and its interaction with PLC-ß. These observations document a modulation of Gq/PLC/IP3-mediated signaling by acute exposure to lithium, reflected in distinct physiological changes in cellular responses.

Nueva variante del gen STAG3 causante de insuficiencia ovárica prematura / New STAG3 gene variant as a cause of premature ovarian insufficiency

RESUMEN Objetivos: describir un caso de falla ovárica secundaria a una variante patogénica homocigota en el gen STAG3 no reportada previamente. Materiales y métodos: paciente de 16 años con amenorrea primaria y ausencia de características sexuales secundarias, en quien se documentó hipotiroidismo autoinmune, pobre desarrollo genital y cintilla gonadal, por lo cual se realizó secuenciación de exorna clínico. Se identificó una variante homocigota patogénica previamente no reportada en el gen STAG3, el cual ha sido relacionado con insuficiencia ovárica prematura (IOP). Conclusiones: en este caso, la realización de exorna clínico fue determinante para identificar una alteración del gen STAG, probablemente asociada a la IOP y el pronóstico a largo plazo de la paciente. Se establece una nueva variante patogénica c.2773delT; p.Ser925Profs*6 del gen STAG3 asociada a la IOP.

ABSTRACT Objectives: To describe a case of ovarian failure secondary to a homozygous pathogenic variant in the STAG3 gene not previously reported. Material and methods: A 16-year-old patient with primary amenorrhea and absence of secondary sexual characteristics, with documented autoimmune hypothyroidism, poor genital and gonadal streak development which prompted the performance of clinical exorne sequencing. A homozygous pathogenic variant not previously reported in the STAG3 gene, which has been associated with premature ovarian insufficiency (POI), was identified. Conclusions: In this case, clinical exorne sequencing was key for identifying a STAG gene abnormality, probably associated with POI and long term prognosis for the patient. A new pathogenic variant c.2773delT; p.Ser925Profs*6 of the STAG3 gene associated with POI was established.

Influence of spatially segregated IP3-producing pathways on spike generation and transmitter release in Purkinje cell axons.

It has been known for a long time that inositol-trisphosphate (IP3) receptors are present in the axon of certain types of mammalian neurons, but their functional role has remained unexplored. Here we show that localized photolysis of IP3 induces spatially constrained calcium rises in Purkinje cell axons. Confocal immunohistology reveals that the axon initial segment (AIS), as well as terminals onto deep cerebellar cells, express specific subtypes of Gα/q and phospholipase C (PLC) molecules, together with the upstream purinergic receptor P2Y1. By contrast, intermediate parts of the axon express another set of Gα/q and PLC molecules, indicating two spatially segregated signaling cascades linked to IP3 generation. This prompted a search for distinct actions of IP3 in different parts of Purkinje cell axons. In the AIS, we found that local applications of the specific P2Y1R agonist MRS2365 led to calcium elevation, and that IP3 photolysis led to inhibition of action potential firing. In synaptic terminals on deep cerebellar nuclei neurons, we found that photolysis of both IP3 and ATP led to GABA release. We propose that axonal IP3 receptors can inhibit action potential firing and increase neurotransmitter release, and that these effects are likely controlled by purinergic receptors. Altogether our results suggest a rich and diverse functional role of IP3 receptors in axons of mammalian neurons.

Juvenile polyautoimmunity in a rheumatology setting.

Overt polyautoimmunity (PolyA) corresponds to the presence of more than one well-defined autoimmune disease (AD) manifested clinically in a single patient. The current study aimed to describe the main characteristics of juvenile PolyA in a pediatric rheumatology setting and analyze the chronological aspects, index cases, familial autoimmunity, and clustering pattern. This was a cross-sectional and multicenter study in which 313 children with overt PolyA were included. Patients were systematically interviewed and their medical records reviewed using a questionnaire that sought information about demographic, clinical, immunological, and familial characteristics. A hierarchical cluster analysis was done to determine similarities between autoimmune diseases based on PolyA. PolyA occurred simultaneously in 138 (44%) patients. Multiple autoimmune syndrome was observed in 62 (19.8%) patients. There were 25 index diseases of which, systemic lupus erythematosus (SLE, n = 134, 42.8%), juvenile idiopathic arthritis (JIA, n = 40, 12.7%), Hashimoto's thyroiditis (HT, n = 24, 7.66%), immune thrombocytopenic purpura (ITP n = 20, 6.39%), antiphospholipid syndrome (APS, n = 15, 4.79%), and vitiligo (VIT, n = 15, 4.79%) were the most frequent and represented 79.23% of the total number of patients. Familial autoimmunity influenced PolyA. A high aggregation of autoimmunity was observed (λr = 3.5). Three main clusters were identified, of which SLE and APS were the most similar pair of diseases (based on the Jaccard index) followed by HT and JIA, which were related to ITP and Sjögren's syndrome. The third cluster was composed of localized scleroderma and VIT. Our findings may assist physicians to make an early diagnosis of this frequent condition. Pediatric patients with ADs should be systematically assessed for PolyA.

Light control of G protein signaling pathways by a novel photopigment.

Channelopsins and photo-regulated ion channels make it possible to use light to control electrical activity of cells. This powerful approach has lead to a veritable explosion of applications, though it is limited to changing membrane voltage of the target cells. An enormous potential could be tapped if similar opto-genetic techniques could be extended to the control of chemical signaling pathways. Photopigments from invertebrate photoreceptors are an obvious choice-as they do not bleach upon illumination -however, their functional expression has been problematic. We exploited an unusual opsin, pScop2, recently identified in ciliary photoreceptors of scallop. Phylogenetically, it is closer to vertebrate opsins, and offers the advantage of being a bi-stable photopigment. We inserted its coding sequence and a fluorescent protein reporter into plasmid vectors and demonstrated heterologous expression in various mammalian cell lines. HEK 293 cells were selected as a heterologous system for functional analysis, because wild type cells displayed the largest currents in response to the G-protein activator, GTP-γ-S. A line of HEK cells stably transfected with pScop2 was generated; after reconstitution of the photopigment with retinal, light responses were obtained in some cells, albeit of modest amplitude. In native photoreceptors pScop2 couples to Go; HEK cells express poorly this G-protein, but have a prominent Gq/PLC pathway linked to internal Ca mobilization. To enhance pScop2 competence to tap into this pathway, we swapped its third intracellular loop-important to confer specificity of interaction between 7TMDRs and G-proteins-with that of a Gq-linked opsin which we cloned from microvillar photoreceptors present in the same retina. The chimeric construct was evaluated by a Ca fluorescence assay, and was shown to mediate a robust mobilization of internal calcium in response to illumination. The results project pScop2 as a potentially powerful optogenetic tool to control signaling pathways.

Molecular and functional identification of a novel photopigment in Pecten ciliary photoreceptors.

The two basic animal photoreceptor types, ciliary and microvillar, use different light-transduction schemes: their photopigments couple to Gt versus Gq proteins, respectively, to either mobilize cyclic nucleotides or trigger a lipid signaling cascade. A third class of photoreceptors has been described in the dual retina of some marine invertebrates; these present a ciliary morphology but operate via radically divergent mechanisms, prompting the suggestion that they comprise a novel lineage of light sensors. In one of these organisms, an uncommon putative opsin was uncovered that was proposed to signal through Go Orthologues subsequently emerged in diverse phyla, including mollusks, echinoderms, and chordates, but the cells in which they express have not been identified, and no studies corroborated their function as visual pigments or their suggested signaling mode. Conversely, in only one invertebrate species, Pecten irradians, have the ciliary photoreceptors been physiologically characterized, but their photopigment has not been identified molecularly. We used the transcriptome of Pecten retina to guide the cloning by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) extensions of a new member of this group of putative opsins. In situ hybridization shows selective transcription in the distal retina, and specific antibodies identify a single band of the expected molecular mass in Western blots and distinctly label ciliary photoreceptors in retina sections. RNA interference knockdown resulted in a reduction in the early receptor current-the first manifestation of light transduction-and prevented the prolonged aftercurrent, which requires a large buildup of activated rhodopsin. We also obtained a full-length clone of the α-subunit of a Go from Pecten retina complementary DNA and localized it by in situ hybridization to the distal photoreceptors. Small interfering RNA targeting this Go caused a specific depression of the photocurrent. These results establish this novel putative opsin as a bona fide visual pigment that couples to Go to convey the light signal.

Esclerodermia localizada juvenil: ¿es una enfermedad benigna? / Juvenile localized scleroderma: is it a benign disease?

Resumen Introducción: La esclerodermia localizada juvenil es una enfermedad polimórfica que ocurre con mayor frecuencia en niñas. Se acompaña de morbilidad importante. El compromiso extradérmico es frecuente y se reportan tasas de poliautoinmunidad de hasta 7%. Al momento, se desconocen las características clínicas de los pacientes colombianos con esta enfermedad. Objetivo: Describir las características clínicas, morbilidades y secuelas en pacientes con diagnóstico de esclerodermia localizada juvenil, en múltiples centros de reumatología pediátrica en Colombia. Materiales y métodos: Estudio descriptivo, retrospectivo y multicéntrico. Pacientes con diagnóstico de esclerodermia localizada juvenil con un mínimo de 1 ario de evolución y 6 meses de seguimiento, en 10 centros de reumatología pediátrica mediante revisión de historias clínicas. Resultados: El n = 88. La distribución por género fue: femenino 2,1; masculino 1. Edad promedio al inicio de la enfermedad 7,1 años (0-14). Promedio de duración de la enfermedad al diagnóstico 16,5 meses (1-96). La distribución por subtipos fue morfea circunscrita (32,9%), mixta (31,8%), linear (21,5%, asciende a 55% al incluir formas mixtas con lesiones lineares) generalizada (11,4%) y panesclerótica (2,3%). Se detectaron alteraciones estéticas en el 91%, alteraciones del crecimiento en 41% y compromiso funcional de articulaciones vecinas en 32%. Se presentó compromiso extradérmico en 22,7% y poliautoinmunidad en 12,5%. Conclusiones: La esclerodermia localizada juvenil es una enfermedad polimórfica e impredecible. En la mayoría de los casos el diagnóstico es tardío. La tasa de compromiso extradérmico sugiere que no es una enfermedad limitada a la piel. Un diagnóstico temprano, tratamiento dinámico y seguimiento cercano permiten prevenir y detectar tempranamente complicaciones derivadas de la enfermedad.

Abstract Introduction: Juvenile localized scleroderma is a polymorphic disease. It is more prevalent in girls and has a significant morbidity. Extra-cutaneous involvement is common, and polyautoimmunity can reach 7%. The clinical characteristics of this disease in Colombian patients are currently unknown. Objective: To describe the clinical characteristics, morbidity and outcomes in patients with juvenile localized scleroderma in different paediatric rheumatology clinics in Colombia. Materials and methods: A descriptive, retrospective, and multicentre study was conducted on patients with juvenile localized scleroderma with a minimum of 1 year of disease onset, and 6 months of follow-up in 10 paediatric rheumatology clinics. Results: The study included 88 patients, with a gender distribution of female 2.1: male 1. Mean age at disease onset was 7.1 years (0-14). Mean disease duration at diagnosis was 16.5 months (1-96). Sub-type distribution was, circumscribed (32.9%), mixed (31.8%), and linear (21.5%, that increased to 55% if linear lesions of the mixed subtype are included), generalised (11.4%), and pan-sclerotic morphea (2.3%). Aesthetic compromise was detected in 91%, with growth disturbances in 41%, and joint functional compromise in 32%. Extra-cutaneous involvement occurred in 22.7% and polyautoimmunity in 12.5%. Conclusions: Juvenile localized scleroderma is a polymorphic and unpredictable disease. It diagnosed late in most of the cases. Extra-cutaneous involvement suggests that is not a disease limited to skin. An early diagnosis, a dynamic treatment and a close follow-up helps to prevent, and detect, complications arising from the disease.

Calcium activates the light-dependent conductance in melanopsin-expressing photoreceptors of amphioxus.

Melanopsin, the photopigment of the "circadian" receptors that regulate the biological clock and the pupillary reflex in mammals, is homologous to invertebrate rhodopsins. Evidence supporting the involvement of phosphoinositides in light-signaling has been garnered, but the downstream effectors that control the light-dependent conductance remain unknown. Microvillar photoreceptors of the primitive chordate amphioxus also express melanopsin and transduce light via phospholipase-C, apparently not acting through diacylglycerol. We therefore examined the role of calcium in activating the photoconductance, using simultaneous, high time-resolution measurements of membrane current and Ca(2+) fluorescence. The light-induced calcium rise precedes the onset of the photocurrent, making it a candidate in the activation chain. Moreover, photolysis of caged Ca elicits an inward current of similar size, time course and pharmacology as the physiological photoresponse, but with a much shorter latency. Internally released calcium thus emerges as a key messenger to trigger the opening of light-dependent channels in melanopsin-expressing microvillar photoreceptors of early chordates.

Dissecting the determinants of light sensitivity in amphioxus microvillar photoreceptors: possible evolutionary implications for melanopsin signaling.

Melanopsin, a photopigment related to the rhodopsin of microvillar photoreceptors of invertebrates, evolved in vertebrates to subserve nonvisual light-sensing functions, such as the pupillary reflex and entrainment of circadian rhythms. However, vertebrate circadian receptors display no hint of a microvillar specialization and show an extremely low light sensitivity and sluggish kinetics. Recently in amphioxus, the most basal chordate, melanopsin-expressing photoreceptors were characterized; these cells share salient properties with both rhabdomeric photoreceptors of invertebrates and circadian receptors of vertebrates. We used electrophysiology to dissect the gain of the light-transduction process in amphioxus and examine key features that help outline the evolutionary transition toward a sensor optimized to report mean ambient illumination rather than mediating spatial vision. By comparing the size of current fluctuations attributable to single photon melanopsin isomerizations with the size of single-channels activated by light, we concluded that the gain of the transduction cascade is lower than in rhabdomeric receptors. In contrast, the expression level of melanopsin (gauged by measuring charge displacements during photo-induced melanopsin isomerization) is comparable with that of canonical visual receptors. A modest amplification in melanopsin-using receptors is therefore apparent in early chordates; the decrease in photopigment expression-and loss of the anatomical correlates-observed in vertebrates subsequently enabled them to attain the low photosensitivity tailored to the role of circadian receptors.

Síndrome de Sjõgren en pediatría: diagnóstico y manejo / Sjõgren's syndrome in children: diagnosis and management

El síndrome de Sjõgren (SS) es una enfermedad autoinmune que compromete las glándulas exocrinasprovocando una respuesta inflamatoria que conduce a una hiposecreción de las mismas.Es de rara ocurrencia en la edad pediátrica. Clínicamente se manifiesta como ojo y boca seca,asociados a una gran variedad de síntomas y signos que pueden simular otras enfermedadesautoinmunes. Puede ser primario o secundario a otras enfermedades autoinmunes.Actualmente, se aceptan los criterios europeo-americanos de 2002, de los cuales son necesarios4 de los 6 criterios y que uno de ellos sea una biopsia de glándula salival positiva o bienAnticuerpos (Ac) anti-Ro/La positivos. El tratamiento abarca 2 aspectos diferentes: por un lado,el tratamiento de la sequedad de ojos y boca (agentes sustitutivos de lágrimas), fármacos agonistasmuscarínicos, etc., por otro lado, el abordaje de las manifestaciones extra glandulares(antiinflamatorios no esteroideos, corticoides, agentes modificadores de la enfermedad, agentescitotóxicos e incluso terapias biológicas).Se describe el caso clínico de una niña de 13 años de edad que cumple con los criterios clínicos,de laboratorio e histopatológicos de síndrome de Sjõgren primário.

Melanopsin-expressing amphioxus photoreceptors transduce light via a phospholipase C signaling cascade.

Melanopsin, the receptor molecule that underlies light sensitivity in mammalian 'circadian' receptors, is homologous to invertebrate rhodopsins and has been proposed to operate via a similar signaling pathway. Its downstream effectors, however, remain elusive. Melanopsin also expresses in two distinct light-sensitive cell types in the neural tube of amphioxus. This organism is the most basal extant chordate and can help outline the evolutionary history of different photoreceptor lineages and their transduction mechanisms; moreover, isolated amphioxus photoreceptors offer unique advantages, because they are unambiguously identifiable and amenable to single-cell physiological assays. In the present study whole-cell patch clamp recording, pharmacological manipulations, and immunodetection were utilized to investigate light transduction in amphioxus photoreceptors. A G(q) was identified and selectively localized to the photosensitive microvillar membrane, while the pivotal role of phospholipase C was established pharmacologically. The photocurrent was profoundly depressed by IP3 receptor antagonists, highlighting the importance of IP3 receptors in light signaling. By contrast, surrogates of diacylglycerol (DAG), as well as poly-unsaturated fatty acids failed to activate a membrane conductance or to alter the light response. The results strengthen the notion that calcium released from the ER via IP3-sensitive channels may fulfill a key role in conveying--directly or indirectly--the melanopsin-initiated light signal to the photoconductance; moreover, they challenge the dogma that microvillar photoreceptors and phoshoinositide-based light transduction are a prerogative of invertebrate eyes.

The light-sensitive conductance of melanopsin-expressing Joseph and Hesse cells in amphioxus.

Two types of microvillar photoreceptors in the neural tube of amphioxus, an early chordate, sense light via melanopsin, the same photopigment as in "circadian" light detectors of higher vertebrates. Because in amphioxus melanopsin activates a G(q)/phospholipase C cascade, like phototransduction in arthropods and mollusks, possible commonalities in the photoconductance were investigated. Unlike other microvillar photoreceptors, reversal of the photocurrent can only be attained upon replacement of extracellular Na(+). In addition to Na(+), Ca(2+) is also permeant, as indicated by the fact that (a) in normal ionic conditions the photocurrent remains inward at V(m) > E(Na); (b) in Na-free solution a small residual inward photocurrent persists at V(m) near resting level, provided that Ca is present; and (c) V(rev) exhibits a modest shift with [Ca](o) manipulations. The unusual reversal is accounted for by an uncommonly low permeability of the light-dependent channels to K(+), as [K](o) only marginally affects the photocurrent amplitude and its reversal. Lanthanum and ruthenium red (RuR), two TRP channel antagonists, reversibly suppress the response to photostimulation of moderate intensity; therefore, the melanopsin-initiated cascade may recruit ion channels of the same family as those of rhabdomeric photoreceptors. With brighter lights, blockage declines, so that both La(3+) and RuR induce a right shift in the sensitivity curve without a reduction of its asymptote. Nonetheless, an effect on the transduction cascade, rather than the channels, was ruled out on the basis of the voltage dependency of the blockade and the lack of effects of intracellular application of the same substances. The mechanisms of action of these antagonists thus entail a state-dependent blockade, with a higher affinity for the channel in the closed conformation. Collectively, the results indicate a kinship of the light-sensitive channels of amphioxus with those of invertebrate rhabdomeric visual cells and support the representation of this lineage of photoreceptors among chordates.

Arrestin in ciliary invertebrate photoreceptors: molecular identification and functional analysis in vivo.

Arrestin was identified in ciliary photoreceptors of Pecten irradians, and its role in terminating the light response was established electrophysiologically. Downstream effectors in these unusual visual cells diverge from both microvillar photoreceptors and rods and cones; the finding that key regulatory mechanisms of the early steps of visual excitation are conserved across such distant lineages of photoreceptors underscores that a common blueprint for phototransduction exists across metazoa. Arrestin was detected by Western blot analysis of retinal lysates, and localized in ciliary photoreceptors by immunostaining of whole-eye cryosections and dissociated cells. Two arrestin isoforms were molecularly identified by PCR; these present the canonical N- and C-arrestin domains, and are identical at the nucleotide level over much of their sequence. A high degree of homology to various ß-arrestins (up to 70% amino acid identity) was found. In situ hybridization localized the two transcripts within the retina, but failed to reveal finer spatial segregation, possibly because of insufficient differences between the riboprobes. Intracellular dialysis of anti arrestin antibodies into voltage-clamped ciliary photoreceptors produced a gradual slow-down of the photocurrent falling phase, leaving a tail that decayed over many seconds after light termination. The antibodies also caused spectrally neutral flashes to elicit prolonged aftercurrents in the absence of large metarhodopsin accumulation; such aftercurrents could be quenched by chromatic illumination that photoconverts metarhodopsin back to rhodopsin. These observations indicate that the antibodies depleted functionally available arrestin, and implicate this molecule in the deactivation of the photoresponse at the rhodopsin level.

Light-transduction in melanopsin-expressing photoreceptors of Amphioxus.

Spatial vision in different organisms is mediated by 2 classes of photoreceptors: microvillar and ciliary. Recently, additional photosensitive cells implicated in nonvisual light-dependent functions have been identified in the mammalian retina. A previously undescribed photopigment, melanopsin, underlies these photoresponses, and it has been proposed that its transduction mechanisms may be akin to the lipid-signaling scheme of invertebrate microvillar receptors, rather than the cyclic-nucleotide cascade of vertebrates. Melanopsin has an ancient origin in deuterostomia, and expresses in 2 morphologically distinct classes of cells in the neural tube of Amphioxus, the most basal extant chordate: pigmented ocelli, and Joseph cells. However, to our knowledge, their physiology and alleged photosensitivity had never been investigated. We dissociated both types of cells, and conclusively demonstrated by patch-electrode recoding that they are primary photoreceptors; their receptor potential is depolarizing, accompanied by an increase in membrane conductance. The action spectrum peaks in the blue region, approximately 470 nm, similar to the absorption of melanopsin in vitro. The light-dependent conductance rectifies inwardly; Na and Ca are differentially implicated in the 2 cell types. Fluorescence Ca imaging reveals that photostimulation rapidly mobilizes calcium from internal stores. Intracellular 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate severely impairs the photoresponse, indicating that light-evoked Ca elevation is an important event in photoexcitation. These observations support the notion that the lineage of microvillar photoreceptors and its associated light-signaling pathway also evolved in the chordates. Thus, Joseph cells and pigmented ocelli of the Amphioxus may represent a link between ancestral rhabdomeric-like light sensors present in prebilaterians and the circadian photoreceptors of higher vertebrates.

Antigenos de VPH en lesiones intraepiteliales escamosas: inmunohistoquímica con P16ink4a, VIROACTIV® y Ki67 / HPV antigens in squamous intraepithelial lesions

El método más utilizado en la actualidad para la detección precoz del cáncer del cérvix uterino (CCU) es la toma de citología cervicovaginal (CCV), la cual ha demostrado en forma amplia su utilidad dada su capacidad para detectar las lesiones precursoras del CCU, lo cual ha contribuido a disminuir de modo notable la morbimortalidad por esta neoplasia. La consecuencia inmediata de los programas de muestreo ha sido el incremento significativo del número de biopsias cervicales. Su análisis histológico es importante pues se considera el patrón de referencia (gold standard) en que el clínico se basa para planificar el tratamiento adecuado para estas pacientes. Sin embargo, existen casos en los que el diagnóstico histopatológico preciso es subjetivo y susceptible de opiniones variadas entre los observadores, hecho que ha incitado la búsqueda de técnicas alternativas como la inmunohistoquímica, para ayudar a establecer un valor pronóstico más confiable en las lesiones preneoplásicas del cérvix. Es por esto que nuestro estudio tiene como objetivo principal determinar la expresión IHQ de antìgenos de VPH (subtipo de alto riesgo) y el índice de proliferación celular en lesiones intraepiteliales escamosas de bajo grado (LIEBG) y lesiones intraepiteliales escamosas de alto grado (LIEAG), utilizando los marcadores biomoleculares Ki67, P16ink4a y Viroactiv®.

Cytology-based screening is currently the most used strategy for early detection of cervical cancer. Cytology has proved to be significantly useful due to its ability to detect precancerous lesions, thus, contributing to a substantial reduction of morbidity and mortality due to this neoplasia. The imme-diate consequence of screening programs has been a significant increase on the number of cervical biopsies performed. The importance of histological analyses of these samples lies on the fact that they are considered the gold standard in which the clinicians base adequate treatment plans for patients. Nevertheless, in some cases a precise histology/pathology interpretation is subjective and susceptible to various opinions among observers. Due to the latter, finding alternative diagnostic techniques suchas immunohistochemistry (IHC) has been encouraged to help establish a more reliable prognostic value in cervical preneoplastic lesions. For this reason, the main purpose of our study is to determi-ne the IHQ expression of HPV antigens (high-risk sub-type) and cell proliferation rate in low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL), using biomolecular markers such as Ki67, Pl6ink4a and Viroactiv ®.

Prevalencia de bajo peso al nacer y factores maternos asociados: Unidad de Atención y Protección Materno Infantil de la Clínica Universitaria Bolivariana, Medellín, Colombia / Low birth-weight prevalence and associated maternal factors at the Bolivariana teaching hospital's Maternal and Infant Protection and Attention Unit in Medellín, Colombia

Objetivo: estudiar la prevalencia de bajo peso al nacer (BPN), parto pretérmino (PPT) y restricción de crecimiento intrauterino (RCIU), los factores maternos asociados y su fracción etiológica en la Unidad de Atención y Protección Materno Infantil de la Clínica Universitaria Bolivariana. Metodología: estudio transversal que analizó 2.672 pares madre-hijo registrados por el Sistema Informático Perinatal. La muestra representó 45 por ciento de los partos atendidos en la Unidad Materno Infantil de la Clínica Universitaria Bolivariana durante el 5 de mayo de 2003 y el 30 de marzo de 2006. Resultados: la prevalencia encontrada fue de 17 por ciento para BPN, 21 por ciento para PPT y 12 por ciento para RCIU. El control prenatal inadecuado, el hábito de fumar y la presencia de alguna patología materna durante la gestación presentaron asociación con BPN. Conclusión: la identificación de factores maternos asociados con el BPN servirá para implantar políticas de promoción y prevención específicas para la población en riesgo. Futuros estudios de base poblacional que permitan extrapolar estos resultados deben ser realizados en Colombia.