Protection against hemorrhagic shock in mice genetically deficient in poly(ADP-ribose)polymerase

Hemorrhagic shock (HS) and resuscitation leads to widespread production of oxidant species. Activation of the enzyme poly(ADP-ribose) polymerase (PARP) has been shown to contribute to cell necrosis and organ failure in various disease conditions associated with oxidative stress. We tested the hypothesis whether PARP activation plays a role in the multiple organ dysfunction complicating HS and resuscitation in a murine model of HS and resuscitation by using mice genetically deficient in PARP (PARP−/−) and their wild-type littermates (PARP+/+). Animals were bled to a mean blood pressure of 45 mmHg (1 mmHg = 133 Pa) and resuscitated after 45 min with isotonic saline (2× volume of shed blood). There was a massive activation of PARP, detected by poly(ADP-ribose) immunohistochemistry, which localized to the areas of the most severe intestinal injury, i.e., the necrotic epithelial cells at the tip of the intestinal villi, and colocalized with tyrosine nitration, an index of peroxynitrite generation. Intestinal PARP activation resulted in gut hyperpermeability, which developed in PARP+/+ but not PARP−/− mice. PARP−/− mice were also protected from the rapid decrease in blood pressure after resuscitation and showed an increased survival time, as well as reduced lung neutrophil sequestration. The beneficial effects of PARP suppression were not related to a modulation of the NO pathway nor to a modulation of signaling through IL-6, which similarly increased in both PARP+/+ and PARP−/− mice exposed to HS. We propose that PARP activation and associated cell injury (necrosis) plays a crucial role in the intestinal injury, cardiovascular failure, and multiple organ damage associated with resuscitated HS.

Temporal dynamics of verbal object comprehension

Knowledge of the stage composition and the temporal dynamics of human cognitive operations is critical for building theories of higher mental activity. This information has been difficult to acquire, even with different combinations of techniques such as refined behavioral testing, electrical recording/interference, and metabolic imaging studies. Verbal object comprehension was studied herein in a single individual, by using three tasks (object naming, auditory word comprehension, and visual word comprehension), two languages (English and Farsi), and four techniques (stimulus manipulation, direct cortical electrical interference, electrocorticography, and a variation of the technique of direct cortical electrical interference to produce time-delimited effects, called timeslicing), in a subject in whom indwelling subdural electrode arrays had been placed for clinical purposes. Electrical interference at a pair of electrodes on the left lateral occipitotemporal gyrus interfered with naming in both languages and with comprehension in the language tested (English). The naming and comprehension deficit resulted from interference with processing of verbal object meaning. Electrocorticography indices of cortical activation at this site during naming started 250–300 msec after visual stimulus presentation. By using the timeslicing technique, which varies the onset of electrical interference relative to the behavioral task, we found that completion of processing for verbal object meaning varied from 450 to 750 msec after current onset. This variability was found to be a function of the subject’s familiarity with the objects.

Mental chronometry using latency-resolved functional MRI

Vascular responses to neural activity are exploited as the basis of a number of brain imaging techniques. The vascular response is thought to be too slow to resolve the temporal sequence of events involved in cognitive tasks, and hence, imaging studies of mental chronometry have relied on techniques such as the evoked potential. Using rapid functional MRI (fMRI) of single trials of two simple behavioral tasks, we demonstrate that while the microvascular response to the onset of neural activity is delayed consistently by several seconds, the relative timing between the onset of the fMRI responses in different brain areas appears preserved. We examined a number of parameters that characterize the fMRI response and determined that its onset time is best defined by the inflection point from the resting baseline. We have found that fMRI onset latencies determined in this manner correlate well with independently measurable parameters of the tasks such as reaction time or stimulus presentation time and can be used to determine the origin of processing delays during cognitive or perceptual tasks with a temporal accuracy of tens of milliseconds and spatial resolution of millimeters.

A New Model for Nuclear Envelope BreakdownV⃞

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia

Neuropathological and brain imaging studies suggest that schizophrenia may result from neurodevelopmental defects. Cytoarchitectural studies indicate cellular abnormalities suggestive of a disruption in neuronal connectivity in schizophrenia, particularly in the dorsolateral prefrontal cortex. Yet, the molecular mechanisms underlying these findings remain unclear. To identify molecular substrates associated with schizophrenia, DNA microarray analysis was used to assay gene expression levels in postmortem dorsolateral prefrontal cortex of schizophrenic and control patients. Genes determined to have altered expression levels in schizophrenics relative to controls are involved in a number of biological processes, including synaptic plasticity, neuronal development, neurotransmission, and signal transduction. Most notable was the differential expression of myelination-related genes suggesting a disruption in oligodendrocyte function in schizophrenia.

Amyloid beta-peptide disrupts carbachol-induced muscarinic cholinergic signal transduction in cortical neurons.

Cholinergic pathways serve important functions in learning and memory processes, and deficits in cholinergic transmission occur in Alzheimer disease (AD). A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C, resulting in the liberation of inositol trisphosphate and Ca2+ release from intracellular stores. We now report that amyloid beta-peptide (Abeta), which forms plaques in the brain in AD, impairs muscarinic receptor activation of G proteins in cultured rat cortical neurons. Exposure of rodent fetal cortical neurons to Abeta25-35 and Abeta1-40 resulted in a concentration and time-dependent attenuation of carbachol-induced GTPase activity without affecting muscarinic receptor ligand binding parameters. Downstream events in the signal transduction cascade were similarly attenuated by Abeta. Carbachol-induced accumulation of inositol phosphates (IP, IP2, IP3, and IP4) was decreased and calcium imaging studies revealed that carbachol-induced release of calcium was severely impaired in neurons pretreated with Abeta. Muscarinic cholinergic signal transduction was disrupted with subtoxic levels of exposure to AP. The effects of Abeta on carbachol-induced GTPase activity and calcium release were attenuated by antioxidants, implicating free radicals in the mechanism whereby Abeta induced uncoupling of muscarinic receptors. These data demonstrate that Abeta disrupts muscarinic receptor coupling to G proteins that mediate induction of phosphoinositide accumulation and calcium release, findings that implicate Abeta in the impairment of cholinergic transmission that occurs in AD.

Morphology of high-luminosity compact radio sources.

High-dynamic range imaging and monitoring with very-long-baseline interferometry reveal a rich morphology of luminous flat-spectrum radio sources. One-sided core-jet structures abound, and superluminal motion is frequently measured. In a few cases, both distinct moving features and diffuse underlying jet emission can be detected. Superluminal motion seen in such sources is typically complex, on curved trajectories or ridge lines, and with variable component velocities, including stationary features. The curved trajectories seen can be modeled by helical motion within the underlying jet flow. The very-long-baseline interferometry properties of the superluminal features in the jet of 3C 345 and other similar sources can be explained by models invoking the emission from shocks, at least within the vicinity of the compact core. Inverse-Compton calculations, constrained by x-ray observations, yield realistic estimates for the physical conditions in the parsec-scale jet. There is evidence for a transition region in this source beyond which other factors (e.g., plasma interactions and nonsynchrotron radiation processes) may become prominent. Multifrequency and polarization imaging (especially at high frequencies) are emerging as critical tools in testing model predictions.

An apical permeability barrier to NH3/NH4+ in isolated, perfused colonic crypts.

Fermentation of nonabsorbed nutrients in the colon generates high concentrations of NH3/NH4+ in the colonic lumen. NH3 is a small, lipophilic neutral weak base that readily permeates almost all cell membranes, whereas its conjugate weak acid NH4+ generally crosses membranes much more slowly. It is not known how colonocytes maintain intracellular pH in the unusual acid-base environment of the colon, where permeant acid-base products of fermentation exist in high concentration. To address this issue, we hand dissected and perfused single, isolated crypts from rabbit proximal colon, adapting techniques from renal-tubule microperfusion. Crypt perfusion permits control of solutions at the apical (luminal) and basolateral (serosal) surfaces of crypt cells. We assessed apical- vs. basolateral-membrane transport of NH3/NH4+ by using fluorescent dyes and digital imaging to monitor intracellular pH of microvacuolated crypt cells as well as luminal pH. We found that, although the basolateral membranes have normal NH3/NH4+ permeability properties, there is no evidence for transport of either NH3 or NH4+ across the apical borders of these crypt cells. Disaggregating luminal mucus did not increase the transport of NH3/NH4+ across the apical border. We conclude that, compared to the basolateral membrane, the apical border of crypt colonocytes has a very low permeability-area product for NH3/NH4+. This barrier may represent an important adaptation for the survival of crypt cells in the environment of the colon.

A mouse model for beta 0-thalassemia.

We have used a "plug and socket" targeting technique to generate a mouse model of beta 0-thalassemia in which both the b1 and b2 adult globin genes have been deleted. Mice homozygous for this deletion (Hbbth-3/Hbbth-3) die perinatally, similar to the most severe form of Cooley anemia in humans. Mice heterozygous for the deletion appear normal, but their hematologic indices show characteristics typical of severe thalassemia, including dramatically decreased hematocrit, hemoglobin, red blood cell counts, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, as well as dramatically increased reticulocyte counts, serum bilirubin concentrations, and red cell distribution widths. Tissue and organ damage typical of beta-thalassemia, such as bone deformities and splenic enlargement due to increased hematopoiesis, are also seen in the heterozygous animals, as is spontaneous iron overload in the spleen, liver, and kidneys. The mice homozygous for the b1 and b2 deletions should be of great value in developing therapies for the treatment of thalassemias in utero. The heterozygous animals will be useful for studying the pathophysiology of thalassemias and have the potential of generating a model of sickle cell anemia when mated with appropriate transgenic animals.

High-affinity binding sites for histone H1 in plasmid DNA.

The interaction of histone H1 isolated from chicken erythrocytes with restriction fragments from plasmids pBR322 and pUC19 was studied by gel electrophoresis. Certain restriction fragments exhibited unusually high affinity for the histone, forming high molecular mass complexes at protein to DNA ratios at which the other fragments did not show evidence for binding. The highly preferred fragments are intrinsically curved, as judged by their electrophoretic mobility in polyacrylamide gels, by computer modeling, and by imaging with scanning force microscopy. However, control experiments with either curved portions of the same fragments or highly curved kinetoplast DNA fragments showed that the presence of curvature alone was not sufficient for preferential binding. By using various restriction fragments centered around the highly preferred sequence, it was found that the high-affinity binding required in addition the presence of specific sequences on both sides of the region of curvature. Thus, both curvature and the presence of specific sites seem to be required to generate high affinity.

Investigação de aquífero fraturado para entendimento de fluxo e transporte de contaminantes clorados: estudo de caso em Valinhos, SP

Casos de contaminação de aquíferos fraturados são bastante complexos, tendo em vista a heterogeneidade das redes de fraturas, e no geral, sua investigação demanda a utilização de técnicas pouco usuais, como por exemplo o imageamento acústico e a perfilagem de velocidade de fluxo de água. Na área de estudo, localizada em Valinhos/SP, o uso inadequado de solventes organoclorados no passado ocasionou a contaminação do aquífero raso em duas áreas, e o aparecimento de concentrações no aquifero profundo levaram a condução do atual trabalho, que teve como principal objetivo a elaboração de um modelo conceitual de fluxo de água e transporte de contaminantes no aquífero cristalino. Previamente à investigação do aquífero fraturado, foi realizada uma análise de trabalhos existentes, incluindo a interpretação de lineamentos, levantamentos geológicos além de perfilagens geofísicas de superfície. Em cada área investigada, foi realizada a perfuração de um poço profundo e aplicadas as técnicas de perfilagens de raios gama, cáliper, flowmeter, imageamento acústico, além da filmagem do poço e realização de ensaios hidráulicos nos dois pontos perfurados. Para caracterização química do aquífero fraturado, foram realizadas coletas de água subterrânea em intervalos selecionados com a utilização de obturadores pneumáticos. As cargas hidráulicas medidas durante a amostragem também auxiliaram no entendimento da direção do fluxo de água. O aquífero cristalino é formado por rochas gnáissicas e se encontra bastante fraturado e intemperizado, principalmente na porção superficial da rocha (até aproximadamente 65,0 m) onde as maiores velocidades de fluxo de água também foram observadas. A rocha sã possui uma menor densidade de fraturas e predominância de minerais mais claros. As fraturas de baixo a médio angulo de mergulho (Grupo 1) são as mais frequentes em ambas as perfurações e possuem direção principal N-S a NE-SW. São observadas, no geral, exercendo grande influência sobre o fluxo de água, principalmente na porção alterada do gnaisse. Fraturas com ângulo elevado de mergulho, classificadas como Grupo 2 (paralelas à foliação) e Grupo 3 (direção NW à W), são também observadas ao longo de toda a perfuração estabelecendo a conexão hidráulica entre as fraturas do Grupo 1. Em menor proporção, são ainda verificadas fraturas com ângulos de mergulho >40 ° pertencente aos Grupos 4 (NE-SW), 5 (E-W), 6 (NW-SE) e 7 (E-W). O fluxo de água subterrânea se mostrou descendente na porção superior da rocha alterada e ascendente na porção mais profunda, possivelmente direcionando a água subterrânea para a região de transição da rocha mais alterada para a rocha sã (entre 61 a 65 m de profundidade). Apesar do fluxo ascendente em profundidade, o bombeamento de poços tubulares existentes no entorno ao longo dos anos, favoreceu a migração dos contaminantes para porções mais profundas. Os contaminantes observados no poço tubular P6 possuem maior semelhança com os contaminantes observados na Área 2, e ambos estão localizados entre lineamentos NW-SE, indicando uma possível influência dos lineamentos no controle sobre o fluxo de água. No entanto, para entendimento do transporte dos contaminantes em área, é necessário um adensamento da rede de monitoramento, levando em consideração a heterogeneidade do meio e as incertezas relacionadas à extrapolação dos dados para áreas não investigadas.

Retrieval of phenological stages of onion fields during the first year of growth by means of C-band polarimetric SAR measurements

The phenological stages of onion fields in the first year of growth are estimated using polarimetric observables and single-polarization intensity channels. Experiments are undertaken on a time series of RADARSAT-2 C-band full-polarimetric synthetic aperture radar (SAR) images collected in 2009 over the Barrax region, Spain, where ground truth information about onion growth stages is provided by the European Space Agency (ESA)-funded agricultural bio/geophysical retrieval from frequent repeat pass SAR and optical imaging (AgriSAR) field campaign conducted in that area. The experimental results demonstrate that polarimetric entropy or copolar coherence when used jointly with the cross-polarized intensity allows unambiguously distinguishing three phenological intervals.

Modifications post-traductionnelles des canaux calciques cardiaques de type L : identification des résidus asparagine qui participent à la glycosylation de la sous-unité auxiliaire CaVα2δ1

Les canaux calciques de type L CaV1.2 sont principalement responsables de l’entrée des ions calcium pendant la phase plateau du potentiel d’action des cardiomyocytes ventriculaires. Cet influx calcique est requis pour initier la contraction du muscle cardiaque. Le canal CaV1.2 est un complexe oligomérique qui est composé de la sous-unité principale CaVα1 et des sous-unités auxiliaires CaVβ et CaVα2δ1. CaVβ joue un rôle déterminant dans l’adressage membranaire de la sous-unité CaVα1. CaVα2δ1 stabilise l’état ouvert du canal mais le mécanisme moléculaire responsable de cette modulation n’a pas été encore identifié. Nous avons récemment montré que cette modulation requiert une expression membranaire significative de CaVα2δ1 (Bourdin et al. 2015). CaVα2δ1 est une glycoprotéine qui possède 16 sites potentiels de glycosylation de type N. Nous avons donc évalué le rôle de la glycosylation de type-N dans l’adressage membranaire et la stabilité de CaVα2δ1. Nous avons d’abord confirmé que la protéine CaVα2δ1 recombinante, telle la protéine endogène, est significativement glycosylée puisque le traitement à la PNGase F se traduit par une diminution de 50 kDa de sa masse moléculaire, ce qui est compatible avec la présence de 16 sites Asn. Il s’est avéré par ailleurs que la mutation simultanée de 6/16 sites (6xNQ) est suffisante pour 1) réduire significativement la densité de surface de! CaVα2δ1 telle que mesurée par cytométrie en flux et par imagerie confocale 2) accélérer les cinétiques de dégradation telle qu’estimée après arrêt de la synthèse protéique et 3) diminuer la modulation fonctionnelle des courants générés par CaV1.2 telle qu’évaluée par la méthode du « patch-clamp ». Les effets les plus importants ont toutefois été obtenus avec les mutants N663Q, et les doubles mutants N348Q/N468Q, N348Q/N812Q, N468Q/N812Q. Ensemble, ces résultats montrent que Asn663 et à un moindre degré Asn348, Asn468 et Asn812 contribuent à la biogenèse et la stabilité de CaVα2δ1 et confirment que la glycosylation de type N de CaVα2δ1 est nécessaire à la fonction du canal calcique cardiaque de type L.

Lethal chondrodysplasia in a family of Holstein cattle is associated with a de novo splice site variant of COL2A1

Background Lethal chondrodysplasia (bulldog syndrome) is a well-known congenital syndrome in cattle and occurs sporadically in many breeds. In 2015, it was noticed that about 12 % of the offspring of the phenotypically normal Danish Holstein sire VH Cadiz Captivo showed chondrodysplasia resembling previously reported bulldog calves. Pedigree analysis of affected calves did not display obvious inbreeding to a common ancestor, suggesting the causative allele was not a rare recessive. The normal phenotype of the sire suggested a dominant inheritance with incomplete penetrance or a mosaic mutation. Results Three malformed calves were examined by necropsy, histopathology, radiology, and computed tomography scanning. These calves were morphologically similar and displayed severe disproportionate dwarfism and reduced body weight. The syndrome was characterized by shortening and compression of the body due to reduced length of the spine and the long bones of the limbs. The vicerocranium had severe dysplasia and palatoschisis. The bones had small irregular diaphyses and enlarged epiphyses consisting only of chondroid tissue. The sire and a total of four affected half-sib offspring and their dams were genotyped with the BovineHD SNP array to map the defect in the genome. Significant genetic linkage was obtained for several regions of the bovine genome including chromosome 5 where whole genome sequencing of an affected calf revealed a COL2A1 point mutation (g.32473300 G > A). This private sequence variant was predicted to affect splicing as it altered the conserved splice donor sequence GT at the 5’-end of COL2A1 intron 36, which was changed to AT. All five available cases carried the mutant allele in heterozygous state and all five dams were homozygous wild type. The sire VH Cadiz Captivo was shown to be a gonadal and somatic mosaic as assessed by the presence of the mutant allele at levels of about 5 % in peripheral blood and 15 % in semen. Conclusions The phenotypic and genetic findings are comparable to a previously reported COL2A1 missense mutation underlying lethal chondrodysplasia in the offspring of a mosaic French Holstein sire (Igale Masc). The identified independent spontaneous splice site variant in COL2A1 most likely caused chondrodysplasia and must have occurred during the early foetal development of the sire. This study provides a first example of a dominant COL2A1 splice site variant as candidate causal mutation of a severe lethal chondrodysplasia phenotype. Germline mosaicism is a relatively frequent mechanism in the origin of genetic disorders and explains the prevalence of a certain fraction of affected offspring. Paternal dominant de novo mutations are a risk in cattle breeding, especially because the ratio of defective offspring may be very high and be associated with significant animal welfare problems.

Plasma Heme Oxygenase-1 in Patients Resuscitated from out-of-Hospital Cardiac Arrest

Heme oxygenase-1 (HO-1) is an enzyme induced by hypoxia and reperfusion injury, and is associated with organ dysfunction in critically ill patients. Patients resuscitated from out-of-hospital cardiac arrest (OHCA) are subjected to hypoxemia, brain injury, and organ dysfunction. Accordingly, we studied HO-1 among these patients. A total of 143 OHCA patients resuscitated from a shockable initial rhythm and admitted to an ICU were included, with plasma HO-1 measured at ICU admission and at 24 h. We analyzed the associations between plasma HO-1 and time to return of spontaneous circulation (ROSC), 90-day mortality, and 12-month Cerebral Performance Category (CPC). HO-1 plasma concentrations were higher after OHCA compared with controls. HO-1 concentrations at admission and on day 1 associated with ROSC (P = 0.002 to P = 0.003). Admission and day 1 HO-1 plasma concentrations were higher in 90-day non-survivors than in survivors (P = 0.017, 0.026). In addition, poor neurological outcome (CPC 3-5) was associated with higher HO-1 plasma levels at admission (P = 0.024). Admission plasma HO-1 levels had an AUC of 0.623 to predict 90-day mortality and an AUC of 0.611 to predict CPC 3 to 5. In conclusion, we found that higher HO-1 plasma levels are associated with longer ROSC and poor long-term outcome.