Amperometric Self-Referencing Ceramic Based Microelectrode Arrays for D-Serine Detection

D-serine is the major D-amino acid in the mammalian central nervous system. As the dominant co-agonist of the endogenous synaptic NMDA receptor, D-serine plays a role in synaptic plasticity, learning, and memory. Alterations in D-serine are linked to neuropsychiatric disorders including schizophrenia. Thus, it is of increasing interest to monitor the concentration of D-serine in vivo as a relevant player in dynamic neuron-glia network activity. Here we present a procedure for amperometric detection of D-serine with self-referencing ceramic-based microelectrode arrays (MEAs) coated with D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). We demonstrate in vitro D-serine recordings with a mean sensitivity of 8.61 ± 0.83 pA/µM to D-serine, a limit of detection (LOD) of 0.17 ± 0.01 µM, and a selectivity ratio of 80:1 or greater for D-serine over ascorbic acid (mean ± SEM; n = 12) that can be used for freely moving studies

Heme Mediated STAT3 Activation in Severe Malaria

The mortality of severe malaria [cerebral malaria (CM), severe malaria anemia (SMA), acute lung injury (ALI) and acute respiratory distress syndrome (ARDS)] remains high despite the availability associated with adequate treatments. Recent studies in our laboratory and others have revealed a hitherto unknown correlation between chemokine CXCL10/CXCR3, Heme/HO-1 and STAT3 and cerebral malaria severity and mortality. Although Heme/HO-1 and CXCL10/CXCR3 interactions are directly involved in the pathogenesis of CM and fatal disease, the mechanism dictating how Heme/HO-1 and CXCL10/CXCR3 are expressed and regulated under these conditions is still unknown. We therefore tested the hypothesis that these factors share common signaling pathways and may be mutually regulated.We first clarified the roles of Heme/HO-1, CXCL10/CXCR3 and STAT3 in CM pathogenesis utilizing a well established experimental cerebral malaria mouse (ECM, P. berghei ANKA) model. Then, we further determined the mechanisms how STAT3 regulates HO-1 and CXCL10 as well as mutual regulation among them in CRL-2581, a murine endothelial cell line.The results demonstrate that (1) STAT3 is activated by P. berghei ANKA (PBA) infection in vivo and Heme in vitro. (2) Heme up-regulates HO-1 and CXCL10 production through STAT3 pathway, and regulates CXCL10 at the transcriptional level in vitro. (3) HO-1 transcription is positively regulated by CXCL10. (4) HO-1 regulates STAT3 signaling.Our data indicate that Heme/HO-1, CXCL10/CXCR3 and STAT3 molecules as well as related signaling pathways play very important roles in the pathogenesis of severe malaria. We conclude that these factors are mutually regulated and provide new opportunities to develop potential novel therapeutic targets that could be used to supplement traditional prophylactics and treatments for malaria and improve clinical outcomes while reducing malaria mortality. Our ultimate goal is to develop novel therapies targeting Heme or CXCL10-related biological signaling molecules associated with development of fatal malaria

The role of the serotonin receptor subtypes 5-HT1A and 5-HT7 and its interaction in emotional learning and memory

Serotonin [5-hydroxytryptamine (5-HT)] is a multifunctional neurotransmitter innervating cortical and limbic areas involved in cognition and emotional regulation. Dysregulation of serotonergic transmission is associated with emotional and cognitive deficits in psychiatric patients and animal models. Drugs targeting the 5-HT system are widely used to treat mood disorders and anxiety-like behaviors. Among the fourteen 5-HT receptor (5-HTR) subtypes, the 5-HT1AR and 5-HT7R are associated with the development of anxiety, depression and cognitive function linked to mechanisms of emotional learning and memory. In rodents fear conditioning and passive avoidance (PA) are associative learning paradigms to study emotional memory. This review assesses the role of 5-HT1AR and 5-HT7R as well as their interplay at the molecular, neurochemical and behavioral level. Activation of postsynaptic 5-HT1ARs impairs emotional memory through attenuation of neuronal activity, whereas presynaptic 5-HT1AR activation reduces 5-HT release and exerts pro-cognitive effects on PA retention. Antagonism of the 5-HT1AR facilitates memory retention possibly via 5-HT7R activation and evidence is provided that 5HT7R can facilitate emotional memory upon reduced 5-HT1AR transmission. These findings highlight the differential role of these 5-HTRs in cognitive/emotional domains of behavior. Moreover, the results indicate that tonic and phasic 5-HT release can exert different and potentially opposing effects on emotional memory, depending on the states of 5-HT1ARs and 5-HT7Rs and their interaction. Consequently, individual differences due to genetic and/or epigenetic mechanisms play an essential role for the responsiveness to drug treatment, e.g., by SSRIs which increase intrasynaptic 5-HT levels thereby activating multiple pre- and postsynaptic 5-HTR subtypes

Amperometric Self-Referencing Ceramic Based Microelectrode Arrays for D-Serine Detection

D-serine is the major D-amino acid in the mammalian central nervous system. As the dominant co-agonist of the endogenous synaptic NMDA receptor, D-serine plays a role in synaptic plasticity, learning, and memory. Alterations in D-serine are linked to neuropsychiatric disorders including schizophrenia. Thus, it is of increasing interest to monitor the concentration of D-serine in vivo as a relevant player in dynamic neuron-glia network activity. Here we present a procedure for amperometric detection of D-serine with self-referencing ceramic-based microelectrode arrays (MEAs) coated with D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). We demonstrate in vitro D-serine recordings with a mean sensitivity of 8.61 +/- 0.83 pA/mu M to D-serine, a limit of detection (LOD) of 0.17 +/- 0.01 mu M, and a selectivity ratio of 80:1 or greater for D-serine over ascorbic acid (mean +/- SEM; n = 12) that can be used for freely moving studies

High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease

An increase in bile ductular structures is observed in diverse human liver diseases. These structures harbour the progenitor cell compartment of the liver. Since ATP-binding cassette (ABC) transporters may have a cytoprotective role in liver disease, an immunohistochemical study was performed on human liver specimens from patients with primary biliary cirrhosis (PBC), chronic hepatitis C virus (HCV) infection, submassive cell necrosis, and normal liver. The expression of MDR1, MDR3, BSEP, MRP1, MRP2, and MRP3 was determined using specific antibodies. Dilution series were constructed to determine the critical staining level in order to estimate the factor of up-regulation. In normal liver, hepatocytes showed canalicular staining for MDR3, BSEP, and MRP2. MDR1 stained the canalicular membrane of hepatocytes as well as that of cholangiocytes. MRP3 showed low immunoreactivity of bile duct epithelial cells and centrilobular hepatocytes only. Normal liver showed no immunoreactivity for MRP1. In diseased liver, the expression of MDR3, BSEP, and MRP2 was relatively stable. In PBC, HCV, and submassive necrosis, the expression levels of MDR1, MRP1, and MRP3 were increased. The strongest immunoreactivity was seen after submassive necrosis, where remaining islands of hepatocytes showed strong canalicular staining for MDR1 and MRP3. Regenerating bile ductules at the interface of portal tracts and necrotic areas stained intensely for MDR1, MRP1, and MRP3. In conclusion, MDR1, MRP1, and MRP3 are up-regulated in hepatocytes in severe human liver disease. Strong MDR1, MRP1, and MRP3 reactivity is seen in regenerating human bile ductule

Reduced Vglut2/Slc17a6 Gene Expression Levels throughout the Mouse Subthalamic Nucleus Cause Cell Loss and Structural Disorganization Followed by Increased Motor Activity and Decreased Sugar Consumption.

The subthalamic nucleus (STN) plays a central role in motor, cognitive, and affective behavior. Deep brain stimulation (DBS) of the STN is the most common surgical intervention for advanced Parkinson's disease (PD), and STN has lately gained attention as target for DBS in neuropsychiatric disorders, including obsessive compulsive disorder, eating disorders, and addiction. Animal studies using STN-DBS, lesioning, or inactivation of STN neurons have been used extensively alongside clinical studies to unravel the structural organization, circuitry, and function of the STN. Recent studies in rodent STN models have exposed different roles for STN neurons in reward-related functions. We have previously shown that the majority of STN neurons express the vesicular glutamate transporter 2 gene (Vglut2/Slc17a6) and that reduction of Vglut2 mRNA levels within the STN of mice [conditional knockout (cKO)] causes reduced postsynaptic activity and behavioral hyperlocomotion. The cKO mice showed less interest in fatty rewards, which motivated analysis of reward-response. The current results demonstrate decreased sugar consumption and strong rearing behavior, whereas biochemical analyses show altered dopaminergic and peptidergic activity in the striatum. The behavioral alterations were in fact correlated with opposite effects in the dorsal versus the ventral striatum. Significant cell loss and disorganization of the STN structure was identified, which likely accounts for the observed alterations. Rare genetic variants of the human VGLUT2 gene exist, and this study shows that reduced Vglut2/Slc17a6 gene expression levels exclusively within the STN of mice is sufficient to cause strong modifications in both the STN and the mesostriatal dopamine system

Hepatic expression of ABC transporters G5 and G8 does not correlate with biliary cholesterol secretion in liver transplant patients

The adenosine triphosphate (ATP)-binding cassette (ABC)-transporters ABCG5 and ABCG8 have been shown to mediate hepatic and intestinal excretion of cholesterol. In various (genetically modified) marine models, a strong relationship was found between hepatic expression of ABCG5/ABCG8 and biliary cholesterol content. Our study aimed to relate levels of hepatic expression of ABCG5 and ABCG8 to biliary excretion of cholesterol in man. From 24 patients who had received a liver transplant, bile samples were collected daily after transplantation over a 2-week period to determine biliary composition. Expression of ABCG5, ABCG8, MDR3, and BSEP was assessed by real-time polymerase chain reaction (PCR) in liver biopsy specimens collected before and after transplantation. Levels of hepatic ABCG5, ABCG8, and MDR3 messenger RNA (mRNA) were strongly correlated. After transplantation, the biliary secretion rate of cholesterol continuously increased, coinciding with gradual increases in bile salt and phospholipid secretion. In contrast, hepatic levels of ABCG5 and ABCG8 mRNA remained unchanged. Surprisingly, no correlation was found between the hepatic expression of ABCG5 and ABCG8 and rates of biliary cholesterol secretion, normalized for biliary phospholipid. secretion. As expected, the concentration of biliary phospholipids correlated well with MDR3 expression. In conclusion the strong relationship between ABCG5 and ABCG8 gene expression is consistent with the coordinate regulation of both genes, and in line with heterodimerization of both proteins into a functional transporter. Hepatic ABCG5/ABCG8 expression, at least during the early phase after transplantation, is not directly related to biliary cholesterol secretion in humans. This finding suggests the existence of alternative pathways for the hepatobiliary transport of cholesterol that are not controlled by ABCG5/ABCG8.</p

Liver transplantation with preservation of the inferior vena cava. A comparison of conventional and piggyback techniques in adults

The aim of this study is to analyse a single centre's experience with two techniques of liver transplantation (OLT), conventional (CON-OLT) and piggyback (PB-ES), and to compare outcome in terms of survival, morbidity, mortality and post-operative liver function as well as operative characteristics. A consecutive series (1994-2000) of 167 adult primary OLT were analysed. Ninety-six patients had CON-OLT and 71 patients had PB-ES. In PB-ES group two revascularization protocols were used. In the first protocol reperfusion of the graft was performed first via the portal vein followed by the arterial anastomosis (PB-seq). In the second protocol the graft was reperfused simultaneously via portal vein and hepatic artery (PB-sim). One-, 3- and 5-yr patient survival in the CON-OLT and PB-ES groups were 90, 83 and 80%, and 83, 78 and 78%, respectively (p = ns). Graft survival at the same time points was 81, 73 and 69%, and 78, 69 and 65%, respectively (p = ns). Apart from the higher number of patients with cholangitis and sepsis in CON-OLT group, morbidity, retransplantation rate and post-operative liver and kidney function were not different between the two groups. The total operation time was not different between both groups (9.4 h in PB-ES vs. 10.0 h in CON-OLT), but in PB-ES group cold and warm ischaemia time (CIT and WIT), revascularization time (REVT), functional and anatomic anhepatic phases (FAHP and AAHP) were significantly shorter (8.9 h vs. 10.7 h, 54 min vs. 63 min, 82 min vs. 114 min, 118 min vs. 160 min and 87 min vs. 114 min, respectively, p <0.05). RBC use in the PB-ES group was lower compared to the CON-OLT group (4.0 min vs. 10.0 units, p <0.05). Except for WIT and REVT there were no differences in operative characteristics between PB-Sim and PB-Seq groups. The WIT was significantly longer in PB-Sim group compared with PB-Seq group (64 min vs. 50 min, p <0.05); however REVT was significantly shorter in PB-Sim group (64 min vs. 97 min, p <0.05). Results of this study show that both techniques are comparable in survival and morbidity; however PB-ES results in shorter AAHP, FAHP, REVT and WIT as well as less RBC use. In the PB-ES group there seems to be no adavantage for any of the revascularization protocols

Expression of heme oxygenase-1 in human livers before transplantation correlates with graft injury and function after transplantation

Upregulation of heme oxygenase-1 (HO-1) has been proposed as an adaptive mechanism protecting against ischemia/reperfusion (I/R) injury. We investigated HO-1 expression in 38 human liver transplants and correlated this with I/R injury and graft function. Before transplantation, median HO-1 mRNA levels were 3.4-fold higher (range: 0.7-9.3) in donors than in normal controls. Based on the median value, livers were divided into two groups: low and high HO-1 expression. These groups had similar donor characteristics, donor serum transaminases, cold ischemia time, HSP-70 expression and the distribution of HO-1 promoter polymorphism. After reperfusion, HO-1 expression increased significantly further in the initial low HO-1 expression group, but not in the high HO-1 group. Postoperatively, serum transaminases were significantly lower and the bile salt secretion was higher in the initial low HO-1 group, compared to the high expression group. Immunofluorescence staining identified Kupffer cells as the main localization of HO-1. In conclusion, human livers with initial low HO-1 expression (<3.4 times controls) are able to induce HO-1 further during reperfusion and are associated with less injury and better function than initial high HO-1 expression (> 3.4 times controls). These data suggest that an increase in HO-1 during transplantation is more protective than high HO-1 expression before transplantation

Lumateperone-mediated effects on prefrontal glutamatergic receptor-mediated neurotransmission : A dopamine D-1 receptor dependent mechanism

Lumateperone is a novel drug approved for the treatment of schizophrenia in adults and depressive episodes associated with bipolar depression in adults, as monotherapy and as adjunctive therapy with lithium or valproate treatment in the United States. Lumateperone simultaneously modulates key neurotransmitters, such as serotonin, dopamine, and glutamate, implicated in serious mental illness. In patients with schizophrenia, lumateperone was shown to improve positive symptoms along with negative and depressive symptoms, while also enhancing prosocial behavior. Moreover, in patients with bipolar I or II disorder, lumateperone improved depressive symptoms as well. To further understand the mechanisms related to lumateperone's clinical response, the aim of this study was to investigate the effect of lumateperone on dopaminergic-and glutamatergic signaling in the rat medial prefrontal cortex (mPFC). We used the conditioned avoidance response (CAR) test to determine the antipsychotic-like effect of lumateper one, electrophysiology in vitro to study lumateperone's effects on NMDA-and AMPA-induced currents in the mPFC, and the neurochemical techniques microdialysis and amperometry to measure dopamine-and glutamate release in the rat mPFC. Our results demonstrate that lu-mateperone; i) significantly suppressed CAR in rats, indicating an antipsychotic-like effect, ii) facilitated NMDA and AMPA receptor-mediated currents in the mPFC, in a dopamine D-1-dependent manner, and iii) significantly increased dopamine and glutamate release in the rat mPFC. To the extent that these findings can be translated to humans, the ability of lumate-perone to activate these pathways may contribute to its demonstrated effectiveness in safely improving symptoms related to neuropsychiatric disorder including mood alterations. (C) 2022 The Author(s). Published by Elsevier B.V