Οι τετρακυκλίνες ως αναστολείς της 3-μερκαπτοπυροσταφυλικής θειοτρανσφεράσης (3MST)

Το H2S κατατάσσεται στην ομάδα των αέριων διαβιβαστών μαζί με το NO και το CO. Παράγεται ενδογενώς κυρίως με ενζυμική διαδικασία από τα ένζυμα β-συνθετάση της κυσταθειονίνης (CBS), γ-λυάση της κυσταθειονίνης (CSE) και 3-μερκαπτοπυροσταφυλική θειοτρανσφεράση (3MST). Το H2S εμπλέκεται σε μια πληθώρα ενδογενών σηματοδοτικών μονοπατιών, ρυθμίζοντας έτσι διάφορες φυσιολογικές λειτουργίες. Η συμβολή του στην ομοιόσταση του οργανισμού γίνεται ιδιαίτερα εμφανής σε περιπτώσεις είτε μειωμένης, είτε αυξημένης ενδογενούς σύνθεσής του, οι οποίες οδηγούν ή συμμετέχουν στην ανάπτυξη νόσων. Με σκοπό την ενδελεχή διερεύνηση του ρόλου του αέριου διαβιβαστή στις διάφορες παθοφυσιολογικές καταστάσεις, καθίσταται απαραίτητη η διαλεύκανση των βιολογικών δράσεων των ενζύμων βιοσύνθεσής του στον οργανισμό. Δεδομένων των ελλιπών βιβλιογραφικών στοιχείων σχετικά με το ένζυμο 3MST -συγκριτικά με τα άλλα δύο ένζυμα παραγωγής υδροθείου- στην παρούσα διπλωματική εργασία επικεντρωθήκαμε στο εν λόγω ένζυμο. Σε πρώτο βήμα, προβήκαμε στη βελτιστοποίηση της παραγωγής του ενζύμου 3MST από βακτηριακά κύτταρα Escherichia coli, τα οποία έφεραν ως φορέα κλωνοποίησης πλασμίδιο με ενσωματωμένη την αλληλουχία του γονιδίου της ανθρώπινης πρωτεΐνης 3MST, ως χιμαιρική πρωτεΐνη με την S-τρανσφεράση της γλουταθειόνης (GST). Σε δεύτερο βήμα, έπειτα από λεπτομερή έρευνα της υπάρχουσας βιβλιογραφίας και αρκετές δοκιμές, καταφέραμε να βελτιστοποιήσουμε τη μέθοδο ‘κυανούν του μεθυλενίου’ (Methylene Blue Assay) για τον έλεγχο της δραστικότητας του συγκεκριμένου ενζύμου. Μέχρι προσφάτως, καμία ένωση δεν είχε χαρακτηριστεί ως αναστολέας του ενζύμου 3MST. Σημειώνεται πως η χρήση μορίων με ανασταλτική ή και ενισχυτική δράση σε in vitro και in vivo πειράματα, μπορεί να αποκαλύψει ενδιαφέροντα αποτελέσματα σχετικά με το ρόλο ενός ενζύμου στη φυσιολογία και παθοφυσιολογία. Με γνώμονα, λοιπόν, την ανακάλυψη μορίων με ανασταλτική δράση επί του ενζύμου ή έστω ‘μορίων-οδηγών’ που θα χρησιμοποιηθούν ως ‘καλούπια’ για τη σχεδίαση ισχυρότερων αναστολέων, προβήκαμε, τέλος, στη μελέτη της επίδρασης των μορίων των τετρακυκλινών στη δραστικότητα του ενζύμου. Τα πρώτα αποτελέσματα αυτής της εργασίας μπορούν να χαρακτηριστούν ως ενθαρρυντικά, με συγκεκριμένες τετρακυκλίνες να αποδεικνύονται αναστολείς του 3MST. Περαιτέρω πειράματα μάς έδωσαν ικανοποιητικά στοιχεία για να διατυπώσουμε υποθέσεις σχετικά με το μηχανισμό ανασταλτικής δράσης αυτών των ενώσεων.Hydrogen sulfide (H2S) is the third gasotransmitter after NO and CO and it is produced endogenously via non-enzymatic or enzymatic pathways, with the latter being the most common. Cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase are the enzymes responsible for its biosynthesis. H2S is involved in a variety of endogenous signaling pathways, thereby regulating various physiological functions. Its contribution to the homeostasis of the human organism becomes particularly evident in cases of either reduced or increased endogenous synthesis, which results in or contributes to different diseases. In order to thoroughly investigate the role of the gaseous transmitter in various pathophysiological conditions, it becomes necessary to elucidate the role of the enzymes of its biosynthesis in mammalian organisms. Given the fact that few bibliographic data is published upon the 3MST enzyme -comparably with the other two enzymes- our thesis focuses on 3MST. Firstly, we optimized the production of the 3MST enzyme from Escherichia coli bacterial cells. The plasmids that were introduced into the bacterial cells, contained the sequence of human 3MST in frame with glutathione S-transferase (GST). Secondly, following a detailed study of the existing literature and several experiments, we succeeded in optimizing the "methylene blue" method in order to examine the activity of this enzyme. It is noteworthy that the 3MST enzyme lacks extensive literature and until recently, no substance was described as its inhibitor. It is also well known that the use of molecules with inhibitory or amplifying activity in in vitro and in vivo experiments may reveal interesting results regarding the role of an enzyme in the physiology and pathophysiology of the studied organism. Therefore, the discovery of molecules with inhibitory activity on this enzyme or even the discovery of hit compounds which could be improved in order for stronger inhibitors to be made, would be of great significance. Given this, we tested the effect of different molecules characterized as tetracyclines on the enzyme activity. Our first results can be described as encouraging, as specific tetracyclines show inhibition of the 3MST. Further experiments allowed us to speculate the mechanism of these inhibitor molecules

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OBJECTIVE: Functional MRI has thus far demonstrated that HIV has an impact on frontal-striatal systems involved in executive functioning. The potential impact of HIV on frontal-striatal systems involved in reward processing has yet to be examined by functional MRI. This study therefore aims to investigate the effects of HIV infection on reward processing by examining the function of the ventral-striatal reward system during a monetary incentive delay task. DESIGN: This is a cross-sectional case-control study. METHODS: Eighteen combined antiretroviral therapy-naive HIV-positive (HIV+) participants, as well as 16 matched healthy controls, performed a monetary incentive delay task. This paradigm assesses behaviour as well as functional brain activity-associated reward anticipation and reward outcome. RESULTS: HIV+ participants showed a general decrease in activation associated with both neutral as well as potentially rewarding cues in their ventral striatum. We found normal activity related to reward outcome in the orbito-frontal cortex. Despite HIV+ participants' reaction times being significantly slower when independently measured from the reward paradigm, this performance deficit normalized during the performance of the reward task. CONCLUSION: HIV caused a decrease in activity during cue processing in the ventral striatum, with normal cortical functioning during reward outcome processing. Our results therefore suggest that HIV not only has an impact on fronto-striatal systems involved in executive functioning, but also has a direct impact on the function of the ventral-striatal reward system

Blood and cerebrospinal fluid biomarker changes in patients with HIV-associated neurocognitive impairment treated with lithium: analysis from a randomised placebo-controlled trial

HIV-associated neurocognitive disorders (HAND) persist in the era of antiretroviral therapy (ART). Thus, ART does not completely halt or reverse the pathological processes behind HAND. Adjuvant mitigating treatments are, therefore, prudent. Lithium treatment is known to promote neuronal brain–derived neurotrophic factors (BDNF). Lithium is also an inhibitor of glycogen synthase kinase-3 beta (GSK-3-β). We analyzed biomarkers obtained from participants in a randomized placebo-controlled trial of lithium in ART-treated individuals with moderate or severe HAND. We assayed markers at baseline and 24 weeks across several pathways hypothesized to be affected by HIV, inflammation, or degeneration. Investigated biomarkers included dopamine, BDNF, neurofilament light chain, and CD8 + lymphocyte activation (CD38 + HLADR +). Alzheimer’s Disease (AD) biomarkers included soluble amyloid precursor protein alpha and beta (sAPPα/β), Aβ38, 40, 42, and ten other biomarkers validated as predictors of mild cognitive impairment and progression in previous studies. These include apolipoprotein C3, pre-albumin, α1-acid glycoprotein, α1-antitrypsin, PEDF, CC4, ICAM-1, RANTES, clusterin, and cystatin c. We recruited 61 participants (placebo = 31; lithium = 30). The age baseline mean was 40 (± 8.35) years and the median CD4 + T-cell count was 498 (IQR: 389–651) cells/μL. Biomarker concentrations between groups did not differ at baseline. However, both groups’ blood dopamine levels decreased significantly after 24 weeks (adj. p < 002). No other marker was significantly different between groups, and we concluded that lithium did not confer neuroprotection following 24 weeks of treatment. However, the study was limited in duration and sample size

Application of economic evaluation to assess feasibility for reimbursement of genomic testing as part of personalized medicine interventions

Background: The incorporation of genomic testing into clinical practice constitutes an opportunity to improve patients’ lives, as it makes possible the implementation of innovative, individualized clinical interventions that maximize efficacy and/or minimize the risk of adverse drug reactions. In order to ensure equal access to genomic testing for all patients, the costs associated with these tests should be reimbursed by their respective national healthcare systems. Given that funding for the public health sector is decreasing in real terms, it is of paramount importance that the emerging interventions are thoroughly evaluated both in terms of their clinical effectiveness and their full economic cost. Objective: The aim of this study was to identify those genome-guided interventions that could be adopted and reimbursed by national healthcare systems. Further, we recorded the underlying factors determining the broad adoption of genome-guided interventions in clinical practice, in order to identify potential reimbursement criteria. Methods: We performed a systematic review of published (PubMed-listed) scientific articles on the economic evaluation of those individualized clinical interventions that include genomic tests. Information on genomic tests reimbursed by the US Medicare program was also included. Subsequently, we correlated the regulatory guidance given for the interventions collated in our systematic review with the corresponding economic evaluation results and policies of the Medicare program. Regulatory guidance information was collected from the PharmGKB online knowledgebase and the Clinical Pharmacogenetics Implementation Consortium (CPIC). Results: Most of the included studies constitute cost-utility analyses, in which the outcome of the interventions has been measured in quality-adjusted life years (QALYs) whereas an estimate of the total cost has been based upon direct medical cost data. Favorable economic evaluation results, as well as concrete evidence demonstrating the clinical utility of pre-emptive genotyping, are considered as prerequisites for the broad adoption and reimbursement of the costs incurred during genomic testing. Indicatively, pre-emptive HLA-B*5701 and TPMT testing before administration of abacavir and azathioprine, respectively, is reimbursed by Medicare based on both economic and efficacy evidence. Likewise, the medical necessary screening for MMR and BRCA1/2 genes are reimbursed for high-risk populations. Conclusions: Our findings further underline the need for further cost-utility analyses within different national healthcare systems, in order to promote the reimbursement of the cost of innovative genome-guided therapeutic interventions

Development of Degradable, pH‐Sensitive Star Vectors for Enhancing the Cytoplasmic Delivery of Nucleic Acids

The report describes the synthesis of degradable, pH‐sensitive, membrane‐destabilizing, star‐shaped polymers where copolymers of hydrophobic hexyl methacrylate (HMA) and 2‐(dimethylamino)ethyl methacrylate (DMAEMA) monomers are grafted from the secondary face of a beta‐cyclodextrin (β‐CD) core via acid‐labile hydrazone linkages using atom transfer radical polymerization. The effect of the graft's molecular weight, HMA/DMAEMA molar ratio, and the fraction of DMAEMA converted to cationic N,N,N‐trimethylaminoethyl methacrylate (TMAEMA) monomers on polymer's transfection capacity is systematically investigated. Results show that all star‐shaped polymers condense anti‐GAPDH silencing RNA (siRNA) into nanosized particles at +/‐ ratio ≤ 4:1. Star polymers with shorter (25kDa) P(HMA‐ co ‐DMAEMA‐ co ‐TMAEMA) grafts are more efficient and less cytotoxic than carriers with longer (40kDa) grafts. The results show that increasing the ratio of hydrophobic HMA monomers in graft's composition higher than 50 mole% dramatically reduces polymer's aqueous solubility and abolishes their transfection capacity. Further, retention of DMAEMA monomers in graft's composition provide a buffering capacity that enhanced the endosomal escape and transfection capacity of the polymers. These systematic studies show that β‐CD‐P(HMA‐ co ‐DMAEMA‐ co ‐TMAEMA) 4.8 polymer with a 25 kDa average graft's molecular weight and a 50/25/25 ratio of HMA/DMAEMA/TMAEMA monomers is the most efficient carrier in delivering the siRNA cargo into the cytoplasm of epithelial cancer cells. A series of degradable, pH‐sensitive, membrane‐destabilizing, star‐shaped polymers is synthesized. Star polymers are engineered to “sense” the drop in endosomal pH, which triggers the hydrolysis of acid‐labile hydrazone linkages and release of membrane‐active grafts that rupture the endosomal membrane and release the loaded siRNA cargo into the cytoplasm to produce the desired knockdown of targeted gene expression at both the mRNA and protein levels.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/99666/1/3885_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/99666/2/adfm_201203762_sm_suppl.pd

HIV Drug Resistance (HIVDR) in Antiretroviral Therapy-Naïve Patients in Tanzania Not Eligible for WHO Threshold HIVDR Survey Is Dramatically High

The World Health Organization (WHO) has recommended guidelines for a HIV drug resistance (HIVDR) survey for resource-limited countries. Eligibility criteria for patients include age below 25 years in order to focus on the prevalence of transmitted HIVDR (tHIVDR) in newly-infected individuals. Most of the participating sites across Africa have so far reported tHIVDR prevalences of below 5%. In this study we investigated whether the rate of HIVDR in patients <25 years is representative for HIVDR in the rest of the therapy-naïve population. HIVDR was determined in 88 sequentially enrolled ART-naïve patients from Mwanza, Tanzania (mean age 35.4 years). Twenty patients were aged <25 years and 68 patients were aged 25-63 years. The frequency of HIVDR in the study population was 14.8% (95%; CI 0.072-0.223) and independent of NVP-resistance induced by prevention of mother-to-child transmission programs. Patients >25 years had a significantly higher HIVDR frequency than younger patients (19.1%; 95% CI 0.095-0.28) versus 0%, P = 0.0344). In 2 out of the 16 patients with HIVDR we found traces of antiretrovirals (ARVs) in plasma. ART-naïve patients aged over 25 years exhibited significantly higher HIVDR than younger patients. Detection of traces of ARVs in individuals with HIVDR suggests that besides transmission, undisclosed misuse of ARVs may constitute a significant factor in the generation of the observed high HIVDR rate. The current WHO tHIVDR survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may therefore result in substantial underestimation of the prevalence of HIVDR in the therapy-naïve population. Similar studies should be performed also in other areas to test whether the so far reported optimistic picture of low HIVDR prevalence in young individuals is really representative for the rest of the ART-naïve HIV-infected population

HIV patients treated with low-dose prednisolone exhibit lower immune activation than untreated patients

HIV-associated general immune activation is a strong predictor for HIV disease progression, suggesting that chronic immune activation may drive HIV pathogenesis. Consequently, immunomodulating agents may decelerate HIV disease progression. In an observational study, we determined immune activation in HIV patients receiving low-dose (5 mg/day) prednisolone with or without highly-active antiretroviral therapy (HAART) compared to patients without prednisolone treatment. Lymphocyte activation was determined by flow cytometry detecting expression of CD38 on CD8(+) T cells. The monocyte activation markers sCD14 and LPS binding protein (LBP) as well as inflammation markers soluble urokinase plasminogen activated receptor (suPAR) and sCD40L were determined from plasma by ELISA. CD38-expression on CD8+ T lymphocytes was significantly lower in prednisolone-treated patients compared to untreated patients (median 55.40% [percentile range 48.76-67.70] versus 73.34% [65.21-78.92], p = 0.0011, Mann-Whitney test). Similarly, we detected lower levels of sCD14 (3.6 μg/ml [2.78-5.12] vs. 6.11 μg/ml [4.58-7.70]; p = 0.0048), LBP (2.18 ng/ml [1.59-2.87] vs. 3.45 ng/ml [1.84-5.03]; p = 0.0386), suPAR antigen (2.17 μg/ml [1.65-2.81] vs. 2.56 μg/ml [2.24-4.26]; p = 0.0351) and a trend towards lower levels of sCD40L (2.70 pg/ml [1.90-4.00] vs. 3.60 pg/ml [2.95-5.30]; p = 0.0782). Viral load in both groups was similar (0.8 × 105 ng/ml [0.2-42.4 × 105] vs. 1.1 × 105 [0.5-12.2 × 105]; p = 0.3806). No effects attributable to prednisolone were observed when patients receiving HAART in combination with prednisolone were compared to patients who received HAART alone.\ud Patients treated with low-dose prednisolone display significantly lower general immune activation than untreated patients. Further longitudinal studies are required to assess whether treatment with low-dose prednisolone translates into differences in HIV disease progression

Structural basis of subtype-selective competitive antagonism for GluN2C/2D-containing NMDA receptors.

N-Methyl-D-aspartate receptors (NMDARs) play critical roles in the central nervous system. Their heterotetrameric composition generates subtypes with distinct functional properties and spatio-temporal distribution in the brain, raising the possibility for subtype-specific targeting by pharmacological means for treatment of neurological diseases. While specific compounds for GluN2A and GluN2B-containing NMDARs are well established, those that target GluN2C and GluN2D are currently underdeveloped with low potency and uncharacterized binding modes. Here, using electrophysiology and X-ray crystallography, we show that UBP791 ((2S*,3R*)-1-(7-(2-carboxyethyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) inhibits GluN2C/2D with 40-fold selectivity over GluN2A-containing receptors, and that a methionine and a lysine residue in the ligand binding pocket (GluN2D-Met763/Lys766, GluN2C-Met736/Lys739) are the critical molecular elements for the subtype-specific binding. These findings led to development of UBP1700 ((2S*,3R*)-1-(7-(2-carboxyvinyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) which shows over 50-fold GluN2C/2D-selectivity over GluN2A with potencies in the low nanomolar range. Our study shows that the L-glutamate binding site can be targeted for GluN2C/2D-specific inhibition

Ginkgo biloba Extract in Alzheimer’s Disease: From Action Mechanisms to Medical Practice

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, is one of the most popular herbal supplements. Numerous preclinical studies have shown the neuroprotective effects of EGb761 and support the notion that it may be effective in the treatment and prevention of neurodegenerative disorders such as Alzheimer’s disease (AD). Despite the preclinical promise, the clinical efficacy of this drug remains elusive. In this review, possible mechanisms underlying neuroprotective actions of EGb761 are described in detail, together with a brief discussion of the problem of studying this herb clinically to verify its efficacy in the treatment and prevention of AD. Moreover, various parameters e.g., the dosage and the permeability of the blood brain barrier (BBB), impacting the outcome of the clinical effectiveness of the extract are also discussed. Overall, the findings summarized in this review suggest that, a better understanding of the neuroprotective mechanisms of EGb761 may contribute to better understanding of the effectiveness and complexity of this herb and may also be helpful for design of therapeutic strategies in future clinical practice. Therefore, in future clinical studies, different factors that could interfere with the effect of EGb761 should be considered