OxyHbMeter-a novel bedside medical device for monitoring cell-free hemoglobin in the cerebrospinal fluid-proof of principle.

Delayed cerebral ischemia (DCI) occurs in up to one third of patients suffering from aneurysmal subarachnoid hemorrhage (aSAH). Untreated, it leads to secondary cerebral infarctions and is frequently associated with death or severe disability. After aneurysm rupture, erythrocytes in the subarachnoid space lyse and liberate free hemoglobin (Hb), a key driver for the development of DCI. Hemoglobin in the cerebrospinal fluid (CSF-Hb) can be analyzed through a two-step procedure of centrifugation to exclude intact erythrocytes and subsequent spectrophotometric quantification. This analysis can only be done in specialized laboratories but not at the bedside in the intensive care unit. This limits the number of tests done, increases the variability of the results and restricts accuracy. Bedside measurements of CSF-Hb as a biomarker with a point of care diagnostic test system would allow for a continuous monitoring for the risk of DCI in the individual patient. In this study, a microfluidic chip was explored that allows to continuously separate blood particles from CSF or plasma based on acoustophoresis. An in vitro test bench was developed to test in-line measurements with the developed microfluidic chip and a spectrometer. The proof of principle for a continuous particle separation device has been established with diluted blood and CSF samples from animals and aSAH patients, respectively. Processing 1 mL of blood in our microfluidic device was achieved within around 70 min demonstrating only minor deviations from the gold standard centrifugation (7% average error of patient samples), while saving several hours of processing time and additionally the reduction of deviations in the results due to manual labor.

Patients' Use of a Standardized Medication List - A Mixed Methods Study.

Purpose: A medication list (ML) is a document listing the patient's entire medication, instructions for use, and indications. In Germany, a national standard was established in 2016 by law. However, data on patients' use of this standardized ML are scarce. We investigated (i) patients' practical use of the ML, (ii) patients' understanding of the ML, (iii) completeness and correctness of the current ML version, and (iv) reasons why patients did not adhere to their ML. Patients and Methods: Community pharmacists recruited patients possessing a standardized ML with ≥5 medications. Information sources to evaluate the ML were: (a) brown bag analysis, (b) practical demonstration, (c) patient interview, and (d) patient file. Data were analyzed using qualitative and quantitative methods. Results: Two hundred and eighty-eight patients (median age: 76 years, range: 27-95) were enrolled. (i) 38.5% of the patients used their ML regularly to prepare their medication and 73.3% to inform their physician. (ii) Overall, patients' understanding of the ML was good, with >80% of the patients being able to identify all relevant information. (iii) While n = 2779 medications were actually taken, n = 2539 were documented on the ML. No ML was fully correct and complete. Regarding particularly relevant items, ie, active ingredient, strength, dosage, medication missing or listed but not taken, 79.2% of ML were incorrect or incomplete. Handwritten modifications on the ML were frequent. (iv) Almost 60% of all patients did not follow their ML with "fear of adverse drug reactions" being the most frequently (n = 50) mentioned reason. Conclusion: Completeness and correctness of the current ML version was poor with handwritten modifications being frequent. Additionally, most of the patients did not adhere to their ML. This indicates that measures that lead to correct and up-to-date ML and improvements in patient counseling about their medication should be developed and implemented into routine practice.

Suppression of Rotational Domains of CuI Employing Sodium Halide Buffer Layers.

The occurrence of rotational domains is a well-known issue for copper iodide (CuI) that naturally occurs for growth on popular substrates like sapphire. However, this has detrimental effects on the thin film quality like increasing surface roughness or deteriorated transport characteristics due to grain boundary scattering. Utilizing pulsed laser deposition and the in situ growth of sodium chloride (NaCl) and sodium bromide (NaBr) template layers, studies were performed on their potential on suppressing the formation of rotational domains of CuI on c-plane sapphire and SrF2(111) substrates. Corresponding samples were investigated concerning their epitaxial properties and further characterized regarding (volume) crystalline, morphological, and electrical properties. Particularly for NaBr template layers, fully single-crystalline growth of CuI thin films was obtained and resulted in significantly reduced surface roughness of the CuI layer.

Analysis of Signal Transduction Pathways Downstream M2 Receptor Activation: Effects on Schwann Cell Migration and Morphology.

BACKGROUND: Schwann cells (SCs) express cholinergic receptors, suggesting a role of cholinergic signaling in the control of SC proliferation, differentiation and/or myelination. Our previous studies largely demonstrated that the pharmacological activation of the M2 muscarinic receptor subtype caused an inhibition of cell proliferation and promoted the expression of pro-myelinating differentiation genes. In order to elucidate the molecular signaling activated downstream the M2 receptor activation, in the present study we investigated the signal transduction pathways activated by the M2 orthosteric agonist arecaidine propargyl ester (APE) in SCs. METHODS: Using Western blot we analyzed some components of the noncanonical pathways involving ß1-arrestin and PI3K/AKT/mTORC1 signaling. A wound healing assay was used to evaluate SC migration. RESULTS: Our results demonstrated that M2 receptor activation negatively modulated the PI3K/Akt/mTORC1 axis, possibly through ß1-arrestin downregulation. The involvement of the mTORC1 complex was also supported by the decreased expression of its specific target p-p70 S6KThr389. Then, we also analyzed the expression of p-AMPKαthr172, a negative regulator of myelination that resulted in reduced levels after M2 agonist treatment. The analysis of cell migration and morphology allowed us to demonstrate that M2 receptor activation caused an arrest of SC migration and modified cell morphology probably by the modulation of ß1-arrestin/cofilin-1 and PKCα expression, respectively. CONCLUSIONS: The data obtained demonstrated that M2 receptor activation in addition to the canonical Gi protein-coupled pathway modulates noncanonical pathways involving the mTORC1 complex and other kinases whose activation may contribute to the inhibition of SC proliferation and migration and address SC differentiation.

The Mechanisms Mediated by α7 Acetylcholine Nicotinic Receptors May Contribute to Peripheral Nerve Regeneration.

Due to the microenvironment created by Schwann cell (SC) activity, peripheral nerve fibers are able to regenerate. Inflammation is the first response to nerve damage and the removal of cellular and myelin debris is essential in preventing the persistence of the local inflammation that may negatively affect nerve regeneration. Acetylcholine (ACh) is one of the neurotransmitters involved in the modulation of inflammation through the activity of its receptors, belonging to both the muscarinic and nicotinic classes. In this report, we evaluated the expression of α7 nicotinic acetylcholine receptors (nAChRs) in rat sciatic nerve, particularly in SCs, after peripheral nerve injury. α7 nAChRs are absent in sciatic nerve immediately after dissection, but their expression is significantly enhanced in SCs after 24 h in cultured sciatic nerve segments or in the presence of the proinflammatory neuropeptide Bradykinin (BK). Moreover, we found that activation of α7 nAChRs with the selective partial agonist ICH3 causes a decreased expression of c-Jun and an upregulation of uPA, MMP2 and MMP9 activity. In addition, ICH3 treatment inhibits IL-6 transcript level expression as well as the cytokine release. These results suggest that ACh, probably released from regenerating axons or by SC themselves, may actively promote through α7 nAChRs activation an anti-inflammatory microenvironment that contributes to better improving the peripheral nerve regeneration.

Cholinergic Modulation of Neuroinflammation: Focus on α7 Nicotinic Receptor.

All nervous system pathologies (e.g., neurodegenerative/demyelinating diseases and brain tumours) develop neuroinflammation, a beneficial process during pathological events, aimed at removing damaged cells, toxic agents, and/or pathogens. Unfortunately, excessive inflammation frequently occurs during nervous system disorders, becoming a detrimental event capable of enhancing neurons and myelinating glial cell impairment, rather than improving their survival and activity. Consequently, targeting the neuroinflammation could be relevant for reducing brain injury and rescuing neuronal and glial cell functions. Several studies have highlighted the role of acetylcholine and its receptors in the regulation of central and peripheral inflammation. In particular, α7 nicotinic receptor has been described as one of the main regulators of the "brain cholinergic anti-inflammatory pathway". Its expression in astrocytes and microglial cells and the ability to modulate anti-inflammatory cytokines make this receptor a new interesting therapeutic target for neuroinflammation regulation. In this review, we summarize the distribution and physiological functions of the α7 nicotinic receptor in glial cells (astrocytes and microglia) and its role in the modulation of neuroinflammation. Moreover, we explore how its altered expression and function contribute to the development of different neurological pathologies and exacerbate neuroinflammatory processes.

Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons.

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

Structural Insights into BET Client Recognition of Endometrial and Prostate Cancer-Associated SPOP Mutants.

BET proteins such as BRD3 are oncogenic transcriptional coactivators. SPOP binding triggers their proteasomal degradation. In both endometrial and prostate cancers, SPOP mutations occur in the MATH domain, but with opposed influence on drug susceptibility. In prostate cancer, SPOP mutations presumably cause increased BET levels, decreasing BET inhibitor drug susceptibility. As opposed, in endometrial cancer, decreased BET levels concomitant with higher BET inhibitor drug susceptibility were observed. Here, we present the to our knowledge first co-crystal structure of SPOP and a bromodomain containing protein (BRD3). Our structural and biophysical data confirm the suggested loss-of-function in prostate cancer-associated SPOP mutants and provide mechanistic explanation. As opposed to previous literature, our data on endometrial cancer-associated SPOP mutants do not show altered binding behavior compared to wild-type SPOP, indicating a more complex regulatory mechanism. SPOP mutation screening may thus be considered a valuable personalized medicine tool for effective antitumor therapy.

The Structure of the SPOP-Pdx1 Interface Reveals Insights into the Phosphorylation-Dependent Binding Regulation.

Pdx1 is a transcription factor crucial for development and maintenance of a functional pancreas. It regulates insulin expression and glucose homeostasis. SPOP is an E3-ubiquitin ligase adaptor protein that binds Pdx1, thus triggering its ubiquitination and proteasomal degradation. However, the underlying mechanisms are not well understood. Here, we present the crystal structure of the SPOP-Pdx1 complex. We show that Pdx1 residues 223-233 bind to SPOP MATH domain with low micromolar affinity. The interface is extended compared to other SPOP-client proteins. Previously, Pdx1 phosphorylation has been proposed to have a regulatory function. In this respect we show that phosphorylation lowers the affinity of Pdx1 to SPOP by isothermal titration calorimetry and nuclear magnetic resonance data. Our data provide insights into a critical protein-protein interaction that regulates cellular Pdx1 levels by SPOP-mediated decay. A reduction of Pdx1 levels in ß cells is linked to apoptosis and considered a hallmark of type 2 diabetes.

Adenosine A2A receptor contributes to the anti-inflammatory effect of the fixed herbal combination STW 5 (Iberogast®) in rat small intestinal preparations.

STW 5 (Iberogast®), an established herbal combination, was effective in randomized, double blind clinical studies in functional dyspepsia and irritable bowel syndrome. Since STW 5 was found to influence intestinal motility and has anti-inflammatory properties, this study investigated the expression of adenosine receptors and characterized their role in the control of the anti-inflammatory action of STW 5 and its fresh plant component STW 6 in inflammation-disturbed rat small intestinal preparations. The inflammation was induced by intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS, 0.01 M). The effects of coincubation with selective receptor agonists and antagonists, STW 5, STW 6, or combinations of these compounds on acetylcholine (ACh)-evoked contraction of ileum/jejunum preparations were tested. Adenosine receptor mRNA expression was examined by reverse transcription-polymerase chain reaction (RT-PCR). In untreated preparations, RT-PCR revealed the presence of all adenosine receptor subtypes. Suppressed expression was detected for all subtypes in inflamed tissues, except for A(2B)R mRNA, which was unaffected. STW 5 reversed these effects and enhanced A(2A)R expression above control levels. Radioligand binding assays confirm the affinity of STW 5 to the A(2A)R, and the A(2A)R antagonist was able to prevent the effect of STW 5 on TNBS-induced attenuation of the ACh contraction. Our findings provide evidence that STW 5, but not STW 6 interacts with A(2A)R, which is involved in the anti-inflammatory action of STW 5. STW 6 did not contribute to adenosine A(2A)R-mediated anti-inflammatory effect of STW 5. Other signaling pathways could be involved in the mechanism of action of STW 6.

Development of Polar Adenosine A2A Receptor Agonists for Inflammatory Bowel Disease: Synergism with A2B Antagonists.

Adenosine A2A receptor agonists for the local treatment of inflammatory bowel disease (IBS) were designed and synthesized. Polar groups were introduced to prevent peroral absorption and subsequent systemic, e.g., hypotensive, side effects. 4-(2-{6-Amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purin-2-ylthio}ethyl)benzenesulfonic acid (7, PSB-0777) was selected for further evaluation in rat ileum/jejunum preparations in ex vivo experiments. Compound 7 significantly improved impaired acetylcholine-induced contractions induced by 2,4,6-trinitrobenzenesulfonic acid and showed synergism with an A2B-selective antagonist. Thus, nonabsorbable, locally active A2A agonists, as a monotherapy or in combination with an A2B antagonist, may be an efficient novel treatment for IBS, preventing the severe systemic side effects of known A2A agonists.

Adenosine A(2A) agonist and A(2B) antagonist mediate an inhibition of inflammation-induced contractile disturbance of a rat gastrointestinal preparation.

Adenosine can show anti-inflammatory as well as pro-inflammatory activities. The contribution of the specific adenosine receptor subtypes in various cells, tissues and organs is complex. In this study, we examined the effect of the adenosine A(2A) receptor agonist CGS 21680 and the A(2B)R antagonist PSB-1115 on acute inflammation induced experimentally by 2,4,6-trinitrobenzenesulfonic acid (TNBS) on rat ileum/jejunum preparations. Pre-incubation of the ileum/jejunum segments with TNBS for 30 min resulted in a concentration-dependent inhibition of acetylcholine (ACh)-induced contractions. Pharmacological activation of the A(2A)R with CGS 21680 (0.1-10 microM) pre-incubated simultaneously with TNBS (10 mM) prevented concentration-dependently the TNBS-induced inhibition of the ACh contractions. Stimulation of A(2B)R with the selective agonist BAY 60-6583 (10 microM) did neither result in an increase nor in a further decrease of ACh-induced contractions compared to the TNBS-induced inhibition. The simultaneous pre-incubation of the ileum/jejunum segments with TNBS (10 mM) and the selective A(2B)R antagonist PSB-1115 (100 microM) inhibited the contraction-decreasing effect of TNBS. The effects of the A(2A)R agonist and the A(2B)R antagonist were in the same range as the effect induced by 1 microM methotrexate. The combination of the A(2A)R agonist CGS 21680 and the A(2B)R antagonist PSB-1115 at subthreshold concentrations of both agents found a significant amelioration of the TNBS-diminished contractility. Our results demonstrate that the activation of A(2A) receptors or the blockade of the A(2B) receptors can prevent the inflammation-induced disturbance of the ACh-induced contraction in TNBS pre-treated small intestinal preparations. The combination of both may be useful for the treatment of inflammatory bowel diseases.

Inhibition of inflammation-induced alterations in rat small intestine by the herbal preparations STW 5 and STW 6.

Inflammation is a common mechanism of many gastrointestinal diseases. Therefore, it is interesting to know, whether complex phytopharmaceuticals known to modulate gastrointestinal motor function reveal also anti-inflammatory properties. We tested the fixed herbal combination product STW 5 (Iberogast((R))) and its main component Iberis amara fresh plant extract (STW 6) to characterize their protective potential in an experimental inflammation model in vitro. The test system consisted of ileum/jejunum segments from male Wistar rats. Inflammation was evoked by intraluminal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) for 30min. Preincubation of TNBS together with STW 5 and STW 6 prevented the TNBS-induced inhibition of ACh-induced contractions. No differences were found between water-dissolved and ethanol-dissolved extracts. STW 5 and STW 6 reduced morphological changes induced by TNBS in mucosal and muscle layers. The IL-10 mRNA measured by qRT-PCR was not influenced by TNBS but increased by STW 5 and STW 6. The TNBS-induced increase in the TNFalpha-mRNA expression was suppressed by STW 5 but not by STW 6. Additionally, STW 5 decreased TNFalpha release in LPS-stimulated human monocytes. STW 6 influenced neither the TNFalpha-mRNA nor the TNFalpha release. These findings demonstrate that STW 5 reduced inflammation-induced alterations in ileum/jejunum segments. The effects were associated with a restoration of the disturbed ACh-induced contraction, pathohistological protection and inhibition of TNFalpha. STW 6 may contribute to the protective effect of STW 5 mainly by increasing IL-10 pathway but not by influencing TNFalpha.