Development and Characterization of an Enzymatic Method for the Rapid Determination of Gamma Hydroxybutyric Acid

Gamma hydroxybutyric acid (GHB) is a regulated therapeutic drug, which naturally occurs in mammalian brain tissues as an intermediate of the GABA (gamma aminobutyric acid) neurotransmitter metabolism. The increasing misuse of GHB as a narcotic or abusing drug in recent years calls for the development of a simple and rapid screening method as an alternative to the currently available, technically demanding diagnostic methods. We have developed a rapid enzymatic assay based on the GHB dehydrogenase of Ralstonia eutropha. The enzyme is expressed as a recombinant protein in Escherichia coli and characterized in terms of reaction mechanism and kinetic parameters for the catalysis of conversion of GHB into succinic semialdehyde (SSA). The concomitant NADH production enables spectrophotometric monitoring of the reaction and the quantification of GHB in physiological fluids depending on initial velocities. We have tested a panel of twelve serum and urine samples containing GHB concentrations from 0.0 to 2.1 mmol/L. GHB dehydrogenase activity obeys a non classical bi bi ping pong mechanism exhibiting substrate inhibition by NAD+. With an optimal NAD+ concentration of 3.7 mmol/L in the reaction, the enzyme yields a KM of 1.0 mmol/L for GHB and a Vmax of 3.37 mmol/min/mg. The assay shows a linear standard curve from 0.1 to at least 1 mmol/L of GHB. Spiking experiments result in mean recoveries of 92% for urine and 114% for serum, respectively. The comparison to an ion chromatographic reference method exhibits a mean difference of 10% divergence from the target values in urine and 9% in serum, respectively

Supplementary data for article : Konstantinović, J. M.; Selaković, M.; Srbljanovic, J.; Djurkovic-Djakovic, O.; Bogojević, K.; Sciotti, R.; Šolaja, B. A. Antimalarials with Benzothiophene Moieties as Aminoquinoline Partners. Molecules 2017, 22 (3). https://doi.org/10.3390/molecules22030343

Supplementary material for: [https://doi.org/10.3390/molecules22030343]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2441

Supplementary data for article : Konstantinović, J. M.; Selaković, M.; Srbljanovic, J.; Djurkovic-Djakovic, O.; Bogojević, K.; Sciotti, R.; Šolaja, B. A. Antimalarials with Benzothiophene Moieties as Aminoquinoline Partners. Molecules 2017, 22 (3). https://doi.org/10.3390/molecules22030343

Supplementary material for: [https://doi.org/10.3390/molecules22030343]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2441

Sleep-wake sensitive mechanisms of adenosine release in the basal forebrain of rodents : an in vitro study

Adenosine acting in the basal forebrain is a key mediator of sleep homeostasis. Extracellular adenosine concentrations increase during wakefulness, especially during prolonged wakefulness and lead to increased sleep pressure and subsequent rebound sleep. The release of endogenous adenosine during the sleep-wake cycle has mainly been studied in vivo with microdialysis techniques. The biochemical changes that accompany sleep-wake status may be preserved in vitro. We have therefore used adenosine-sensitive biosensors in slices of the basal forebrain (BFB) to study both depolarization-evoked adenosine release and the steady state adenosine tone in rats, mice and hamsters. Adenosine release was evoked by high K+, AMPA, NMDA and mGlu receptor agonists, but not by other transmitters associated with wakefulness such as orexin, histamine or neurotensin. Evoked and basal adenosine release in the BFB in vitro exhibited three key features: the magnitude of each varied systematically with the diurnal time at which the animal was sacrificed; sleep deprivation prior to sacrifice greatly increased both evoked adenosine release and the basal tone; and the enhancement of evoked adenosine release and basal tone resulting from sleep deprivation was reversed by the inducible nitric oxide synthase (iNOS) inhibitor, 1400 W. These data indicate that characteristics of adenosine release recorded in the BFB in vitro reflect those that have been linked in vivo to the homeostatic control of sleep. Our results provide methodologically independent support for a key role for induction of iNOS as a trigger for enhanced adenosine release following sleep deprivation and suggest that this induction may constitute a biochemical memory of this state

A case report of a patient with upper extremity symptoms: differentiating radicular and referred pain

<p>Abstract</p> <p>Background</p> <p>Similar upper extremity symptoms can present with varied physiologic etiologies. However, due to the multifaceted nature of musculoskeletal conditions, a definitive diagnosis using physical examination and advanced testing is not always possible. This report discusses the diagnosis and case management of a patient with two episodes of similar upper extremity symptoms of different etiologies.</p> <p>Case Presentation</p> <p>On two separate occasions a forty-four year old female patient presented to a chiropractic office with a chief complaint of insidious right-sided upper extremity symptoms. During each episode she reported similar pain and parasthesias from her neck and shoulder to her lateral forearm and hand.</p> <p>During the first episode the patient was diagnosed with a cervical radiculopathy. Conservative treatment, including manual cervical traction, spinal manipulation and neuromobilization, was initiated and resolved the symptoms.</p> <p>Approximately eighteen months later the patient again experienced a severe acute flare-up of the upper extremity symptoms. Although the subjective complaint was similar, it was determined that the pain generator of this episode was an active trigger point of the infraspinatus muscle. A diagnosis of myofascial referred pain was made and a protocol of manual trigger point therapy and functional postural rehabilitative exercises improved the condition.</p> <p>Conclusion</p> <p>In this case a thorough physical evaluation was able to differentiate between radicular and referred pain. By accurately identifying the pain generating structures, the appropriate rehabilitative protocol was prescribed and led to a successful outcome for each condition. Conservative manual therapy and rehabilitative exercises may be an effective treatment for certain cases of cervical radiculopathy and myofascial referred pain.</p

Discrepancy between prevalence and perceived effectiveness of treatment methods in myofascial pain syndrome: Results of a cross-sectional, nationwide survey

Background: Myofascial pain is a common dysfunction with a lifetime prevalence affecting up to 85% of the general population. Current guidelines for the management of myofascial pain are not available. In this study we investigated how physicians on the basis of prescription behaviour evaluate the effectiveness of treatment options in their management of myofascial pain. Methods: We conducted a cross-sectional, nationwide survey with a standardized questionnaire among 332 physicians (79.8% male, 25.6% female, 47.5 +/- 9.6 years) experienced in treating patients with myofascial pain. Recruitment of physicians took place at three German meetings of pain therapists, rheumatologists and orthopaedists, respectively. Physicians estimated the prevalence of myofascial pain amongst patients in their practices, stated what treatments they used routinely and then rated the perceived treatment effectiveness on a six-point scale (with 1 being excellent). Data are expressed as mean +/- standard deviation. Results: The estimated overall prevalence of active myofascial trigger points is 46.1 +/- 27.4%. Frequently prescribed treatments are analgesics, mainly metamizol/paracetamol (91.6%), non-steroidal anti-inflammatory drugs/coxibs (87.0%) or weak opioids (81.8%), and physical therapies, mainly manual therapy (81.1%), TENS (72.9%) or acupuncture (60.2%). Overall effectiveness ratings for analgesics (2.9 +/- 0.7) and physical therapies were moderate (2.5 +/- 0.8). Effectiveness ratings of the various treatment options between specialities were widely variant. 54.3% of all physicians characterized the available treatment options as insufficient. Conclusions: Myofascial pain was estimated a prevalent condition. Despite a variety of commonly prescribed treatments, the moderate effectiveness ratings and the frequent characterizations of the available treatments as insufficient suggest an urgent need for clinical research to establish evidence-based guidelines for the treatment of myofascial pain syndrome

Supplementary data for article: Opsenica, I.; Burnett, J. C.; Gussio, R.; Opsenica, D. M.; Todorović, N.; Lanteri, C. A.; Sciotti, R. J.; Gettayacamin, M.; Basilico, N.; Taramelli, D.; et al. A Chemotype That Inhibits Three Unrelated Pathogenic Targets: The Botulinum Neurotoxin Serotype A Light Chain, P. Falciparum Malaria, and the Ebola Filovirus. Journal of Medicinal Chemistry 2011, 54 (5), 1157–1169. https://doi.org/10.1021/jm100938u

Supporting information for: [https://doi.org/10.1021/jm100938u]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1157

Euclid preparation. TBD. Forecast impact of super-sample covariance on 3x2pt analysis with Euclid

Deviations from Gaussianity in the distribution of the fields probed by large-scale structure surveys generate additional terms in the data covariance matrix, increasing the uncertainties in the measurement of the cosmological parameters. Super-sample covariance (SSC) is among the largest of these non-Gaussian contributions, with the potential to significantly degrade constraints on some of the parameters of the cosmological model under study -- especially for weak lensing cosmic shear. We compute and validate the impact of SSC on the forecast uncertainties on the cosmological parameters for the Euclid photometric survey, obtained with a Fisher matrix analysis, both considering the Gaussian covariance alone and adding the SSC term -- computed through the public code PySSC. The photometric probes are considered in isolation and combined in the `3$\times$2pt' analysis. We find the SSC impact to be non-negligible -- halving the Figure of Merit of the dark energy parameters ($w_0$, $w_a$) in the 3$\times$2pt case and substantially increasing the uncertainties on $\Omega_{{\rm m},0}, w_0$, and $\sigma_8$ for cosmic shear; photometric galaxy clustering, on the other hand, is less affected due to the lower probe response. The relative impact of SSC does not show significant changes under variations of the redshift binning scheme, while it is smaller for weak lensing when marginalising over the multiplicative shear bias nuisance parameters, which also leads to poorer constraints on the cosmological parameters. Finally, we explore how the use of prior information on the shear and galaxy bias changes the SSC impact. Improving shear bias priors does not have a significant impact, while galaxy bias must be calibrated to sub-percent level to increase the Figure of Merit by the large amount needed to achieve the value when SSC is not included.Comment: 22 pages, 13 figure