Exploring the interplay between extracellular pH and Dronedarone's pharmacological effects on cardiac function.

Dronedarone (DRN) is a clinically used drug to mitigate arrhythmias with multichannel block properties, including the sodium channel Nav1.5. Extracellular acidification is known to change the pharmacological properties of several antiarrhythmic drugs. Here, we explore how modification in extracellular pH (pHe) shapes the pharmacological profile of DRN upon Nav1.5 sodium current (INa) and in the ex vivo heart preparation. Embryonic human kidney cells (HEK293T/17) were used to transiently express the human isoform of Nav1.5 α-subunit. Patch-Clamp technique was employed to study INa. Neurotoxin-II (ATX-II) was used to induce the late sodium current (INaLate). Additionally, ex vivo Wistar male rat preparations in the Langendorff system were utilized to study electrocardiogram (ECG) waves. DRN preferentially binds to the closed state inactivation mode of Nav1.5 at pHe 7.0. The recovery from INa inactivation was delayed in the presence of DRN in both pHe 7.0 and 7.4, and the use-dependent properties were distinct at pHe 7.0 and 7.4. However, the potency of DRN upon the peak INa, the voltage dependence for activation, and the steady-state inactivation curves were not altered in both pHe tested. Also, the pHe did not change the ability of DRN to block INaLate. Lastly, DRN in a concentration and pH dependent manner modulated the QRS complex, QT and RR interval in clinically relevant concentration. Thus, the pharmacological properties of DRN upon Nav1.5 and ex vivo heart preparation partially depend on the pHe. The pHe changed the biological effect of DRN in the heart electrical function in relevant clinical concentration.

Contrasting leaf intercellular space development in sorghum and maize modulates different tolerance capacity to water limitation.

The objective of this study was to evaluate the relationship between intercellular spaces and leaf gas exchange and the effect of total intercellular space on the growth of maize and sorghum under water restriction. The experiments were conducted in a greenhouse in a 2 × 3 factorial arrangement (two plant types and three water conditions: field capacity (FC = 100%), 75%FC, and 50%FC) with 10 replicates. The lack of water was a limiting factor for maize because it showed reductions in leaf area, leaf thickness, biomass, and gas exchange parameters, while sorghum remained unchanged, maintaining its water-use efficiency. This maintenance was correlated with the growth of intercellular spaces in sorghum leaves because the increased internal volume led to better CO2 control and prevented excessive water loss under drought stress. In addition, sorghum had more stomata than maize. These characteristics contributed to the drought tolerance of sorghum, while maize could not make the same adjustments. Therefore, changes in intercellular spaces promoted adjustments to avoid water loss and may have improved CO2 diffusion, characteristics that are important for drought-tolerant plants.

CB1R activation in nucleus accumbens core promotes stress-induced reinstatement of cocaine seeking by elevating extracellular glutamate in a drug-paired context.

Preclinical models of stress-induced relapse to drug use have shown that the dysregulation of glutamatergic transmission within the nucleus accumbens (NA) contributes notably to the reinstatement of cocaine-seeking behavior in rodents. In this sense, there has been increasing interest in the cannabinoid type-1 receptor (CB1R), due to its crucial role in modulating glutamatergic neurotransmission within brain areas involved in drug-related behaviors. This study explored the involvement of CB1R within the NA subregions in the restraint stress-induced reinstatement of cocaine-conditioned place preference (CPP), as well as in the regulation of glutamatergic transmission, by using a pharmacological approach and the in vivo microdialysis sampling technique in freely moving rats. CB1R blockade by the antagonist/inverse agonist AM251 (5 nmol/0.5 µl/side) or CB1R activation by the agonist ACEA (0.01 fmol/0.5 µl/side), prevented or potentiated restraint stress-induced reinstatement of cocaine-CPP, respectively, after local administration into NAcore, but not NAshell. In addition, microdialysis experiments demonstrated that restraint stress elicited a significant increase in extracellular glutamate in NAcore under reinstatement conditions, with the local administration of AM251 or ACEA inhibiting or potentiating this, respectively. Interestingly, this rise specifically corresponded to the cocaine-associated CPP compartment. We also showed that this context-dependent change in glutamate paralleled the expression of cocaine-CPP, and disappeared after the extinction of this response. Taken together, these findings demonstrated the key role played by CB1R in mediating reinstatement of cocaine-CPP after restraint stress, through modulation of the context-specific glutamate release within NAcore. Additionally, CB1R regulation of basal extracellular glutamate was demonstrated and proposed as the underlying mechanism.

Targeting the pH Paradigm at the Bedside: A Practical Approach.

The inversion of the pH gradient in malignant tumors, known as the pH paradigm, is increasingly becoming accepted by the scientific community as a hallmark of cancer. Accumulated evidence shows that this is not simply a metabolic consequence of a dysregulated behavior, but rather an essential process in the physiopathology of accelerated proliferation and invasion. From the over-simplification of increased lactate production as the cause of the paradigm, as initially proposed, basic science researchers have arrived at highly complex and far-reaching knowledge, that substantially modified that initial belief. These new developments show that the paradigm entails a different regulation of membrane transporters, electrolyte exchangers, cellular and membrane enzymes, water trafficking, specialized membrane structures, transcription factors, and metabolic changes that go far beyond fermentative glycolysis. This complex world of dysregulations is still shuttered behind the walls of experimental laboratories and has not yet reached bedside medicine. However, there are many known pharmaceuticals and nutraceuticals that are capable of targeting the pH paradigm. Most of these products are well known, have low toxicity, and are also inexpensive. They need to be repurposed, and this would entail shorter clinical studies and enormous cost savings if we compare them with the time and expense required for the development of a new molecule. Will targeting the pH paradigm solve the "cancer problem"? Absolutely not. However, reversing the pH inversion would strongly enhance standard treatments, rendering them more efficient, and in some cases permitting lower doses of toxic drugs. This article's goal is to describe how to reverse the pH gradient inversion with existing drugs and nutraceuticals that can easily be used in bedside medicine, without adding toxicity to established treatments. It also aims at increasing awareness among practicing physicians that targeting the pH paradigm would be able to improve the results of standard therapies. Some clinical cases will be presented as well, showing how the pH gradient inversion can be treated at the bedside in a simple manner with repurposed drugs.

Decreased Equilibrative Nucleoside Transporter 1 (ENT1) Activity Contributes to the High Extracellular Adenosine Levels in Mesenchymal Glioblastoma Stem-Like Cells.

Glioblastoma multiforme is one of the most malignant types of cancer. This is mainly due to a cell subpopulation with an extremely aggressive potential, called glioblastoma stem-like cells (GSCs). These cells produce high levels of extracellular adenosine which has been associated with increased chemoresistance, migration, and invasion in glioblastoma. In this study, we attempted to elucidate the mechanisms that control extracellular adenosine levels in GSC subtypes. By using primary and U87MG-derived GSCs, we associated increased extracellular adenosine with the mesenchymal phenotype. [3H]-adenosine uptake occurred mainly through the equilibrative nucleoside transporters (ENTs) in GSCs, but mesenchymal GSCs have lower expression and ENT1-mediated uptake activity than proneural GSCs. By analyzing expression and enzymatic activity, we determined that ecto-5'-nucleotidase (CD73) is predominantly expressed in proneural GSCs, driving AMPase activity. While in mesenchymal GSCs, both CD73 and Prostatic Acid Phosphatase (PAP) contribute to the AMP (adenosine monophosphate) hydrolysis. We did not observe significant differences between the expression of proteins involved in the metabolization of adenosine among the GCSs subtypes. In conclusion, the lower expression and activity of the ENT1 transporter in mesenchymal GSCs contributes to the high level of extracellular adenosine that these GSCs present.

A new insight of Platelet-Rich Fibrin clots morphology and their elemental composition.

This study analyzed the architecture of Platelet-Rich Fibrin (PRF) clots and assessed their elemental composition in order to provide new insight into this biomaterial. Five surplus PRF clots (2,700 RPM, 12 min.) donated by patients (63.6 ±â€¯12.3 years old) were prepared for use in dental clinical procedures. The internal three-dimensional morphology of the red zones and the thirds of the yellow zones of the clots were analyzed by Variable Pressure Scanning Electron Microscope (VPSEM) after sample preparation by two methods: 1. Fixation (2.5% gluataraldehyde); and 2. Fixation with subsequent partial removal of extracellular elements (8 N, HCl). Semi-quantitative elemental analysis was performed by energy-dispersive X-ray spectrometry (EDX). VPSEM analysis showed erythrocytes in both the red zone and the yellow zone, which consisted mainly of fibrin. Removal of extracellular elements enriched the morphology of both zones; the organization of the fibrin was observed to differ in the thirds of the yellow zone, with increasing density and organization to distal. The elements that compose organic substances (C-Carbon, N-Nitrogen, O-Oxygen, Na-Sodium and P-Phosphorus) and halogens (Cl-Chloride and S-Sulfur) were detected; the highest concentrations were of C, followed by O (p < 0.05), in the proximal region of the fibrin. The results of the present study suggest organization of fibrin in the PRF clot, and also reveal the distribution of the elements present in the different regions of the clot. Improved understanding of these characteristics may favor the use of this biomaterial by increasing its efficiency and functionality.

Initial biophysical characterization of Amynthas gracilis giant extracellular hemoglobin (HbAg).

The aim of the present work was the biophysical characterization of the Amynthas gracilis hemoglobin (HbAg). The oxy-HbAg optical absorption data, with Soret and Q bands centered at 415, 540 and 575 nm, were stable and unchanged at pH 7.0. An increase in pH promotes decrease in the intensity in the optical absorption bands, suggesting an oligomeric dissociation and partial oxidation. Identical stability at pH 7.0 was observed in DLS results that presented a hydrodynamic diameter of 28 nm, characteristic of the whole oligomer. DLS shows that HbAg undergoes oligomeric dissociation and an aggregation/denaturation process that corroborates spectroscopic data. Our results showed that the monomer d presents four isoforms with molecular mass (MM) ranging from 16,244 to 16,855 Da; the trimer subunit presents two isoforms, (abc)1 and (abc)2, with MM of 51,415 ± 20 Da and 51,610 ± 14 Da, respectively, and a less intense species, at 67,793 Da, assigned to the tetramer abcd. Monomeric chains a, obtained from reduction of the disulfide-bonded trimer abc, present four isoforms with MM 17,015 Da, 17,061 Da, 17,138 Da and 17,259 Da. DLS and LSI revealed an isoeletric point (pI) of oxy-HbAg of 6.0 ± 0.3 and 5.5, respectively. Data analysis by IEF-SDS-PAGE revealed that the pI of oxy-HbAg is 6.11, correlating with DLS and LSI data. These studies indicate that oxy-HbAg is very stable, at pH 7.0, and has differing properties from orthologous giant hemoglobins.

Characterization of the apo-form of extracellular hemoglobin of Glossoscolex paulistus (HbGp) and its stability in the presence of urea.

The structural study of small heme-containing proteins, such as myoglobin, in the apo-form lacking heme has been extensively described, but the characterization and stability of the giant Glossoscolex paulistus hemoglobin (HbGp), in the absence of heme groups, has not been studied. Spectroscopic data show efficient extraction of the heme groups from the hemoglobin, with relatively small secondary and tertiary structural changes in apo-HbGp noticed compared to oxy-HbGp. Electrophoresis shows a partial precipitation of the trimer abc (significantly lower intensity of the corresponding band in the gel), due to extraction of heme groups, and the predominance of the intense monomeric d band, as well as of two linker bands. AUC and DLS data agree with SDS-PAGE in showing that the apo-HbGp undergoes dissociation into the d and abc subunits. Subunits d and abc are characterized by sedimentation coefficients and percentage contributions of 2.0 and 3.0 S and 76 and 24%, respectively. DLS data suggest that the apo-HbGp is unstable, and two populations are present in solution: one with a diameter around 6.0 nm, identified with the dissociated species, and a second one with diameter 100-180 nm, due to aggregated protein. Finally, the presence of urea promotes the exposure of the fluorescent probes, extrinsic ANS and intrinsic protein tryptophans to the aqueous solvent due to the unfolding process. An understanding of the effect of heme extraction on the stability of hemoproteins is important for biotechnological approaches such as the introduction of non-native prosthetic groups and development of artificial enzymes with designed properties.

Immortalization of Mesenchymal Stromal Cells by TERT Affects Adenosine Metabolism and Impairs their Immunosuppressive Capacity.

Mesenchymal stromal cells (MSCs) are promising candidates for cell-based therapies, mainly due to their unique biological properties such as multipotency, self-renewal and trophic/immunomodulatory effects. However, clinical use has proven complex due to limitations such as high variability of MSCs preparations and high number of cells required for therapies. These challenges could be circumvented with cell immortalization through genetic manipulation, and although many studies show that such approaches are safe, little is known about changes in other biological properties and functions of MSCs. In this study, we evaluated the impact of MSCs immortalization with the TERT gene on the purinergic system, which has emerged as a key modulator in a wide variety of pathophysiological conditions. After cell immortalization, MSCs-TERT displayed similar immunophenotypic profile and differentiation potential to primary MSCs. However, analysis of gene and protein expression exposed important alterations in the purinergic signaling of in vitro cultured MSCs-TERT. Immortalized cells upregulated the CD39/NTPDase1 enzyme and downregulated CD73/NT5E and adenosine deaminase (ADA), which had a direct impact on their nucleotide/nucleoside metabolism profile. Despite these alterations, adenosine did not accumulate in the extracellular space, due to increased uptake. MSCs-TERT cells presented an impaired in vitro immunosuppressive potential, as observed in an assay of co-culture with lymphocytes. Therefore, our data suggest that MSCs-TERT have altered expression of key enzymes of the extracellular nucleotides/nucleoside control, which altered key characteristics of these cells and can potentially change their therapeutic effects in tissue engineering in regenerative medicine.

Associations between Cardiac Magnetic Resonance T1 Mapping Parameters and Ventricular Arrhythmia in Patients with Chagas Disease.

Chronic Chagas disease can progress to myocardial involvement with intense fibrosis, which may predispose patients to sudden cardiac death through ventricular arrhythmia. The associations of myocardial fibrosis detected by cardiac magnetic resonance (CMR) parameters with non-sustained ventricular tachycardia (NSVT) were evaluated. This cross-sectional study included patients in early stages of Chagas disease (n = 47) and a control group (n = 15). Patients underwent cardiac evaluation, including CMR examination. Myocardial fibrosis assessment by CMR with measurement of late gadolinium enhancement (LGE), native T1, and extracellular volume (ECV) was performed. There was an increase in myocardial fibrosis CMR parameters and ventricular arrhythmias among different stages of Chagas disease, combined with a decrease in the left ventricular ejection fraction (LVEF) by CMR and also in the right ventricular systolic function by S' wave on tissue Doppler. Fibrosis mass and ECV were associated with the Rassi score, ventricular extrasystole, and E/e' ratio in a logistic regression model adjusted for age and gender. The ECV maintained an association with the presence of NSVT, even after adjustments for fibrosis mass and LVEF assessed by CMR. The receiver-operating characteristic area under the curve for global ECV (0.85; 95% CI: 0.71-0.99) and NSVT was greater than that for fibrosis mass (0.75; 95% CI: 0.54-0.96), although this difference was not statistically significant. Extracellular volume could be an early marker of increased risk of ventricular arrhythmia in Chagas disease, presenting an independent association with NSVT in the initial stages of chronic Chagas cardiomyopathy, even after adjustment for fibrosis mass and LVEF.

Administration of low doses of the 5-HT1A receptor agonist 8-OH-DPAT attenuates the discriminative signal of amphetamine in the conditioned taste aversion procedure.

Several studies have reported that low doses of the 5-HT1A receptor agonist 8-OH-DPAT reduce cocaine-induced locomotor activity. However, it has also been reported that high doses of 8-OH-DPAT do not substitute for or alter the discriminative signal of cocaine (COC) or amphetamine (AMPH). This study aimed to evaluate the effects of low and high doses of the 5-HT1A agonist 8-OH-DPAT on the discriminative signal of AMPH using conditioned taste aversion as a drug discrimination procedure. Additionally, to establish a correlation between the behavioral effects in drug discrimination and changes in dopamine (DA) and gamma-aminobutyric acid (GABA) concentrations, we evaluated the effect of systemic administration of low or high doses of the 5-HT1A receptor agonist 8-OH-DPAT and of the 5-HT1A receptor antagonist WAY100135 on DA and GABA extracellular concentrations in the nucleus accumbens (nAcc) and ventral tegmental area (VTA), respectively, using cerebral microdialysis. The behavioral results showed that low but not high doses of 8-OH-DPAT produced a reduction in the AMPH-induced discriminative signal, while WAY100135 administration prevented such effects. The microdialysis results showed that a low dose of 8-OH-DPAT decreased extracellular DA concentrations in the nAcc and increased GABA concentrations in the VTA. Pretreatment with WAY100135 prevented these effects. These data support the hypothesis that 5-HT1A receptors modulate the behavioral effects of psychostimulant drugs, such as AMPH, through somatodendritic 5-HT1A autoreceptors in the raphe nucleus indicating that 5-HT1A receptors may be an important target for the development of pharmacological treatments for psychostimulant addiction.

Extracellular tRNAs and tRNA-derived fragments.

Fragmentation of tRNAs generates a family of small RNAs collectively known as tRNA-derived fragments. These fragments vary in sequence and size but have been shown to regulate many processes involved in cell homoeostasis and adaptations to stress. Additionally, the field of extracellular RNAs (exRNAs) is rapidly growing because exRNAs are a promising source of biomarkers in liquid biopsies, and because exRNAs seem to play key roles in intercellular and interspecies communication. Herein, we review recent descriptions of tRNA-derived fragments in the extracellular space in all domains of life, both in biofluids and in cell culture. The purpose of this review is to find consensus on which tRNA-derived fragments are more prominent in each extracellular fraction (including extracellular vesicles, lipoproteins and ribonucleoprotein complexes). We highlight what is becoming clear and what is still controversial in this field, in order to stimulate future hypothesis-driven studies which could clarify the role of full-length tRNAs and tRNA-derived fragments in the extracellular space.

Peri/Epicellular Thiol Oxidoreductases as Mediators of Extracellular Redox Signaling.

Significance: Supracellular redox networks regulating cell-extracellular matrix (ECM) and organ system architecture merge with structural and functional (catalytic or allosteric) properties of disulfide bonds. This review addresses emerging evidence that exported thiol oxidoreductases (TORs), such as thioredoxin, protein disulfide isomerases (PDIs), quiescin sulfhydryl oxidases (QSOX)1, and peroxiredoxins, composing a peri/epicellular (pec)TOR pool, mediate relevant signaling. pecTOR functions depend mainly on kinetic and spatial regulation of thiol-disulfide exchange reactions governed by redox potentials, which are modulated by exported intracellular low-molecular-weight thiols, together conferring signal specificity. Recent Advances: pecTOR redox-modulates several targets including integrins, ECM proteins, surface molecules, and plasma components, although clear-cut documentation of direct effects is lacking in many cases. TOR catalytic pathways, displaying common patterns, culminate in substrate thiol reduction, oxidation, or isomerization. Peroxiredoxins act as redox/peroxide sensors, contrary to PDIs, which are likely substrate-targeted redox modulators. Emerging evidence suggests important pecTOR roles in patho(physio)logical processes, including blood coagulation, vascular remodeling, mechanosensing, endothelial function, immune responses, and inflammation. Critical Issues: Effects of pecPDIs supporting thrombosis/platelet activation have been well documented and reached the clinical arena. Roles of pecPDIA1 in vascular remodeling/mechanosensing are also emerging. Extracellular thioredoxin and pecPDIs redox-regulate immunoinflammation. Routes of TOR externalization remain elusive and appear to involve Golgi-independent routes. pecTORs are particularly accessible drug targets. Future Directions: Further understanding mechanisms of thiol redox reactions and developing assays for assessing pecTOR redox activities remain important research avenues. Also, addressing pecTORs as disease markers and achieving more efficient/specific drugs for pecTOR modulation are major perspectives for diagnostic/therapeutic improvements.

Are thick leaves, large mesophyll cells and small intercellular air spaces requisites for CAM?

BACKGROUND AND AIMS: It is commonly accepted that the leaf of a crassulacean acid metabolism (CAM) plant is thick, with large mesophyll cells and vacuoles that can accommodate the malic acid produced during the night. The link between mesophyll characteristics and CAM mode, whether obligate or C3/CAM, was evaluated. METHODS: Published values of the carbon isotopic ratio (δ 13C) as an indicator of CAM, leaf thickness, leaf micrographs and other evidence of CAM operation were used to correlate cell density, cell area, the proportion of intercellular space in the mesophyll (IAS) and the length of cell wall facing the intercellular air spaces (Lmes/A) with CAM mode. KEY RESULTS: Based on 81 species and relatively unrelated families (15) belonging to nine orders, neither leaf thickness nor mesophyll traits helped explain the degree of CAM expression. A strong correlation was found between leaf thickness and δ 13C in some species of Crassulaceae and between leaf thickness and nocturnal acid accumulation in a few obligate CAM species of Bromeliaceae but, when all 81 species were pooled together, no significant changes with δ 13C were observed in cell density, cell area, IAS or Lmes/A. CONCLUSIONS: An influence of phylogeny on leaf anatomy was evidenced in a few cases but this precluded generalization for widely separate taxa containing CAM species. The possible relationships between leaf anatomy and CAM mode should be interpreted cautiously.

Large Intracellular Domain-Dependent Effects of Positive Allosteric Modulators on Glycine Receptors.

Glycine receptors (GlyRs) are members of the pentameric ligand-gated ionic channel family (pLGICs) and mediate fast inhibitory neurotransmission in the brain stem and spinal cord. The function of GlyRs can be modulated by positive allosteric modulators (PAMs). So far, it is largely accepted that both the extracellular (ECD) and transmembrane (TMD) domains constitute the primary target for many of these PAMs. On the other hand, the contribution of the intracellular domain (ICD) to the PAM effects on GlyRs remains poorly understood. To gain insight about the role of the ICD in the pharmacology of GlyRs, we examined the contribution of each domain using a chimeric receptor. Two chimeras were generated, one consisting of the ECD of the prokaryotic homologue Gloeobacter violaceus ligand-gated ion channel (GLIC) fused to the TMD of the human α1GlyR lacking the ICD (Lily) and a second with the ICD (Lily-ICD). The sensitivity to PAMs of both chimeric receptors was studied using electrophysiological techniques. The Lily receptor showed a significant decrease in the sensitivity to four recognized PAMs. Remarkably, the incorporation of the ICD into the Lily background was sufficient to restore the wild-type α1GlyR sensitivity to these PAMs. Based on these data, we can suggest that the ICD is necessary to form a pLGIC having full sensitivity to positive allosteric modulators.

iPSCs-derived nerve-like cells from familial Alzheimer's disease PSEN 1 E280A reveal increased amyloid-beta levels and loss of the Y chromosome.

Alzheimer's disease (AD) is a progressive, degenerative disorder that mainly results in memory loss and a cognitive disorder. Although the cause of AD is still unknown, a minor percentage of AD cases are produced by genetic mutations in the presenilin-1 (PSEN1) gene. Differentiated neuronal cells derived from induced pluripotent stem cells (iPSCs) of patients can recapitulate key pathological features of AD in vitro; however, iPSCs studies focused on the p.E280 A mutation, which afflicts the largest family in the world with familial AD, have not been carried out yet. Although a link between the loss of the Y (LOY) chromosome in peripheral blood cells and risk for AD has been reported, LOY-associated phenotype has not been previously studied in PSEN1 E280 A carriers. Here, we report the reprogramming of fibroblast cells into iPSCs from a familial AD patient with the PSEN1 E280 A mutation, followed by neuronal differentiation into neural precursor cells (NPCs), and the differentiation of NPCs into differentiated neurons that lacked a Y chromosome. Although the PSEN1 E280 A iPSCs and NPCs were successfully obtained, after 8 days of differentiation, PSEN1 E280 A differentiated neurons massively died reflected by release and/ or activation of death markers, and failed to reach complete neural differentiation compared to PSEN 1 wild type cells.

Extracellular Galectin-3 Induces Accelerated Oligodendroglial Differentiation Through Changes in Signaling Pathways and Cytoskeleton Dynamics.

Galectin-3 (Gal-3) is a chimeric protein structurally composed of unusual tandem repeats of proline and short glycine-rich segments fused onto a carbohydrate recognition domain. Our studies have previously demonstrated that Gal-3 drives oligodendrocyte (OLG) differentiation to control myelin integrity and function. The cytoskeleton plays a key role in OLG maturation: the initial stage of OLG process extension requires dynamic actin filament assembly, while subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins which are dependent on myelin basic protein (MPB) expression. In this context, the aim of the present work was to elucidate the mechanism by which recombinant Gal-3 (rGal-3) induces OLG maturation, giving special attention to the actin cytoskeleton. Our results show that rGal-3 induced early actin filament assembly accompanied by Erk signaling deactivation, which led to a decrease in the number of platelet-derived growth factor receptor α (PDGFRα)+ cells concomitantly with an increase in the number of 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase)+ cells at 1 day of treatment (TD1), and Akt signaling activation at TD1 and TD3. Strikingly, rGal-3 induced an accelerated shift from polymerized to depolymerized actin between TD3 and TD5, accompanied by a significant increase in MBP, gelsolin, Rac1, Rac1-GTP, and ß-catenin expression at TD5. These results were strongly supported by assays using Erk 1/2 and Akt inhibitors, indicating that both pathways are key to rGal-3-mediated effects. Erk 1/2 inhibition in control-treated cells resembled an rGal-3 like state characterized by an increase in MBP, ß-catenin, and gelsolin expression. In contrast, Akt inhibition in rGal-3-treated cells reduced MBP, ß-catenin, and gelsolin expression, indicating a blockade of rGal-3 effects. Taken together, these results indicate that rGal-3 accelerates OLG maturation by modulating signaling pathways and protein expression which lead to changes in actin cytoskeleton dynamics.

Chemical biomarkers of epileptogenesis and ictogenesis in experimental epilepsy.

Epilepsy produces chronic chemical changes induced by altered cellular structures, and acute ones produced by conditions leading into individual seizures. Here, we aim to quantify 24 molecules simultaneously at baseline and during periods of lowered seizure threshold in rats. Using serial hippocampal microdialysis collections starting two weeks after the pilocarpine-induced status epilepticus, we evaluated how this chronic epilepsy model affects molecule levels and their interactions. Then, we quantified the changes occurring when the brain moves into a pro-seizure state using a novel model of physiological ictogenesis. Compared with controls, pilocarpine animals had significantly decreased baseline levels of adenosine, homovanillic acid, and serotonin, but significantly increased levels of choline, glutamate, phenylalanine, and tyrosine. Step-wise linear regression identified that choline, homovanillic acid, adenosine, and serotonin are the most important features to characterize the difference in the extracellular milieu between pilocarpine and control animals. When increasing the hippocampal seizure risk, the concentrations of normetanephrine, serine, aspartate, and 5-hydroxyindoleacetic acid were the most prominent; however, there were no specific, consistent changes prior to individual seizures.

Understanding the intracellular-to-extracellular localization switch of polyhydroxybutyrate polymerase in pseudomonas backgrounds as a microevolutionary process.

After gene duplication, paralogous genes evolve independently, and consequently, the new proteins encoded by these duplicated genes are exposed to changes in their subcellular location. Although there are increasing evidence that phylogenetically related proteins play different functions in different subcellular compartments, the number of evolutionary steps required for the emergence of a novel protein with a novel subcellular localization remains unclear. Regarding this intriguing topic, here we examine in depth our previous reports describing both intracellular and extracellular polyhydroxybutyrate polymerases (PhaC) in the Pseudomonadales group. The recapitulation of the intracellular-to-extracellular localization switch of PhaC in these strains shows a gradual evolution from a simple cytosolic PhaC form to a complex extracellular PhaC form specifically secreted via the type 1 secretion system. This gradual evolution includes several adaptive and pre-adaptive changes at the genomic, genetic and enzymatic levels, which are intimately related to the lifestyle of organisms during the evolution of protein localization. We conclude that the protein localization switch can be an extremely complex process in nature.