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.

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.

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.

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.

The Extracellular cAMP-Adenosine Pathway in Airway Smooth Muscle.

In the respiratory tract, intracellular cAMP has a key role in the smooth muscle relaxation induced by the ß2-adrenoceptor/Gs protein/adenylyl cyclase axis. In other tissues, cAMP also works as an extracellular messenger, after its efflux and interstitial conversion to adenosine by ectoenzymes. The aim of this study was to identify cAMP efflux and the "extracellular cAMP-adenosine pathway" in the airway smooth muscle. First, we tested the ability of ß2-adrenoceptor agonists formoterol or fenoterol to increase the extracellular cAMP in isolated tracheal rings from adult male Wistar rats. The effects of adenosine, cAMP, 8-Br-cAMP, fenoterol, or formoterol were also evaluated in the isometric contraction of control or carbachol (CCh) precontracted tracheas, normalized as the percentage of CCh-induced response. Fenoterol and formoterol induced 70%-80% relaxation and increased extracellular cAMP levels by up to 280%-450%. Although exogenous cAMP or adenosine evoked phasic contractions, the membrane-permeable cAMP analog 8-Br-cAMP induced relaxation of CCh-precontracted tracheas. The simultaneous inhibition of adenosine degradation/uptake with EHNA [erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride] plus uridine increased by 3-fold the maximum cAMP-induced contraction, whereas it was significantly reduced by AMPCP [adenosine 5'-(α,ß-methylene)diphosphate; an ecto-5'-nucleotidase inhibitor], and by adenosine receptor antagonists CGS-15943 (nonselective) or DPCPX (8-cyclopentyl-1,3-dipropylxanthine) (A1 selective). Finally, CGS-15943 shifted to the left the concentration-relaxation curve for fenoterol. In conclusion, our results show that airway smooth muscle expresses the extracellular cAMP-adenosine pathway associated with contracting effects mediated by A1 receptors. The cAMP efflux triggered by fenoterol/formoterol indicates that the extracellular cAMP-adenosine pathway may play a role in balancing the relaxant effects of ß2-adrenoceptor agonists in airways, which may impact their bronchodilation effects.

mus-52 disruption and metabolic regulation in Neurospora crassa: Transcriptional responses to extracellular phosphate availability.

Advances in the understanding of molecular systems depend on specific tools like the disruption of genes to produce strains with the desired characteristics. The disruption of any mutagen sensitive (mus) genes in the model fungus Neurospora crassa, i.e. mus-51, mus-52, or mus-53, orthologous to the human genes KU70, KU80, and LIG4, respectively, provides efficient tools for gene targeting. Accordingly, we used RNA-sequencing and reverse transcription-quantitative polymerase chain reaction amplification techniques to evaluate the effects of mus-52 deletion in N. crassa gene transcriptional modulation, and thus, infer its influence regarding metabolic response to extracellular availability of inorganic phosphate (Pi). Notably, the absence of MUS-52 affected the transcription of a vast number of genes, highlighting the expression of those coding for transcription factors, kinases, circadian clocks, oxi-reduction balance, and membrane- and nucleolus-related proteins. These findings may provide insights toward the KU molecular mechanisms, which have been related to telomere maintenance, apoptosis, DNA replication, and gene transcription regulation, as well as associated human conditions including immune system disorders, cancer, and aging.

Detection and Analysis of Non-vesicular Extracellular RNA.

Although extracellular vesicles (EVs) are by far the most studied carriers of extracellular small RNAs involved in cell-to-cell communication, most extracellular small RNAs are actually present as soluble vesicle-free supramolecular complexes. Several proteins have been described as the counterparts of these RNase-protected complexes. Here we describe a method for the purification and analysis of non-vesicular extracellular RNA derived from the conditioned media of mammalian cell culture. Focus on this fraction will increase our understanding on extracellular RNA biology, while serving as a source for biomarker discovery complementary to EVs.

Vitamin C modulates glutamate transport and NMDA receptor function in the retina.

Vitamin C (in the reduced form ascorbate or in the oxidized form dehydroascorbate) is implicated in signaling events throughout the central nervous system (CNS). In the retina, a high-affinity transport system for ascorbate has been described and glutamatergic signaling has been reported to control ascorbate release. Here, we investigated the modulatory role played by vitamin C upon glutamate uptake and N-methyl-d-aspartate (NMDA) receptor activation in cultured retinal cells or in intact retinal tissue using biochemical and imaging techniques. We show that both forms of vitamin C, ascorbate or dehydroascorbate, promote an accumulation of extracellular glutamate by a mechanism involving the inhibition of glutamate uptake. This inhibition correlates with the finding that ascorbate promotes a decrease in cell surface levels of the neuronal glutamate transporter excitatory amino acid transporter 3 in retinal neuronal cultures. Interestingly, vitamin C is prone to increase the activity of NMDA receptors but also promotes a decrease in glutamate-stimulated [3 H] MK801 binding and decreases cell membrane content of NMDA receptor glutamate ionotropic receptor subunit 1 (GluN1) subunits. Both compounds were also able to increase cAMP response element-binding protein phosphorylation in neuronal nuclei in a glutamate receptor and calcium/calmodulin kinase-dependent manner. Moreover, the effect of ascorbate is not blocked by sulfinpyrazone and then does not depend on its uptake by retinal cells. Overall, these data indicate a novel molecular and functional target for vitamin C impacting on glutamate signaling in retinal neurons.

Serotonin-gated inward currents are three times more frequent in rat hairy skin sensory afferents than in those innervating the skeletal muscle.

Tight whole-cell patch clamp was performed in 191 DiI (1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate) retrogradely labeled rat sensory afferents from skin shoulders ( n = 93) and biceps femoris muscles ( n = 98). 5-HT-gated inward currents were evoked with 50-µM serotonin (5-HT; 5-hydroxytryptamine), and their frequency and current densities were compared between skin and skeletal muscle sensory afferents. To evaluate if 5-HT-gated inward currents coexist with other ligand-gated currents, the skin and skeletal muscle sensory afferents were also sequentially exposed to external solution at pH 6.8, ATP (50 µM), and capsaicin (1 µM). 5-HT evoked inward currents in 72% (67 of 93) of hairy skin sensory afferents and in only 24% (24 of 98) of skeletal muscle sensory afferents, and this difference was statistically significant ( p < 0.0000, chi-square test). The current densities obtained in hairy skin and skeletal muscle sensory afferents were not significantly different. They were -45.8 ± 7.7 and -32.4 ± 10.5 pA/pF, respectively (mean ± SEM, p < 0.30734). These results indicate that 5-HT-gated inward currents are three times more frequently evoked in small- to medium-sized sensory afferents (25-40 µm) innervating the hairy skin than on those innervating the skeletal muscle. When cells were gathered in two clusters, the difference was four times larger in the small-sized cluster (25-32 µm) and two times larger in the medium-sized cluster (33-40 µm). The results can be explained if the superficial somatic (cutaneous) nociceptive system is more exposed than the deep somatic nociceptive system (musculoskeletal) to physical and chemical stimuli inducing 5-HT-mediated inflammatory pain.

Deficient Insulin-mediated Upregulation of the Equilibrative Nucleoside Transporter 2 Contributes to Chronically Increased Adenosine in Diabetic Glomerulopathy.

Deficient insulin signaling is a key event mediating diabetic glomerulopathy. Additionally, diabetic kidney disease has been related to increased levels of adenosine. Therefore, we tested a link between insulin deficiency and dysregulated activity of the equilibrative nucleoside transporters (ENTs) responsible for controlling extracellular levels of adenosine. In ex vivo glomeruli, high D-glucose decreased nucleoside uptake mediated by ENT1 and ENT2 transporters, resulting in augmented extracellular levels of adenosine. This condition was reversed by exposure to insulin. Particularly, insulin through insulin receptor/PI3K pathway markedly upregulated ENT2 uptake activity to restores the extracellular basal level of adenosine. Using primary cultured rat podocytes as a cellular model, we found insulin was able to increase ENT2 maximal velocity of transport. Also, PI3K activity was necessary to maintain ENT2 protein levels in the long term. In glomeruli of streptozotocin-induced diabetic rats, insulin deficiency leads to decreased activity of ENT2 and chronically increased extracellular levels of adenosine. Treatment of diabetic rats with adenosine deaminase attenuated both the glomerular loss of nephrin and proteinuria. In conclusion, we evidenced ENT2 as a target of insulin signaling and sensitive to dysregulation in diabetes, leading to chronically increased extracellular adenosine levels and thereby setting conditions conducive to kidney injury.

The Lymphocytes Stimulation Induced DNA Release, a Phenomenon Similar to NETosis.

The release of DNA into the extracellular milieu by neutrophil during a process called NETosis has been postulated as an additional source of autoantigens; a process believed to be important in the pathogenesis of some autoimmune disease, such as systemic lupus erythematosus (SLE). However, it is not established if the B and T cells undergo the release of DNA to the extracellular milleu, in response to different stimuli. In this study, it was observed that the treatment of B and T cells with PMA, ionomycin and the serum from patients with SLE induced the extracellular DNA presence in B and T cells. These findings suggest that the phenomenon were similar to those observed in neutrophil's Etosis; B and T cells also released their DNA into the extracellular milieu. The findings express that serum from patients with SLE and SLEDAI ≤ 8 triggers the release of extracellular DNA in neutrophils, B and T cells, that suggested the presence of soluble factors in the serum that favoured this phenomenon.

Biofilm extracellular polysaccharides degradation during starvation and enamel demineralization.

This study was conducted to evaluate if extracellular polysaccharides (EPS) are used by Streptococcus mutans (Sm) biofilm during night starvation, contributing to enamel demineralization increasing occurred during daily sugar exposure. Sm biofilms were formed during 5 days on bovine enamel slabs of known surface hardness (SH). The biofilms were exposed to sucrose 10% or glucose + fructose 10.5% (carbohydrates that differ on EPS formation), 8x/day but were maintained in starvation during the night. Biofilm samples were harvested during two moments, on the end of the 4th day and in the morning of the 5th day, conditions of sugar abundance and starvation, respectively. The slabs were also collected to evaluate the percentage of surface hardness loss (%SHL). The biofilms were analyzed for EPS soluble and insoluble and intracellular polysaccharides (IPS), viable bacteria (CFU), biofilm architecture and biomass. pH, calcium and acid concentration were determined in the culture medium. The data were analyzed by two-way ANOVA followed by Tukey's test or Student's t-test. The effect of the factor carbohydrate treatment for polysaccharide analysis was significant (p < 0.05) but not the harvest moment (p > 0.05). Larger amounts of soluble and insoluble EPS and IPS were formed in the sucrose group when compared to glucose + fructose group (p < 0.05), but they were not metabolized during starvation time (S-EPS, p = 0.93; I-EPS, p = 0.11; and IPS = 0.96). Greater enamel %SHL was also found for the sucrose group (p < 0.05) but the demineralization did not increase during starvation (p = 0.09). In conclusion, the findings suggest that EPS metabolization by S. mutans during night starvation do not contribute to increase enamel demineralization occurred during the daily abundance of sugar.

Enduring attenuation of norepinephrine synaptic availability and augmentation of the pharmacological and behavioral effects of desipramine by repeated immobilization stress.

Here we provide evidence that repeated immobilization stress (RIS) in rats induces a persistent increase in noradrenergic activity in the anterior aspects of the anterolateral bed nucleus of the stria terminalis (alBNST). This increase in noradrenergic activity results from both enhanced synthesis and reuptake of norepinephrine (NE). It leads to a decrease in the synaptic availability of NE, which elicits an augmented noradrenergic response to the inhibitors of NE reuptake (NRIs), such as desipramine (DMI), an antidepressant. The enduring depression-like behavior and the augmentation of the climbing behavior seen in repeatedly stressed rats following subchronic administration of DMI in the forced swimming test (FST) might be explained by a dysregulation of noradrenergic transmission observed in alBNST. Taken together, we propose that dysregulation of noradrenergic transmission such as the one described in the present work may represent a mechanism underlying major depressive disorders (MDD) with melancholic features in humans.

Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris.

BACKGROUND: Pichia pastoris shows physiological advantages in producing recombinant proteins, compared to other commonly used cell factories. This yeast is mostly grown in dynamic cultivation systems, where the cell's environment is continuously changing and many variables influence process productivity. In this context, a model capable of explaining and predicting cell behavior for the rational design of bioprocesses is highly desirable. Currently, there are five genome-scale metabolic reconstructions of P. pastoris which have been used to predict extracellular cell behavior in stationary conditions. RESULTS: In this work, we assembled a dynamic genome-scale metabolic model for glucose-limited, aerobic cultivations of Pichia pastoris. Starting from an initial model structure for batch and fed-batch cultures, we performed pre/post regression diagnostics to ensure that model parameters were identifiable, significant and sensitive. Once identified, the non-relevant ones were iteratively fixed until a priori robust modeling structures were found for each type of cultivation. Next, the robustness of these reduced structures was confirmed by calibrating the model with new datasets, where no sensitivity, identifiability or significance problems appeared in their parameters. Afterwards, the model was validated for the prediction of batch and fed-batch dynamics in the studied conditions. Lastly, the model was employed as a case study to analyze the metabolic flux distribution of a fed-batch culture and to unravel genetic and process engineering strategies to improve the production of recombinant Human Serum Albumin (HSA). Simulation of single knock-outs indicated that deviation of carbon towards cysteine and tryptophan formation improves HSA production. The deletion of methylene tetrahydrofolate dehydrogenase could increase the HSA volumetric productivity by 630%. Moreover, given specific bioprocess limitations and strain characteristics, the model suggests that implementation of a decreasing specific growth rate during the feed phase of a fed-batch culture results in a 25% increase of the volumetric productivity of the protein. CONCLUSION: In this work, we formulated a dynamic genome scale metabolic model of Pichia pastoris that yields realistic metabolic flux distributions throughout dynamic cultivations. The model can be calibrated with experimental data to rationally propose genetic and process engineering strategies to improve the performance of a P. pastoris strain of interest.

eHSP70/iHSP70 and divergent functions on the challenge: effect of exercise and tissue specificity in response to stress.

Heat-shock proteins including HSP70 are stress-related proteins that have been reported in cell protection and survival. In contrast to this, the increase in circulating levels of HSP70 (eHSP70) is associated with cellular damage and inflammatory factors. Physical stress, like exercise, is effective to induce both iHSP70 and eHSP70 in several tissues and cell types, which have different behaviours in response to stress. The different functions of HSP70 before the challenge are dependent of intracellular localization and subsequent molecular chaperone action, but when present in the extracellular space, it activates pro-inflammatory pathways. The different forms in which tissues and cells respond to stress like physical exercise, as well as the optimal intensity of the stress, are determinants for the beneficial effects or as an indicator of dangerous conditions, summoning immune cells as a warning sign to the body.