Amino Acid Transporters Proteins Involved in the Glutamate-Glutamine Cycle and Their Alterations in Murine Models of Alzheimer's Disease.

The brain's ability to integrate external stimuli and generate responses is highly complex. While these mechanisms are not completely understood, current evidence suggests that alterations in cellular metabolism and microenvironment are involved in some dysfunctions as complex as Alzheimer's disease. This pathology courses with defects in the establishment of chemical synapses, which is dependent on the production and supply of neurotransmitters like glutamate and its recycling through the glutamate-glutamine cycle. Alterations in the expression and function of the amino acid transporters proteins involved in this cycle have recently been reported in different stages of Alzheimer's disease. Most of these data come from patients in advanced stages of the disease or post-mortem, due to the ethical and technical limitations of human studies. Therefore, genetically modified mouse models have been an excellent tool to analyze metabolic and even behavioral parameters that are very similar to those that develop in Alzheimer's disease, even at presymptomatic stages. Hence, this paper analyzes the role of glutamate metabolism and its intercellular trafficking in excitatory synapses from different approaches using transgenic mouse models; such an analysis will contribute to our present understanding of AD.

Filtración marginal y contracción en la polimerización en nuevas resinas Bulk Fill: una revisión de la literatura / Marginal leakage and polymerization shrinkage in new Bulk Fill resins: a review of the literature

A pesar de los avances e innovaciones de los materiales dentales, la microfiltración marginal y la contracción durante la polimerización continúan siendo una de las causas principales del fracaso de los tratamientos en odontología restauradora. Un sellado marginal correcto será posible cuando las fuerzas de adhesión superen las fuerzas generadas por la contracción de polimerización y las fuerzas generadas por los cambios dimensionales térmicos posteriores a la polimerización, por lo que investigaciones previas demostraron que estas limitaciones pueden ser superadas con el uso de resinas Bulk Fill como material de relleno de cavidades extensas y profundas de dientes posteriores. Estas resinas. de relleno masivo, están recibiendo atención, principalmente porque se pueden colocar, a diferencia de las resinas convencionales, en incrementos de 4 mm sin afectar la contracción de la polimerización, la adaptación de la cavidad o el grado de conversión. El objetivo de la presente revisión bibliográfica es describir la contracción de polimerización y la consecuente filtración marginal que sufren las resinas compuestas para el sector posterior Bulk Fill con base de datos de la literatura (AU)

Despite advances and innovations in dental materials, marginal microfiltration and shrinkage during polymerization continue to be one of the main causes of treatment failure in Restorative Dentistry. A correct marginal seal will be possible when the adhesion forces exceed the forces generated by polymerization contraction and the forces generated by post-polymerization thermal dimensional changes, for which previous research has shown that these limitations can be overcome with the use of Bulk Fill resins as filling material for large and deep posterior tooth cavities, these massive filling resins are receiving attention mainly because they can be placed, unlike conventional resins, in 4 mm increments without affecting polymerization shrinkage. , cavity adaptation or degree of conversion. The objective of the present bibliographic review is to describe the polymerization contraction and consequent marginal filtration suffered by Composite Resins for the Bulk Fill posterior sector with a literature database (AU)

Effect of an Electronic Order Set on Newborn Hepatitis B Immunization Rates.

Objective: Hepatitis B is an infectious deoxyribonucleic acid virus which can cause significant morbidity and mortality. There is no current definitive treatment, however in the United States immunization is widely available. A paper published by the Advisory Committee on Immunization Practices/Centers for Disease Control (ACIP/CDC) in 2018 made updated recommendations regarding vaccination practices in the United States. The most notable change made was that all healthy newborns weighing ≥2000 g with a negative hepatitis B-status mother should receive hepatitis B immunization within 24 hours of birth. This quality improvement project studied the effect of the electronic medical record newborn admission order set, altered to reflect current societal recommendations, and the resulting newborn hepatitis B immunization rates. Methods: The electronic medical record admission order set was modified to reflect the most recent recommendations made by ACIP/CDC. Hepatitis B immunization rates were then analyzed prior to and following the order set changes. Results: The most significant effect was seen in the overall rate of hepatitis B immunization achieved prior to hospital discharge. In the 12 months before order set modifications were implemented the rate was 9.5%. Following electronic medical record changes it improved to over 90%. In addition, the immunization rate performed within the first 24 hours increased from 74.1% to 91.1%. Finally, these records were made accessible to outpatient providers via a statewide immunization database. Conclusions: This project serves as an example of how modifying order sets can have a dramatic effect on ordering practices and therefore allows for quality improvement.

RON2, a novel gene in Babesia bigemina, contains conserved, immunodominant B-cell epitopes that induce antibodies that block merozoite invasion.

Bovine babesiosis is the most important protozoan disease transmitted by ticks. In Plasmodium falciparum, another Apicomplexa protozoan, the interaction of rhoptry neck protein 2 (RON2) with apical membrane antigen-1 (AMA-1) has been described to have a key role in the invasion process. To date, RON2 has not been described in Babesia bigemina, the causal agent of bovine babesiosis in the Americas. In this work, we found a ron2 gene in the B. bigemina genome. RON2 encodes a protein that is 1351 amino acids long, has an identity of 64% (98% coverage) with RON2 of B. bovis and contains the CLAG domain, a conserved domain in Apicomplexa. B. bigemina ron2 is a single copy gene and it is transcribed and expressed in blood stages as determined by RT-PCR, Western blot, and confocal microscopy. Serum samples from B. bigemina-infected bovines were screened for the presence of RON2-specific antibodies, showing the recognition of conserved B-cell epitopes. Importantly, in vitro neutralization assays showed an inhibitory effect of RON2-specific antibodies on the red blood cell invasion by B. bigemina. Therefore, RON2 is a novel antigen in B. bigemina and contains conserved B-cell epitopes, which induce antibodies that inhibit merozoite invasion.

Revisión de resinas Bulk Fill: estado actual / Revision of Bulk Fill resins: an update

Las restauraciones directas con resinas compuestas han evolucionado en cuanto a la cantidad de carga que poseen ­su formato, composición y distribución­, con el fin mejorar sus propiedades físicas, mecánicas y ópticas para proporcionar mejores resultados estéticos, biológicos y funcionales. En la actualidad el mercado dispone de una resina de relleno único o resinas Bulk Fill, cuya aplicación se realiza en incrementos de 4 mm, acortando el tiempo clínico de trabajo, mediante una técnica simple, rápida y práctica. Sin embargo, se necesitan más estudios clínicos para valorar sus propiedades y su duración en boca y consecuentemente el éxito clínico de la restauración (AU)

Direct restorations with composite resins have evolved in terms of the amount of charge they have, their format, composition and distribution in order to improve their physical, mechanical and optical properties, to provide better aesthetic, biological and functional results. Currently, a single fill resin or Bulk Fill resin is available in the market, which is applied in 4 mm increments, shortening the clinical work time, with a simple, fast and practical technique. However, more clinical studies are needed to assess their properties and their duration in the mouth and consequently the clinical success of the restoration (AU)

Getting the hypertension Dx right: Patient positioning matters.

Taking blood pressure with the patient seated on the edge of an exam table led to misclassification of prehypertension or hypertension in 13.2% of patients.

Glutamine/Glutamate Transporters in Glial Cells: Much More Than Participants of a Metabolic Shuttle.

Glial glutamine and glutamate transporters play an important role in glial/neuronal interactions. An excellent model to establish the role of these membrane proteins is the cerebellum. The most abundant glutamatergic synapse in the central nervous system is present in the molecular layer of the cerebellar cortex, and it is entirely wrapped by Bergmann glial cells. The recycling of glutamate involves glutamate and glutamine transporters enriched in these radial glial processes. The functional properties of amino acid glial transporters allow, in an activity-dependent manner, the conformation of protein complexes important for the adequate support of glutamatergic neurotransmission. A detailed description of the most important features of glial glutamate and glutamine transporters follows, and a working model of the molecular mechanisms by which these glutamate and glutamine binding proteins interact, and by these means might modulate cerebellar glutamatergic transactions, is presented.

Expression of the System N transporter (SNAT5/SN2) during development indicates its plausible role in glutamatergic neurotransmission.

Solute neutral amino acid transporter 5 (SNAT5/SN2) is a member of the System N family, expressed in glial cells in the adult brain, able to transport glutamine, histidine or glycine among other substrates. Its tight association with synapses and its electroneutral mode of operation that allows the bidirectional movement of substrates, supports the idea that this transporter participates in the function of the glutamine-glutamate cycle between neurons and glia. Moreover, SNAT5/SN2 might contribute to the regulation of glycine concentration in glutamatergic synapses and, therefore, to the functioning of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors. Ontogenic maturation of these synapses occurs postnatally through the coordinate expression of a large number of receptors, transporters, structural and regulatory proteins that ensure the correct operation of the excitatory pathways in the central nervous system. Since the temporal pattern of expression of SNAT5/SN2 is unknown, we analyzed it by immunoblot and immunohistochemical techniques. Results indicate that the expression of SNAT5/SN2 is triggered between the second and third postnatal week in the cerebral cortex, in parallel to the expression of the vesicular glutamate transporter vGLUT1 and the glial glutamate transporter GLT1/EAAT2. In the cerebellum, this process occurs about one week later than in the cerebral cortex. Immunohistochemical staining of cortical sections shows that from postnatal day 14 to adulthood the transporter was expressed exclusively in glial cells. Our results are consistent with the idea that SNAT5/SN2 expression is coordinated with that of other proteins necessary for the operation of glutamatergic synapses and reinforce the existence of a regulatory cross-talk between neurons and glia that orchestrates the building up of these synapses.

GLAST/EAAT1-induced glutamine release via SNAT3 in Bergmann glial cells: evidence of a functional and physical coupling.

Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodium-dependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium-dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so-called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time-dependent Na⁺-dependent glutamate/aspartate transporter/EAAT1-induced System N-mediated glutamine release could be demonstrated. Furthermore, D-aspartate, a specific glutamate transporter ligand, was capable of enhancing the co-immunoprecipitation of Na⁺-dependent glutamate/aspartate transporter and Na⁺-dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron-derived glutamate through their contribution to the neurotransmitter turnover.

Serotonin receptors in hippocampus.

Serotonin is an ancient molecular signal and a recognized neurotransmitter brainwide distributed with particular presence in hippocampus. Almost all serotonin receptor subtypes are expressed in hippocampus, which implicates an intricate modulating system, considering that they can be localized as autosynaptic, presynaptic, and postsynaptic receptors, even colocalized within the same cell and being target of homo- and heterodimerization. Neurons and glia, including immune cells, integrate a functional network that uses several serotonin receptors to regulate their roles in this particular part of the limbic system.

Distribution of the purinegic receptors P2X(4) and P2X(6) during rat gut development.

The purinergic receptors P2X(4) and P2X(6) are ion channels activated by ATP. These receptors are present in the gastrointestinal tract, and they are involved in synaptic transmission, taste sensation, and pain, among other functions. In this work, we studied the distribution of P2X(4) and P2X(6) receptors in proximal and distal regions of the gut newborn and adult rats. Using immunohistochemistry, purinergic receptors were found in gut epithelial cells and capillary vessels. In both proximal and distal regions of newborn rats, we observed P2X(4) signal in epithelial cells, whereas P2X(6) was present in capillary vessels in the proximal region and to a lesser extent in the distal region. In both regions of adult gut, we observed P2X(4) and P2X(6) immunostain in the capillary vessels. Semi-quantification indicated a significant difference in the amount of P2X(4) between proximal regions, whereas the P2X(6) content of both newborn regions differed from that in adult proximal gut. We conclude that P2X(4) and P2X(6) purinoreceptors are present in the gut from birth and that they are differentially distributed among regions.

Expression of the SNAT2 amino acid transporter during the development of rat cerebral cortex.

The sodium-coupled neutral amino acid transporter 2 (SNAT2) is a protein that is expressed ubiquitously in mammalian tissues and that displays Na(+), voltage and pH dependent activity. This transporter mediates the passage of small zwitterionic amino acids across the cell membrane and regulates the cell homeostasis and its volume. We have examined the expression of SNAT2 mRNA and protein during the development of the rat cerebral cortex, from gestation through the postnatal stages to adulthood. Our data reveal that SNAT2 mRNA and protein expression is higher during embryogenesis, while it subsequently diminishes during postnatal development. Moreover, during embryonic period SNAT2 colocalizes with the radial glial cells marker GLAST, while in postnatal period it is mainly detected in neuronal dendrites. These findings suggest a relevant role for amino acid transport through SNAT2 in the developing embryonic brain.

Serotonin receptor 5-HT5A in rat hippocampus decrease by leptin treatment.

5-Hydroxytryptamine (5-HT) is involved in a variety of different physiological processes and behaviors through the activation of equally diverse receptors subtypes. In this work we studied the changes on the expression of 5-HT(5A) receptors in rat hippocampus induced by leptin, an adipocyte-derived hormone that has been reported to participate in the modulation of food intake and in adult hippocampal neurogenesis. To study the effect of leptin on the 5-HT(5A) receptor gene expression a qRT-PCR was used and the distribution of those receptors in the hippocampus was visualized by immunohistochemistry. Rats were separated in four groups: control (untreated rats), leptin-treated, serotonin-treated and leptin+serotonin treated. The results showed that even though the 5-HT(5A) gene expression did not change in the hippocampus of any of the treated groups, in the rats treated with leptin and serotonin, the specific immunostaining for the 5-HT(5A) serotonin receptor decreased significantly in the dentate gyrus.

Characterization of differentiated quiescent and nonquiescent cells in yeast stationary-phase cultures.

Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and nonquiescent (NQ) fractions before entering stationary phase. To understand this differentiation, Q and NQ cells from 101 deletion-mutant strains were tested for viability and reproductive capacity. Eleven mutants that affected one or both phenotypes in Q or NQ fractions were identified. NQ fractions exhibit a high level of petite colonies, and nine mutants affecting this phenotype were identified. Microarray analysis revealed >1300 mRNAs distinguished Q from NQ fractions. Q cell-specific mRNAs encode proteins involved in membrane maintenance, oxidative stress response, and signal transduction. NQ-cell mRNAs, consistent with apoptosis in these cells, encode proteins involved in Ty-element transposition and DNA recombination. More than 2000 protease-released mRNAs were identified only in Q cells, consistent with these cells being physiologically poised to respond to environmental changes. Our results indicate that Q and NQ cells differentiate significantly, with Q cells providing genomic stability and NQ cells providing nutrients to Q cells and a regular source of genetic diversity through mutation and transposition. These studies are relevant to chronological aging, cell cycle, and genome evolution, and they provide insight into complex responses that even simple organisms have to starvation.

Mature-onset obesity and insulin resistance in mice deficient in the signaling adapter p62.

Signaling cascades that control adipogenesis are essential in the regulation of body weight and obesity. The adaptor p62 controls pathways that modulate cell differentiation. We report here that p62(-/-) mice develop mature-onset obesity, leptin resistance, as well as impaired glucose and insulin intolerance. The metabolic rate was significantly reduced in p62(-/-) nonobese mice, which displayed increased mRNA levels of PPAR-gamma and reduced levels of UCP-1 in adipose tissue. Basal activity of ERK was enhanced in fat from nonobese mutant mice. Embryo fibroblasts from p62(-/-) mice differentiated better than the wild-type controls into adipocytes, which was abrogated by pharmacological inhibition of the ERK pathway. p62 is induced during adipocyte differentiation and inhibits ERK activation by direct interaction. We propose that p62 normally antagonizes basal ERK activity and adipocyte differentiation and that its loss leads to the hyperactivation of ERK that favors adipogenesis and obesity.

An automated, pressure-driven sampling device for harvesting from liquid cultures for genomic and biochemical analyses.

Here we describe an automated, pressure-driven, sampling device for harvesting 10 to 30 ml samples, in replicate, with intervals as short as 10 s. Correlation between biological replicate time courses measured by microarrays was extremely high. The sampler enables sampling at intervals within the range of many important biological processes.

Crosstalk between PKCzeta and the IL4/Stat6 pathway during T-cell-mediated hepatitis.

PKCzeta is required for nuclear factor kappa-B (NF-kappaB) activation in several cell systems. NF-kappaB is a suppressor of liver apoptosis during development and in concanavalin A (ConA)-induced T-cell-mediated hepatitis. Here we show that PKCzeta-/- mice display inhibited ConA-induced NF-kappaB activation and reduced damage in liver. As the IL-4/Stat6 pathway is necessary for ConA-induced hepatitis, we addressed here the potential role of PKCzeta in this cascade. Interestingly, the loss of PKCzeta severely attenuated serum IL-5 and liver eotaxin-1 levels, two critical mediators of liver damage. Stat6 tyrosine phosphorylation and Jak1 activation were ablated in the liver of ConA-injected PKCzeta-/- mice and in IL-4-stimulated PKCzeta-/- fibroblasts. PKCzeta interacts with and phosphorylates Jak1 and PKCzeta activity is required for Jak1 function. In contrast, Par-4-/- mice have increased sensitivity to ConA-induced liver damage and IL-4 signaling. This unveils a novel and critical involvement of PKCzeta in the IL-4/Stat6 signaling pathway in vitro and in vivo.

Genomic analysis of stationary-phase and exit in Saccharomyces cerevisiae: gene expression and identification of novel essential genes.

Most cells on earth exist in a quiescent state. In yeast, quiescence is induced by carbon starvation, and exit occurs when a carbon source becomes available. To understand how cells survive in, and exit from this state, mRNA abundance was examined using oligonucleotide-based microarrays and quantitative reverse transcription-polymerase chain reaction. Cells in stationary-phase cultures exhibited a coordinated response within 5-10 min of refeeding. Levels of >1800 mRNAs increased dramatically (>or=64-fold), and a smaller group of stationary-phase mRNAs decreased in abundance. Motif analysis of sequences upstream of genes clustered by VxInsight identified an overrepresentation of Rap1p and BUF (RPA) binding sites in genes whose mRNA levels rapidly increased during exit. Examination of 95 strains carrying deletions in stationary-phase genes induced identified 32 genes essential for survival in stationary-phase at 37 degrees C. Analysis of these genes suggests that mitochondrial function is critical for entry into stationary-phase and that posttranslational modifications and protection from oxidative stress become important later. The phylogenetic conservation of stationary-phase genes, and our findings that two-thirds of the essential stationary-phase genes have human homologues and of these, many have human homologues that are disease related, demonstrate that yeast is a bona fide model system for studying the quiescent state of eukaryotic cells.

Identification and removal of contaminating fluorescence from commercial and in-house printed DNA microarrays.

Microarray analysis is a critically important technology for genome-enabled biology, therefore it is essential that the data obtained be reliable. Current software and normalization techniques for microarray analysis rely on the assumption that fluorescent background within spots is essentially the same throughout the glass slide and can be measured by fluorescence surrounding the spots. This assumption is not valid if background fluorescence is spot-localized. Inaccurate estimates of background fluorescence under the spot create a source of error, especially for low expressed genes. We have identified spot-localized, contaminating fluorescence in the Cy3 channel on several commercial and in-house printed microarray slides. We determined through mock hybridizations (without labeled target) that pre-hybridization scans could not be used to predict the contribution of this contaminating fluorescence after hybridization because the change in spot-to-spot fluorescence after hybridization was too variable. Two solutions to this problem were identified. First, allowing 4 h of exposure to air prior to printing on to Corning UltraGAPS slides significantly reduced contaminating fluorescence intensities to approximately the value of the surrounding glass. Alternatively, application of a novel, hyperspectral imaging scanner and multivariate curve resolution algorithms, allowed the spectral contributions of Cy3 signal, glass, and contaminating fluorescence to be distinguished and quantified after hybridization.

Citogenética en enfermedad de Hodgkin (EH) y en Linfoma no Hodgkin (LNH) / Cytogenetics in Hodgkin Disease and no Hodgkin lymphoma

En este trabajo, 34 pacientes con Linfoma (L) fueron estudiados prospectivamente, para determinar el valor diagnóstico de las alteraciones citogenéticas (AC) observadas en linfocitos en sangre periférica, médula ósea, ganglio linfático y líquido ascítico. La mayoría de los 11 pacientes con EH eran adolescentes o adultos jóvenes en estadio avanzado de la enfermedad. Los 23 pacientes con LNH eran mayores de 16 años, con una gran variedad de formas histológicas y se encontraban en estadio IV de la enfermedad. Más del 80 por ciento de los pacientes mostraron AC; las aneuploidías, translocaciones, delecciones y otros cambios estructurales cromosómicos; 3 pacientes con EH y 2 con LNH mostraron cromosoma Philadhelphia positivo, pareciendo tener este hallazgo un significado pronóstico sombrío en este último grupo. En los pacientes que fallecieron se observó en EH en forma simultánea, monosomía 22 y 7, y en LNH alteraciones estructurales y monosomía 22