Backbone modifications in peptidic inhibitors of flaviviral proteases.

The NS2B-NS3 protease is a promising target for the development of drugs against dengue virus (DENV), West Nile virus (WNV) and related flaviviruses. We report the systematic variation of the peptide backbone of the two lead compounds Bz-Arg-Lys-d-Phg-NH2 and Bz-Arg-Lys-d-Phg(OBn)-NH2. While inhibitory activity against WNV protease was generally decreased, the inhibitory potency against DENV protease could be conserved and increased in several peptidomimetics, particularly in those containing a (NMe)arginine fragment or an N-terminal α-keto amide. Methylation at the α-position of the C-terminal phenylglycine led to a 6-fold higher potency against DENV protease. Peptidomimetics with modified backbone showed increased resistance against hydrolytic attack by trypsin and α-chymotrypsin.

Streamlined open-source gel dosimetry analysis in 3D slicer.

Three dimensional dosimetry is being used in an increasingly wide variety of clinical applications as more gel and radiochromic plastic dosimeters become available. However, accessible 3D dosimetry analysis tools have not kept pace. 3D dosimetry data analysis is time consuming and laborious, creating a barrier to entry for busy clinical environments. To help in the adoption of 3D dosimetry, we have produced a streamlined, open-source dosimetry analysis system by developing a custom extension in 3D Slicer, called the Gel Dosimetry Analysis slicelet, which enables rapid and accurate data analysis. To assist those interested in adopting 3D dosimetry in their clinic or those unfamiliar with what is involved in a 3D dosimeter experiment, we first present the workflow of a typical gel dosimetry experiment. This is followed by the results of experiments used to validate, step-wise, each component of our software. Overall, our software has made a full 3D gel dosimeter analysis roughly 20 times faster than previous analysis systems.

Leuco-crystal-violet micelle gel dosimeters: II. Recipe optimization and testing.

In this study, recipe optimization of Leuco Crystal Violet (LCV) micelle gels made with the surfactant Cetyl Trimethyl Ammonium Bromide (CTAB) and the chemical sensitizer 2,2,2-trichloroethanol (TCE) was aided by a two-level three-factor designed experiment. The optimized recipe contains 0.75 mM LCV, 17.0 mM CTAB, 120 mM TCE, 25.0 mM tri-chloro acetic acid (TCAA), 4 wt% gelatin and ~96 wt% water. Dose sensitivity of the optimized gel is 1.5 times higher than that of Jordan's standard LCV micelle gel. Spatial integrity of the 3D dose distribution information in 1L phantoms filled with this recipe is maintained for >120 d. Unfortunately, phantoms made using the optimized recipe showed dose-rate dependence (14% difference in optical attenuation at the peak dose using electron beam irradiations at 100 and 400 MU min(-1)). Further testing suggests that the surfactant CTAB is the cause of this dose rate behaviour.

Chemical differentiation of two taste variants of Gynostemma pentaphyllum by using UPLC-Q-TOF-MS and HPLC-ELSD.

To differentiate the sweet and bitter taste variants of a Chinese medicinal tea Gynostemma pentaphyllum (GP), a method for the quantitative analysis of ginsenosides Rb(1), Rb(3), Rd, and F(2) in GP by using UPLC-Q-TOF-MS was developed. According to the different contents of the four ginsenosides, chemical differentiation of the two taste variants of GP was achieved by principal component analysis (PCA). A supplementary quantitative analysis method of using HPLC-ELSD for determination of 20(S)-panaxadiol in the hydrolysates of GP was also developed. Similarly, chemical differentiation based on different amounts of 20(S)-panaxadiol was established and the result was well consistent with that based on the analysis of the four ginsenosides. It was found that the amounts of the four ginsenosides and 20(S)-panaxadiol in the sweet taste variant were significantly higher than those in the bitter one. The significant difference between the sweet and bitter taste variants of GP was easily visualized in 3D-PCA score plots. The PCA loading plot also indicated the contributions among the four ginsenosides (Rd > Rb(3) > F(2) > Rb(1)) for distinguishing the two taste variants. This is the first report to describe the use of these two quantitative methods (UPLC-Q-TOF-MS and HPLC-ELSD) for the accurate authentication and quality control of GP.

Smoking is associated with an age-related decline in exhaled nitric oxide.

Age-related declines in forced expiratory volume in one second are accelerated in smokers. Smoking is associated with decreased exhaled nitric oxide fraction (F(eNO)). The aim of the present study was to determine the impact of age on F(eNO) in otherwise healthy smokers and nonsmokers. F(eNO) and serum cotinine levels were measured in 994 healthy subjects aged 18-40 yrs. American Thoracic Society questionnaire data on smoking habits was used to validate serum cotinine levels as a surrogate marker for categorisation of smokers and nonsmokers in the cohort. Serum cotinine levels were a good discriminator of smokers (n = 99) and nonsmokers (n = 895). F(eNO) levels were significantly lower in otherwise healthy smokers compared with nonsmokers. There was an inverse correlation of serum cotinine levels with F(eNO). No correlation of age with F(eNO) was found in nonsmokers but an inverse correlation of F(eNO) with age in smokers was found. F(eNO) was significantly lower in smokers aged 21-40 yrs compared with nonsmokers aged 21-40 yrs, but was not lower in smokers aged 18-20 yrs compared with nonsmokers of the same age. Smoking was associated with decreased exhaled nitric oxide. The greatest smoking-related declines in exhaled nitric oxide occurred in older subjects. This suggests that smoking is associated with age-related declines in exhaled nitric oxide and justifies future mechanistic studies that address the impact of exhaled nitric oxide decline on lung function.

Metabolic, but not respiratory, acidosis increases bone PGE(2) levels and calcium release.

A decrease in blood pH may be due to either a reduction in bicarbonate concentration ([HCO(3)(-)]; metabolic acidosis) or to an increase in PCO(2) (respiratory acidosis). In mammals, metabolic, but not respiratory, acidosis increases urine calcium excretion without altering intestinal calcium absorption, indicating that the additional urinary calcium is derived from bone. In cultured bone, chronic metabolic, but not respiratory, acidosis increases net calcium efflux (J(Ca)), decreases osteoblastic collagen synthesis, and increases osteoclastic bone resorption. Metabolic acidosis increases bone PGE(2) production, which is correlated with J(Ca), and inhibition of PGE(2) production inhibits this acid-induced J(Ca). Given the marked differences in the osseous response to metabolic and respiratory acidosis, we hypothesized that incubation of neonatal mouse calvariae in medium simulating respiratory acidosis would not increase medium PGE(2) levels, as observed during metabolic acidosis. To test this hypothesis, we determined medium PGE(2) levels and J(Ca) from calvariae incubated at pH approximately 7.1 to model either metabolic (Met; [HCO(3)(-)] approximately 11 mM) or respiratory (Resp; PCO(2) approximately 83 Torr) acidosis, or at pH approximately 7.5 as a control (Ntl). We found that after 24-48 and 48-51 h in culture, periods when cell-mediated J(Ca) predominates, medium PGE(2) levels and J(Ca) were increased with Met, but not Resp, compared with Ntl, and there was a direct correlation between medium PGE(2) levels and J(Ca). Thus metabolic, but not respiratory, acidosis induces the release of bone PGE(2), which mediates J(Ca) from bone.

Pyridopyrimidine analogues as novel adenosine kinase inhibitors.

A novel series of pyridopyrimidine analogues 9 was identified as potent adenosine kinase inhibitors based on the SAR and computational studies. Substitution of the C7 position of the pyridopyrimidino core with C2' substituted pyridino moiety increased the in vivo potency and enhanced oral bioavailability of these adenosine kinase inhibitors.

Structure-activity studies of 5-substituted pyridopyrimidines as adenosine kinase inhibitors.

The synthesis and SAR of a novel series of non-nucleoside pyridopyrimidine inhibitors of the enzyme adenosine kinase (AK) are described. It was found that pyridopyrimidines with a broad range of medium and large non-polar substituents at the 5-position potently inhibited AK activity. A narrower range of analogues was capable of potently inhibiting adenosine phosphorylation in intact cells indicating an enhanced ability of these analogues to penetrate cell membranes. Potent AK inhibitors were found to effectively reduce nociception in animal models of thermal hyperalgesia and persistent pain.

Prostaglandins regulate acid-induced cell-mediated bone resorption.

Metabolic acidosis induces bone calcium efflux initially by physicochemical dissolution and subsequently by cell-mediated mechanisms involving inhibition of osteoblasts and stimulation of osteoclasts. In rat kidney, acidosis increases endogenous prostaglandin synthesis, and in bone, prostaglandins are important mediators of resorption. To test the hypothesis that acid-induced bone resorption is mediated by prostaglandins, we cultured neonatal mouse calvariae in neutral or physiologically acidic medium with or without 0.56 microM indomethacin to inhibit prostaglandin synthesis. We measured net calcium efflux and medium PGE(2) levels. Compared with neutral pH medium, acid medium led to an increase in net calcium flux and PGE(2) levels after both 48 h and 51 h, a time at which acid-induced net calcium flux is predominantly cell mediated. Indomethacin inhibited the acid-induced increase in both net calcium flux and PGE(2). Net calcium flux was correlated directly with medium PGE(2) (r = 0.879, n = 29, P < 0.001). Exogenous PGE(2), at a level similar to that found after acid incubation, induced net calcium flux in bones cultured in neutral medium. Acid medium also stimulated an increase in PGE(2) levels in isolated bone cells (principally osteoblasts), which was again inhibited by indomethacin. Thus acid-induced stimulation of cell-mediated bone resorption appears to be mediated by endogenous osteoblastic PGE(2) synthesis.

Epidemiology of participation: an Australian community study.

STUDY OBJECTIVE: To determine the levels of participation in social and civic community life in a metropolitan region, and to assess differential levels of participation according to demographic, socioeconomic and health status. To contribute to policy debates on community participation, social capital and health using these empirical data. DESIGN: Cross sectional, postal, self completed survey on health and participation. SETTING: Random sample of the population from the western suburbs of Adelaide, the capital city of South Australia, a population of approximately 210 000. PARTICIPANTS: 2542 respondents from a sample of 4000 people aged 18 years and over who were registered on the electoral roll. MAIN RESULTS: The response rate to the survey was 63.6% (n=2542). Six indices of participation, on range of social and civic activities, with a number of items in each, were created. Levels of participation were highest in the informal social activities index (46.7-83.7% for individual items), and lowest in the index of civic activities of a collective nature (2.4-5.9% for individual items). Low levels of involvement in social and civic activities were reported more frequently by people of low income and low education levels. CONCLUSIONS: Levels of participation in social and civic community life in an urban setting are significantly influenced by individual socioeconomic status, health and other demographic characteristics. An understanding of the pattern of participation is important to inform social and health policy making. Increasing levels of participation will reduce social exclusion and is likely to improve the overall quality of community life.

Pharmacological characterization of recombinant human and rat P2X receptor subtypes.

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.

Differences in static balance and weight distribution between normal subjects and subjects with chronic unilateral low back pain.

Balance reactions are not routinely evaluated in patients with low back pain. The purpose of this study was to determine if there were differences in static balance and weight distribution between subjects with unilateral low back pain (N = 15) and pain-free controls (N = 15). Measurements included limits of stability (%LOS), target sway, weight distribution on each lower extremity in quiet standing, and center of gravity with measurements of maximal excursion in anterior/posterior and medial/lateral directions. Independent t tests were used to compare data between groups. Compared with control subjects, subjects with low back pain demonstrated greater anterior-posterior center of gravity excursion and total center of gravity excursion with eyes open and greater anterior-posterior, medial-lateral, and total center of gravity excursion, target sway, and %LOS with eyes closed. There was no difference in the weight-bearing distribution between groups. This study suggests that static balance in patients with chronic low back pain may be impaired and should be thoroughly evaluated and integrated into physical therapy treatment programs.

Inhibition of adenosine kinase during oxygen-glucose deprivation in rat cortical neuronal cultures.

Adenosine kinase (AK) inhibitors potentiate the actions of endogenous adenosine (ADO) and ameliorate cerebral ischemic damage in animal models. The present study examined the effects of the AK inhibitor, 5-iodotubercidin (5-IT) in an in vitro model of neuronal ischemia, specifically, combined oxygen-glucose deprivation of rat cortical mixed neuronal-glial cultures. Oxygen-glucose deprivation caused extensive neuronal loss which was accompanied by a marked increase in ADO release into the extracellular medium, was ameliorated by exogenous ADO (10 microM(-1) mM), and was exacerbated by a high concentration of the selective A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 10 microM). 5-IT (1 microM) had no effect on extracellular ADO levels nor on neuronal loss. However, AK activity in these cultures was markedly suppressed during oxygen-glucose deprivation. Taken together, these data demonstrate a marked down-regulation of AK activity during oxygen-glucose deprivation in this in vitro model, providing an endogenous mechanism contributing to the accumulation of extracellular ADO, which exerts neuroprotective effects by activating the ADO A1 receptor.

Nitroaromatic amino acids as inhibitors of neuronal nitric oxide synthase.

Nitric oxide (NO.) is an important biomodulator of many physiological processes. The inhibition of inappropriate production of NO. by the isoforms of nitric oxide synthase (NOS) has been proposed as a therapeutic approach for the treatment of stroke, inflammation, and other processes. In this study, certain 2-nitroaryl-substituted amino acid analogues were discovered to inhibit NOS. Analogues bearing a 5-methyl substituent on the aromatic ring demonstrated maximal inhibitory potency. For two selected inhibitors, investigation of the kinetics of the enzyme showed the inhibition to be competitive with l-arginine. Additionally, functional NOS inhibition in tissue preparations was demonstrated.

Role of progesterone on the control of scent marking in Suncus murinus viridescens (Blyth).

The effect of castration and administration of progesterone in different doses on the specialized integumentary glands and scent marking behavior in male musk shrew, Suncus murinus viridescens were studied. Castration effected a considerable atrophy of the secretory epithelial tissues of the flank, oral lip and perineal glands with marked regression in their secretory output. Further, the scent marking frequencies were also reduced and attained a minimum level by the end of 4 weeks after castration. Progesterone administration in effective doses reactivated all these specialized integumentary glands and the scent marking frequency in male shrews within a period of three weeks.

A study on the physiology and biochemistry of the flank gland of the musk shrew, Suncus murinus viridescens (Blyth).

The flank gland becomes functional by about the 9th day after parturition and exhibits a secretory rhythm with a nocturnal peak. Lipids constitute the major biochemical constituent of the total solids. Relatively, cholesterol, ascorbic acid and alkaline phosphatase contents of the flank gland were much higher than those of the control skin samples. Disc electrophoretic pattern of glandular secretion reveal that male and female shrews have 6 & 8 protein fractions respectively. The flank gland was observed to be androgen-dependent in male shrews. Ethological observations indicate that secretions of this gland are used for marking purposes.