Tcf4 dysfunction alters dorsal and ventral cortical neurogenesis in Pitt-Hopkins syndrome mouse model showing sexual dimorphism.

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by haploinsufficiency of transcription factor 4 (TCF4). In this work, we focused on the cerebral cortex and investigated in detail the progenitor cell dynamics and the outcome of neurogenesis in a PTHS mouse model. Labeling and quantification of progenitors and newly generated neurons at various time points during embryonic development revealed alterations affecting the dynamic of cortical progenitors since the earliest stages of cortex formation in PTHS mice. Consequently, establishment of neuronal populations and layering of the cortex were found to be altered in heterozygotes subjects at birth. Interestingly, defective layering process of pyramidal neurons was partially rescued by reintroducing TCF4 expression using focal in utero electroporation in the cerebral cortex. Coincidentally with a defective dorsal neurogenesis, we found that ventral generation of interneurons was also defective in this model, which may lead to an excitation/inhibition imbalance in PTHS. Overall, sex-dependent differences were detected with more marked effects evidenced in males compared with females. All of this contributes to expand our understanding of PTHS, paralleling the advances of research in autism spectrum disorder and further validating the PTHS mouse model as an important tool to advance preclinical studies.

Degenerative Cervical Myelopathy induces sex-specific dysbiosis in mice.

Degenerative Cervical Myelopathy (DCM) is the most common cause of spinal cord impairment in elderly populations. It describes a spectrum of disorders that cause progressive spinal cord compression, neurological impairment, loss of bladder and bowel functions, and gastrointestinal dysfunction. The gut microbiota has been recognized as an environmental factor that can modulate both the function of the central nervous system and the immune response through the microbiota-gut-brain axis. Changes in gut microbiota composition or microbiota-producing factors have been linked to the progression and development of several pathologies. However, little is known about the potential role of the gut microbiota in the pathobiology of DCM. Here, DCM was induced in C57BL/6 mice by implanting an aromatic polyether material underneath the C5-6 laminae. The extent of DCM-induced changes in microbiota composition was assessed by 16S rRNA sequencing of the fecal samples. The immune cell composition was assessed using flow cytometry. To date, several bacterial members have been identified using BLAST against the largest collection of metagenome-derived genomes from the mouse gut. In both, female and males DCM caused gut dysbiosis compared to the sham group. However, dysbiosis was more pronounced in males than in females, and several bacterial members of the families Lachnospiraceae and Muribaculaceae were significantly altered in the DCM group. These changes were also associated with altered microbe-derived metabolic changes in propionate-, butyrate-, and lactate-producing bacterial members. Our results demonstrate that DCM causes dynamic changes over time in the gut microbiota, reducing the abundance of butyrate-producing bacteria, and lactate-producing bacteria to a lesser extent. Genome-scale metabolic modeling using gapseq successfully identified pyruvate-to-butanoate and pyruvate-to-propionate reactions involving genes such as Buk and ACH1, respectively. These results provide a better understanding of the sex-specific molecular effects of changes in the gut microbiota on DCM pathobiology.

Impaired communication at the neuromotor axis during Degenerative Cervical Myelopathy.

Degenerative Cervical Myelopathy (DCM) is a progressive neurological condition characterized by structural alterations in the cervical spine, resulting in compression of the spinal cord. While clinical manifestations of DCM are well-documented, numerous unanswered questions persist at the molecular and cellular levels. In this study, we sought to investigate the neuromotor axis during DCM. We use a clinically relevant mouse model, where after 3 months of DCM induction, the sensorimotor tests revealed a significant reduction in both locomotor activity and muscle strength compared to the control group. Immunohistochemical analyses showed alterations in the gross anatomy of the cervical spinal cord segment after DCM. These changes were concomitant with the loss of motoneurons and a decrease in the number of excitatory synaptic inputs within the spinal cord. Additionally, the DCM group exhibited a reduction in the endplate surface, which correlated with diminished presynaptic axon endings in the supraspinous muscles. Furthermore, the biceps brachii (BB) muscle exhibited signs of atrophy and impaired regenerative capacity, which inversely correlated with the transversal area of remnants of muscle fibers. Additionally, metabolic assessments in BB muscle indicated an increased proportion of oxidative skeletal muscle fibers. In line with the link between neuromotor disorders and gut alterations, DCM mice displayed smaller mucin granules in the mucosa layer without damage to the epithelial barrier in the colon. Notably, a shift in the abundance of microbiota phylum profiles reveals an elevated Firmicutes-to-Bacteroidetes ratio-a consistent hallmark of dysbiosis that correlates with alterations in gut microbiota-derived metabolites. Additionally, treatment with short-chain fatty acids stimulated the differentiation of the motoneuron-like NSC34 cell line. These findings shed light on the multifaceted nature of DCM, resembling a synaptopathy that disrupts cellular communication within the neuromotor axis while concurrently exerting influence on other systems. Notably, the colon emerges as a focal point, experiencing substantial perturbations in both mucosal barrier integrity and the delicate balance of intestinal microbiota.

Acute Systemic White Blood Cell Changes following Degenerative Cervical Myelopathy (DCM) in a Mouse Model.

Degenerative cervical myelopathy (DCM) is caused by age-related degeneration of the cervical spine, causing chronic spinal cord compression and inflammation. The aim of this study was to assess whether the natural progression of DCM is accompanied by hematological changes in the white blood cell composition. If so, these changes can be used for diagnosis complementing established imaging approaches and for the development of treatment strategies, since peripheral immunity affects the progression of DCM. Gradual compression of the spinal cord was induced in C57B/L mice at the C5-6 level. The composition of circulating white blood cells was analyzed longitudinally at four time points after induction of DCM using flow cytometry. At 12 weeks, serum cytokine levels were measured using a Luminex x-MAP assay. Neurological impairment in the mouse model was also assessed using the ladder walk test and CatWalk. Stepping function (* p < 0.05) and overground locomotion (*** p < 0.001) were impaired in the DCM group. Importantly, circulating monocytes and T cells were affected primarily at 3 weeks following DCM. T cells were two-fold lower in the DCM group (*** p < 0.0006), whereas monocytes were four-fold increased (*** p < 0.0006) in the DCM compared with the sham group. Our data suggest that changes in white blood cell populations are modest, which is unique to other spinal cord pathologies, and precede the development of neurobehavioral symptoms.

Genome Sequencing Variations in the Octodon degus, an Unconventional Natural Model of Aging and Alzheimer's Disease.

The degu (Octodon degus) is a diurnal long-lived rodent that can spontaneously develop molecular and behavioral changes that mirror those seen in human aging. With age some degu, but not all individuals, develop cognitive decline and brain pathology like that observed in Alzheimer's disease including neuroinflammation, hyperphosphorylated tau and amyloid plaques, together with other co-morbidities associated with aging such as macular degeneration, cataracts, alterations in circadian rhythm, diabetes and atherosclerosis. Here we report the whole-genome sequencing and analysis of the degu genome, which revealed unique features and molecular adaptations consistent with aging and Alzheimer's disease. We identified single nucleotide polymorphisms in genes associated with Alzheimer's disease including a novel apolipoprotein E (Apoe) gene variant that correlated with an increase in amyloid plaques in brain and modified the in silico predicted degu APOE protein structure and functionality. The reported genome of an unconventional long-lived animal model of aging and Alzheimer's disease offers the opportunity for understanding molecular pathways involved in aging and should help advance biomedical research into treatments for Alzheimer's disease.

Theil Entropy as a Non-Lineal Analysis for Spectral Inequality of Physiological Oscillations.

Theil entropy is a statistical measure used in economics to quantify income inequalities. However, it can be applied to any data distribution including biological signals. In this work, we applied different spectral methods on heart rate variability signals and cellular calcium oscillations previously to Theil entropy analysis. The behavior of Theil entropy and its decomposable property was investigated using exponents in the range of [-1, 2], on the spectrum of synthetic and physiological signals. Our results suggest that the best spectral decomposition method to analyze the spectral inequality of physiological oscillations is the Lomb-Scargle method, followed by Theil entropy analysis. Moreover, our results showed that the exponents that provide more information to describe the spectral inequality in the tested signals were zero, one, and two. It was also observed that the intra-band component is the one that contributes the most to total inequality for the studied oscillations. More in detail, we found that in the state of mental stress, the inequality determined by the Theil entropy analysis of heart rate increases with respect to the resting state. Likewise, the same analytical approach shows that cellular calcium oscillations present on developing interneurons display greater inequality distribution when inhibition of a neurotransmitter system is in place. In conclusion, we propose that Theil entropy is useful for analyzing spectral inequality and to explore its origin in physiological signals.

Gut Microbiota Interaction with the Central Nervous System throughout Life.

During the last years, accumulating evidence has suggested that the gut microbiota plays a key role in the pathogenesis of neurodevelopmental and neurodegenerative diseases via the gut-brain axis. Moreover, current research has helped to elucidate different communication pathways between the gut microbiota and neural tissues (e.g., the vagus nerve, tryptophan production, extrinsic enteric-associated neurons, and short chain fatty acids). On the other hand, altering the composition of gut microbiota promotes a state known as dysbiosis, where the balance between helpful and pathogenic bacteria is disrupted, usually stimulating the last ones. Herein, we summarize selected findings of the recent literature concerning the gut microbiome on the onset and progression of neurodevelopmental and degenerative disorders, and the strategies to modulate its composition in the search for therapeutical approaches, focusing mainly on animal models studies. Readers are advised that this is a young field, based on early studies, that is rapidly growing and being updated as the field advances.

Glycine Receptor Inhibition Differentially Affect Selected Neuronal Populations of the Developing Embryonic Cortex, as Evidenced by the Analysis of Spontaneous Calcium Oscillations.

The embryonic developing cerebral cortex is characterized by the presence of distinctive cell types such as progenitor pools, immature projection neurons and interneurons. Each of these cell types is diverse on itself, but they all take part of the developmental process responding to intrinsic and extrinsic cues that can affect their calcium oscillations. Importantly, calcium activity is crucial for controlling cellular events linked to cell cycle progression, cell fate determination, specification, cell positioning, morphological development and maturation. Therefore, in this work we measured calcium activity in control conditions and in response to neurotransmitter inhibition. Different data analysis methods were applied over the experimental measurements including statistical methods entropy and fractal calculations, and spectral and principal component analyses. We found that developing projection neurons are differentially affected by classic inhibitory neurotransmission as a cell type and at different places compared to migrating interneurons, which are also heterogeneous in their response to neurotransmitter inhibition. This reveals important insights into the developmental role of neurotransmitters and calcium oscillations in the forming brain cortex. Moreover, we present an improved analysis proposing a Gini coefficient-based inequality distribution and principal component analysis as mathematical tools for understanding the earliest patterns of brain activity.

Optogenetic Manipulation of Postsynaptic cAMP Using a Novel Transgenic Mouse Line Enables Synaptic Plasticity and Enhances Depolarization Following Tetanic Stimulation in the Hippocampal Dentate Gyrus.

cAMP is a positive regulator tightly involved in certain types of synaptic plasticity and related memory functions. However, its spatiotemporal roles at the synaptic and neural circuit levels remain elusive. Using a combination of a cAMP optogenetics approach and voltage-sensitive dye (VSD) imaging with electrophysiological recording, we define a novel capacity of postsynaptic cAMP in enabling dentate gyrus long-term potentiation (LTP) and depolarization in acutely prepared murine hippocampal slices. To manipulate cAMP levels at medial perforant path to granule neuron (MPP-DG) synapses by light, we generated transgenic (Tg) mice expressing photoactivatable adenylyl cyclase (PAC) in DG granule neurons. Using these Tg(CMV-Camk2a-RFP/bPAC)3Koka mice, we recorded field excitatory postsynaptic potentials (fEPSPs) from MPP-DG synapses and found that photoactivation of PAC during tetanic stimulation enabled synaptic potentiation that persisted for at least 30 min. This form of LTP was induced without the need for GABA receptor blockade that is typically required for inducing DG plasticity. The paired-pulse ratio (PPR) remained unchanged, indicating the cAMP-dependent LTP was likely postsynaptic. By employing fast fluorescent voltage-sensitive dye (VSD: di-4-ANEPPS) and fluorescence imaging, we found that photoactivation of the PAC actuator enhanced the intensity and extent of dentate gyrus depolarization triggered following tetanic stimulation. These results demonstrate that the elevation of cAMP in granule neurons is capable of rapidly enhancing synaptic strength and neuronal depolarization. The powerful actions of cAMP are consistent with this second messenger having a critical role in the regulation of synaptic function.

Mice lacking neuronal calcium sensor-1 show social and cognitive deficits.

Neuronal calcium sensor-1 or Frequenin is a calcium sensor widely expressed in the nervous system, with roles in neurotransmission, neurite outgrowth, synaptic plasticity, learning, and motivated behaviours. Neuronal calcium sensor-1 has been implicated in neuropsychiatric disorders including autism spectrum disorder, schizophrenia, and bipolar disorder. However, the role of neuronal calcium sensor-1 in behavioural phenotypes and brain changes relevant to autism spectrum disorder have not been evaluated. We show that neuronal calcium sensor-1 deletion in the mouse leads to a mild deficit in social approach and impaired displaced object recognition without affecting social interactions, behavioural flexibility, spatial reference memory, anxiety-like behaviour, or sensorimotor gating. Morphologically, neuronal calcium sensor-1 deletion leads to increased dendritic arbour complexity in the frontal cortex. At the level of hippocampal synaptic plasticity, neuronal calcium sensor-1 deletion leads to a reduction in long-term potentiation in the dentate gyrus, but not area Cornu Ammonis 1. Metabotropic glutamate receptor-induced long-term depression was unaffected in both dentate and Cornu Ammonis 1. These studies identify roles for neuronal calcium sensor-1 in specific subregions of the brain including a phenotype relevant to neuropsychiatric disorders.

Early Actions of Neurotransmitters During Cortex Development and Maturation of Reprogrammed Neurons.

The development of the brain is shaped by a myriad of factors among which neurotransmitters play remarkable roles before and during the formation and maturation of synaptic circuits. Cellular processes such as neurogenesis, morphological development, synaptogenesis and maturation of synapses are temporary and spatially regulated by the local or distal influence of neurotransmitters in the developing cortex. Thus, research on this area has contributed to the understanding of fundamental mechanisms of brain development and to shed light on the etiology of various human neurodevelopmental disorders such as autism and Rett syndrome (RTT), among others. Recently, the field of neuroscience has been shaken by an explosive advance of experimental approaches linked to the use of induced pluripotent stem cells and reprogrammed neurons. This new technology has allowed researchers for the first time to model in the lab the unique events that take place during early human brain development and to explore the mechanisms that cause synaptopathies. In this context, the role of neurotransmitters during early stages of cortex development is beginning to be re-evaluated and a revision of the state of the art has become necessary in a time when new protocols are being worked out to differentiate stem cells into functional neurons. New perspectives on reconsidering the function of neurotransmitters include opportunities for methodological advances, a better understanding of the origin of mental disorders and the potential for development of new treatments.

Cerebral Cortical Circuitry Formation Requires Functional Glycine Receptors.

The development of the cerebral cortex is a complex process that requires the generation, migration, and differentiation of neurons. Interfering with any of these steps can impair the establishment of connectivity and, hence, function of the adult brain. Neurotransmitter receptors have emerged as critical players to regulate these biological steps during brain maturation. Among them, α2 subunit-containing glycine receptors (GlyRs) regulate cortical neurogenesis and the present work demonstrates the long-term consequences of their genetic disruption on neuronal connectivity in the postnatal cerebral cortex. Our data indicate that somatosensory cortical neurons of Glra2 knockout mice (Glra2KO) have more dendritic branches with an overall increase in total spine number. These morphological defects correlate with a disruption of the excitation/inhibition balance, thereby increasing network excitability and enhancing susceptibility to epileptic seizures after pentylenetetrazol tail infusion. Taken together, our findings show that the loss of embryonic GlyRα2 ultimately impairs the formation of cortical circuits in the mature brain.

Microleakage of the prosthetic abutment/implant interface with internal and external connection: in vitro study.

OBJECTIVE: This study determined the degree of marginal microleakage of the abutment-implant interface on platforms with Morse taper connection and external connection. MATERIALS AND METHODS: For this in vitro study, 42 implants, 21 with external connection and 21 with Morse taper connection, were used, immersed in acrylic resin cylinders. Each implant was joined by a prosthetic abutment screw tightened at different degrees, forming the six study groups: (1) External connection, manual tightening (2) External connection, 20 Newton (N) tightening (3) External connection, 30 N tightening (4) Morse taper connection, manual tightening (5) Morse taper connection, 20 N tightening (6) orse taper connection, 30 N tightening. All samples were subjected to load cycling and thermocycling. Then, they were submerged in a solution of 0.2% methylene blue for 24 h. Finally, the microleakage was measured via 20× optical microscopy in each study group, average was obtained, and Mann-Whitney test was applied. RESULTS: Statistically significant differences (P < 0.001) were found between the levels of microleakage presented in the Morse taper connection implants (1.48) and external connection implants (2.8) in all three types of tightening. Microleakage levels decreases when increasing torque is applied to the screws. CONCLUSION: Morse taper connection implants showed lower levels of microleakage than external connection implants; also, it was observed that microleakage decreases in the way torque increases.

Glycine receptors and brain development.

Glycine receptors (GlyRs) are ligand-gated chloride ion channels that mediate fast inhibitory neurotransmission in the spinal cord and the brainstem. There, they are mainly involved in motor control and pain perception in the adult. However, these receptors are also expressed in upper regions of the central nervous system, where they participate in different processes including synaptic neurotransmission. Moreover, GlyRs are present since early stages of brain development and might influence this process. Here, we discuss the current state of the art regarding GlyRs during embryonic and postnatal brain development in light of recent findings about the cellular and molecular mechanisms that control brain development.

Glycine receptor α2 subunit activation promotes cortical interneuron migration.

Glycine receptors (GlyRs) are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

Complex invasion pattern of the cerebral cortex bymicroglial cells during development of the mouse embryo.

Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac. Although it was suggested that they invade the cortex at early developmental stages in the embryo, their invasion pattern remains largely unknown. To address this issue we analyzed the pattern of cortical invasion by microglial cells in mouse embryos at the onset of neuronal cell migration using in vivo immunohistochemistry and ex vivo time-lapse analysis of microglial cells. Microglial cells begin to invade the cortex at 11.5 days of embryonic age (E11.5). They first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. The invasion of the cortical parenchyma occurs in different phases. First, there is a gradual increase of microglial cells between E10.5 and E14.5. From E14.5 to E15.5 there is a rapid phase with a massive increase in microglia, followed by a slow phase again from E15.5 until E17.5. At early stages, many peripheral microglia are actively proliferating before entering the parenchyma. Remarkably, activated microglia accumulate in the choroid plexus primordium, where they are in the proximity of dying cells. Time-lapse analysis shows that embryonic microglia are highly dynamic cells.

Experimental early-life febrile seizures induce changes in GABA(A) R-mediated neurotransmission in the dentate gyrus.

PURPOSE: Febrile seizures (FS), the most frequent seizure type during childhood, have been linked to temporal lobe epilepsy (TLE) in adulthood. Yet, underlying mechanisms are still largely unknown. Altered γ-aminobutyric acid (GABA)ergic neurotransmission in the dentate gyrus (DG) circuit has been hypothesized to be involved. This study aims at analyzing whether experimental FS change inhibitory synaptic input and postsynaptic GABA(A) R function in dentate granule cells. METHODS: We applied an immature rat model of hyperthermia (HT)-induced FS. GABA(A) R-mediated neurotransmission was studied using whole-cell patch-clamp recordings from dentate granule neurons in hippocampal slices within 6-9 days post-HT. KEY FINDINGS: Frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) were reduced in HT rats that had experienced seizures, whereas sIPSC amplitudes were enhanced. Whole-cell GABA responses revealed a doubled GABA(A) R sensitivity in dentate granule cells from HT animals, compared to that of normothermic (NT) controls. Analysis of sIPSCs and whole-cell GABA responses showed similar kinetics in postsynaptic GABA(A) Rs of HT and NT rats. quantitative real-time polymerase chain reaction (qPCR) experiments indicated changes in DG GABA(A) R subunit expression, which was most pronounced for the α3 subunit. SIGNIFICANCE: The data support the hypothesis that FS persistently alter neuronal excitability.

Molecular requirements for ethanol differential allosteric modulation of glycine receptors based on selective Gbetagamma modulation.

It is now believed that the allosteric modulation produced by ethanol in glycine receptors (GlyRs) depends on alcohol binding to discrete sites within the protein structure. Thus, the differential ethanol sensitivity of diverse GlyR isoforms and mutants was explained by the presence of specific residues in putative alcohol pockets. Here, we demonstrate that ethanol sensitivity in two ligand-gated ion receptor members, the GlyR adult α(1) and embryonic α(2) subunits, can be modified through selective mutations that rescued or impaired Gßγ modulation. Even though both isoforms were able to physically interact with Gßγ, only the α(1) GlyR was functionally modulated by Gßγ and pharmacological ethanol concentrations. Remarkably, the simultaneous switching of two transmembrane and a single extracellular residue in α(2) GlyRs was enough to generate GlyRs modulated by Gßγ and low ethanol concentrations. Interestingly, although we found that these TM residues were different to those in the alcohol binding site, the extracellular residue was recently implicated in conformational changes important to generate a pre-open-activated state that precedes ion channel gating. Thus, these results support the idea that the differential ethanol sensitivity of these two GlyR isoforms rests on conformational changes in transmembrane and extracellular residues within the ion channel structure rather than in differences in alcohol binding pockets. Our results describe the molecular basis for the differential ethanol sensitivity of two ligand-gated ion receptor members based on selective Gßγ modulation and provide a new mechanistic framework for allosteric modulations of abuse drugs.

Recidiva del grado de clareamiento dental por té: in vitro / Relapse of dental bleaching after the consumption of tea: in vitro study

INTRODUCCION: El referente estético en nuestra sociedad viene determinado por varios factores, el que más insatisfacción produce es un color anómalo del diente, por eso que al mejorar su color dental, los pacientes esperan buenos resultados y duraderos. OBJETIVO: Determinar la recidiva de color experimentado por piezas dentarias clareadas y posteriormente sumergidas en solución de té. METODOLOGIA: Utilizamos 42 incisivos centrales sanos de bovinos de 3 y 4 años, preparados en probetas cilíndricas acrílicas, mantenidos en saliva artificial a 37ºC, a 100% de humedad, divididas en: grupo experimental (GC), tratado con peróxido de carbamida al 35% gel por 30 minutos y Grupo Control (GNC) no tratado. A ambos medimos color inicial con espectrofómetro Vita Easyshade® y sumergieron en solución de Té por 10 minutos. Medimos el nuevo color, realizando este procedimiento 50 veces. RESULTADOS: Analizados con, Pruebas T y Análisis de Regresión Lineal, tanto el GC como el GNC presentan el primer cambio de color con la primera inmersión en té. El segundo cambio para el GE (p < 0,029) es en la segunda inmersión y para el GC entre la tercera y cuarta. El cambio de color máximo fue alcanzado por el GNC entre las 20 a 30 inmersiones, sin embargo, el GC lo hace entre la 10 y 20 inmersión. CONCLUSIONES: Las piezas clareadas presentan mayor regresión del color que las piezas no tratadas. Además un color inicial claro determina cambios de más intensidad en menor tiempo y sin un patrón definido de progresión.

INTRODUCTION: Esthetic model in our society comes determined by several factors, one of the more important dissatisfaction is an anomalous color of the tooth for that reason patients are wating for good and long lasting results when improving the color of their teeth. OBJETIVE: To determine the color regression experienced by bleached teeth that were later plunged in a solution of tea. METHODOLOGY: 42 central permanent healthy incisor teeth from 3-4 year old bovine, supported in cylindrical acrylic and maintain in artificial saliva at 37ºC for 100% of humidity, were divided in: experimental group (CG), treated with Carbamide Peroxide 35% gel for 30 minutes and Control Group (NCG) not treated. In both, the initial color was measured with Vita Easyshade® Instruments and then plunged into Tea solution for 10 minutes. The new color was measured, and the procedure was repeated 50 times. RESULTS: Analyzed with, T tests and Analysis of Lineal Regression, both the GG and the NCG present the first change of color with the first dip in Tea. The second change for the CG (p < 0.029) is in the second dip and for the CG between the third and fourth dip. The change of maximum color was reached by the NCG between 20 to 30 dips, nevertheless, the CG dip does it between the 10 to 20 immersions. CONCLUSIONS: Bleached teeth present the highest return regression of the color that the not treated. In addition an initial clear color determines changes of more intensity in less time and without a definite form of progression.

Blockade of ethanol-induced potentiation of glycine receptors by a peptide that interferes with Gbetagamma binding.

The large intracellular loop (IL) of the glycine receptor (GlyR) interacts with various signaling proteins and plays a fundamental role in trafficking and regulation of several receptor properties, including a direct interaction with Gbetagamma. In the present study, we found that mutation of basic residues in the N-terminal region of the IL reduced the binding of Gbetagamma to 21 +/- 10% of control. Two basic residues in the C-terminal region, on the other hand, contributed to a smaller extent to Gbetagamma binding. Using docking analysis, we found that both basic regions of the IL bind in nearby regions to the Gbetagamma dimer, within an area of high density of amino acids having an electronegative character. Thereafter, we generated a 17-amino acid peptide with the N-terminal sequence of the wild-type IL (RQH) that was able to inhibit the in vitro binding of Gbetagamma to GlyRs to 57 +/- 5% of control in glutathione S-transferase pull-down assays using purified proteins. More interestingly, when the peptide was intracellularly applied to human embryonic kidney 293 cells, it inhibited the Gbetagamma-mediated modulations of G protein-coupled inwardly rectifying potassium channel by baclofen (24 +/- 14% of control) and attenuated the GlyR potentiation by ethanol (51 +/- 10% versus 10 +/- 3%).