Zoonotic origin and transmission of Middle East respiratory syndrome coronavirus in the UAE.

Since the emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, there have been a number of clusters of human-to-human transmission. These cases of human-to-human transmission involve close contact and have occurred primarily in healthcare settings, and they are suspected to result from repeated zoonotic introductions. In this study, we sequenced whole MERS-CoV genomes directly from respiratory samples collected from 23 confirmed MERS cases in the United Arab Emirates (UAE). These samples included cases from three nosocomial and three household clusters. The sequences were analysed for changes and relatedness with regard to the collected epidemiological data and other available MERS-CoV genomic data. Sequence analysis supports the epidemiological data within the clusters, and further, suggests that these clusters emerged independently. To understand how and when these clusters emerged, respiratory samples were taken from dromedary camels, a known host of MERS-CoV, in the same geographic regions as the human clusters. Middle East respiratory syndrome coronavirus genomes from six virus-positive animals were sequenced, and these genomes were nearly identical to those found in human patients from corresponding regions. These data demonstrate a genetic link for each of these clusters to a camel and support the hypothesis that human MERS-CoV diversity results from multiple zoonotic introductions.

1,4-Benzodiazepine peripheral cholecystokinin (CCK-A) receptor agonists.

A series of 1,4-benzodiazepines, N-1-substituted with an N-isopropyl-N-phenylacetamide moiety, was synthesized and screened for CCK-A agonist activity. In vitro agonist activity on isolated guinea pig gallbladder along with in vivo induction of satiety following intraperitoneal administration in a rat feeding assay was demonstrated.

A comparison of teaching strategies for integrating information technology into clinical nursing education.

As health care becomes more information-intensive and diverse, there is a need to integrate information technology (IT) into clinical education. Little is known, however, about how to design instructional strategies for integrating information technology into clinical nursing education. This article outlines the instructional strategies used by faculty in five nursing programs who taught students to use a point-of-care information technology system. The article also reports students' computer acceptance and summarizes IT clinical teaching recommendations.

Use of constitutive G protein-coupled receptor activity for drug discovery.

This article describes the behavior of transiently transfected human receptors into melanophores and the potential use of constitutive receptor activity to screen for new drug entities. Specifically, transient transfection of melanophores with different concentrations of receptor cDNA presumably leads to increased levels of receptor expression. This leads to an increased response to agonists (both maxima and potency) and, in some cases, an agonist-independent constitutive receptor activity. Transfections with increasing concentrations of the G(s) protein-coupled human calcitonin receptor type 2 (hCTR2) cDNA produced sufficient levels of constitutively activated receptor to cause elevated basal cellular responses. This was observed as a decrease in the transmittance of light through melanophores (consistent with G(s) protein activation) and increased response to human calcitonin. The receptor-mediated nature of this response was confirmed by its reversal with the hCTR2 peptide inverse agonist AC512. A collection of ligands for hCTR2 either increased or decreased constitutive hCTR2 activity, suggesting that the constitutive system was a sensitive discriminator of positive and negative ligand efficacy. Similar results were obtained with G(i)-protein-coupled receptors. Transient transfection of NPY1, NPY2, NPY4, CXCR4, and CCR5 cDNA produced increased light transmittance through melanophores (consistent with G(i)-protein activation). NPY1 cDNA produced little constitutive response on transfection, whereas maximal levels of constitutive activity ranging from 30 to 45% were observed for the other G(i)-protein-coupled receptors. Responses to agonists for these receptors increased (both maxima and potency) with increasing cDNA transfection. The receptor/G(i)-protein nature of both the constitutive and agonist-mediated responses was confirmed by elimination with pertussis toxin pretreatment. These data are discussed in terms of the theoretical aspects of constitutive receptor activity and the applicability of this approach for the general screening of G protein-coupled orphan receptors.

Constitutive receptor systems for drug discovery.

This paper discusses the use of constitutively active G-protein-coupled receptor systems for drug discovery. Specifically, the ternary complex model is used to define the two major theoretical advantages of constitutive receptor screening-namely, the ability to detect antagonists as well as agonists directly and the fact that constitutive systems are more sensitive to agonists. In experimental studies, transient transfection of Chinese hamster ovary cyclic AMP response element (CRE) luciferase reporter cells with cDNA for human parathyroid hormone receptor, glucagon receptor, and glucagon-like peptide (GLP-1) receptor showed cDNA concentration-dependent constitutive activity with parathyroid hormone (PTH-1) and glucagon. In contrast, no constitutive activity was observed for GLP-1 receptor, yet responses to GLP-1 indicated that receptor expression had taken place. In another functional system, Xenopus laevi melanophores transfected with cDNA for human calcitonin receptor showed constitutive activity. Nine ligands for the calcitonin receptor either increased or decreased constitutive activity in this assay. The sensitivity of the system to human calcitonin increased with increasing constitutive activity. These data indicate that, for those receptors which naturally produce constitutive activity, screening in this mode could be advantageous over other methods.

Chronic groin pain in an athlete: an unusual presentation.

Persistent disabling groin pain in an active sportsman is a frustrating diagnostic and management problem for both the athlete and physician. After clinical examination and investigation there remains a group of patients who have unexplained groin pain, and may undergo lengthy periods of conservative management with numerous radiological investigations. Here we highlight such a case.

Optimization of 3-(1H-indazol-3-ylmethyl)-1,5-benzodiazepines as potent, orally active CCK-A agonists.

We previously described a series of 3-(1H-indazol-3-ylmethyl)-1,5-benzodiazepine CCK-A agonists exemplified by compound 1 (GW 5823), which is the first reported binding selective CCK-A full agonist demonstrating oral efficacy in a rat feeding model. In this report we describe analogs of compound 1 designed to explore changes to the C3 and N1 pharmacophores and their effect on agonist activity and receptor selectivity. Agonist efficacy in this series was affected by stereoelectronic factors within the C3 moiety. Binding affinity for the CCK-A vs CCK-B receptor showed little dependence on the structure of the C3 moiety but was affected by the nature of the second substituent at C3. Structure-activity relationships at the N1-anilidoacetamide "trigger" moiety within the C3 indazole series were also investigated. Both agonist efficacy and binding affinity within this series were modulated by variation of substituents on the N1-anilidoacetamide moiety. Evaluation of several analogs in an vivo mouse gallbladder emptying assay revealed compound 1 to be the most potent and efficacious of all the analogs tested. The pharmacokinetic and pharmacodynamic profile of 1 in rats is also discussed.

Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity (II): Optimization of the C3 amino substituent.

Analogs of the previously reported 1,5-benzodiazepine peripheral cholecystokinin (CCK-A) receptor agonist 1 were prepared which explore substitution and/or replacement of the C-3 phenyl urea moiety. Agonist efficacy on the isolated guinea pig gallbladder (GPGB) was retained with a variety of substituted ureas and amide analogs. Three compounds were identified which were orally active in the mouse gallbladder emptying assay (MGBE). The 2-indolamide (52) and N-(carboxymethyl)-2-indolamide (54) derivatives had improved affinity for the human CCK-A receptor but reduced agonist efficacy on the GPGB. Neither indolamide was orally active in a rat feeding assay. In contrast, the (3-carboxyphenyl)urea derivative (29, GW7854) had moderately increased affinity for the human CCK-B receptor but was a potent full agonist on the GPGB and was orally active in both the MGBE and rat feeding assays. GW7854 was a full agonist (EC50 = 60 nM) for calcium mobilization on CHO K1 cells expressing hCCK-A receptors and a potent antagonist of CCK-8 (pA2 = 9.1) on CHO K1 cells expressing hCCK-B receptors. GW7854 is a potent mixed CCK-A agonist/CCK-B antagonist which is orally active in two in vivo models of CCK-A-mediated agonist activity.

A mathematical model for analysis of pharmacologically induced changes in the kinetics of cardiac muscle.

A mathematical model of the isometric contraction of cardiac muscle is developed and utilized to characterize the inotropic and lusitropic effects of cardioactive compounds in isolated guinea pig left atria. In contrast to metrics that are based on minima and maxima of an isometric twitch and its derivative function, the entire time course of the twitch is used to quantify the kinetics of the contraction-relaxation cycle. The model relates observed tension to a time-dependent activation function that describes generation of internal force and a coupling function that determines mechanical response to the activation function. The model is structured so that it is suitable for nonlinear curve fitting to observed data. Results obtained using the model for fitting experimental data from tissues treated with different classes of cardioactive compounds agree with more qualitative results presented by other authors. Experiments using the model to fit data over an extended (90 min) time course revealed differences in the kinetic profiles of milrinone and forskolin. Computer simulations that demonstrate the effect of each model parameter on twitch kinetics are presented, and the relationships between the model and other theoretical and empirical models of cardiac muscle are discussed. The mathematical model is useful to enable a more quantitative understanding of the kinetics of cardiac muscle contraction and relaxation and identify compounds that may be selective for inotropic or lusitropic effects.

Structure activity relationships of a series of buspirone analogs at alpha-1 adrenoceptors: further evidence that rat aorta alpha-1 adrenoceptors are of the alpha-1D-subtype.

The activity of a series of busprione analogs at recombinant and rat thoracic aorta alpha-1 adrenoceptors was investigated. Compound affinity for recombinant alpha-1A, alpha-1B and alpha-1D adrenoceptors from human and animal sources was determined by radioligand binding assays using membranes prepared from rat-1 fibroblasts expressing recombinant receptors with ( +/- )-[125l]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone as the radioligand. Compound affinity and functional activity at rat aortic alpha-1 adrenoceptors were determined using endothelium denuded rings contracted with phenylephrine. BMY 7378 ¿8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]-ehtyl)-8-azaspiro [4,5]decane-7,9-dione dihydrochloride¿ and MDL 73005EF ¿8-[2-(1,4-benzodioxan-2-ylmethylamino) ethyl]-8-azaspiro[4,5]decane-7,9-dione hydrochloride¿ were found to have significant selectivity for the alpha-1D-subtype and were high affinity antagonists of the alpha-1 adrenoceptors in the rat aorta. Leverage plot analysis of affinities of the buspirone analogs and a series of structurally diverse alpha-1 antagonists for recombinant alpha-1 adrenoceptors and rat aorta alpha-1 adrenoceptors demonstrate that the alpha-1 adrenoceptors in the rat aorta are predominantly of the alpha-1D subtype.

3-[2-(N-phenylacetamide)]-1,5-benzodiazepines: orally active, binding selective CCK-A agonists.

A series of modifications were made to the C-3 substituent of the 1,5-benzodiazepine CCK-A agonist 1. Replacement of the inner urea NH and addition of a methyl group to generate a C-3 quaternary carbon resulted in acetamide 6, which showed CCK-A receptor binding selectivity and sub-micromolar agonist activity in vitro. Benzodiazepine 6 was active in an in vivo mouse gallbladder emptying assay and represents a novel orally active, binding selective CCK-A agonist.

Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity. 1. Optimization of the agonist "trigger".

Directed screening of compounds selected from the Glaxo registry file for contractile activity on the isolated guinea pig gallbladder (GPGB) identified a series of 1,5-benzodiazepines with peripheral cholecystokinin (CCK) receptor agonist activity. Agonist efficacy within this series was modulated by variation of substituents on the N1-anilinoacetamide moiety. Remarkably, a single methyl group confers agonist activity, with an N-isopropyl substituent providing optimal efficacy. Hydrophilic substituents on the anilino nitrogen abolish agonist activity or produce antagonists of CCK. In contrast, hydrophilic electron-donating groups at the para-position of the anilino ring enhance or maintain in vitro and in vivo agonist activity. Despite decreased affinity for the human CCK-A receptor, relative to CCK-8, some of these compounds are equipotent to CCK as anorectic agents in rats following intraperitoneal administration.

CCK-A receptor selective antagonists derived from the CCK-A receptor selective tetrapeptide agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (A-71623).

Analogs of the CCK-A receptor selective agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (A-71623) were prepared in which the lysine residue was replaced with L-4-aminophenylalanine and D-or L-3-aminophenylalanine. These new analogs were moderately potent antagonists of CCK-8 in the isolated guinea pig gallbladder with exceptional CCK-A receptor selectivity as evaluated in membrane preparations from CHO K1 cells stably transfected with human CCK-A and CCK-B receptors.

Meeting affective needs of at-risk adolescents.

This study examined an intervention that addressed affective needs of 27 adolescents, 13 to 19 years old, who attended an alternative school, thereby improving their academic performance. No control interventions were employed.

Trophic control of lung development by sympathetic neurons: effects of neonatal sympathectomy with 6-hydroxydopamine.

The onset of peripheral sympathetic neuronal function is thought to provide trophic regulatory signals for development of adrenergic target tissues. In the current study, we examined the effects on lung development of neonatal sympathectomy with 6-hydroxydopamine. The completeness of the lesion and effectiveness in reducing sympathetic input to the tissue were confirmed by direct measurement of norepinephrine levels and turnover. Despite the denervation, no evidence of beta-receptor up-regulation was found; in fact, receptor binding sites tended to be reduced throughout development. The cyclic AMP response to isoproterenol challenge was initially suppressed in the lesioned animals, but became supersensitive even in the face of reduced receptor binding capabilities. Evidence was also obtained for ontogenetic abnormalities in the ornithine decarboxylase/polyamine system, which is partially controlled by beta-adrenergic input and which regulates macromolecule synthesis in replicating and differentiating cells. Eventually, the alterations were reflected in aberrant developmental patterns of DNA, RNA and protein in the lung. These results indicate that sympathetic neurons influence the biochemical development of the lung and may serve to program permanently the relationships among receptor sites, receptor coupling to cellular function, and control of cell maturation.

Role of sympathetic neurons in biochemical and functional development of the kidney: neonatal sympathectomy with 6-hydroxydopamine.

Renal sympathetic function develops over the first 3 weeks of postnatal life in the rat. In the current study, the effects of neonatal sympathectomy with 6-hydroxydopamine were examined on renal biochemical and functional development. The completeness and persistence of sympathetic nerve loss were confirmed by direct measurement of norepinephrine levels and turnover. Evidence was obtained for adverse effects on cellular maturation, as shown by perturbations in the ornithine decarboxylase/polyamine system, which is controlled partially by beta adrenergic input and which regulates macromolecule synthesis in developing cells. A later phase of 6-hydroxydopamine-induced alterations in renal development was seen during the period in which synaptogenesis is prominent and sympathetic tone is high (end of the 2nd postnatal week to end of the 3rd week): the denervated kidneys displayed supersensitivity of beta adrenergically mediated cyclic AMP responses without changes in receptor binding. The alterations in biochemical indices of cellular maturation were accompanied by abnormalities of renal function. 6-Hydroxydopamine caused an increase in the fractional excretion of sodium and deficits in physiological responsiveness of the kidney to a vasopressin analog. Later on, alterations in glomerular filtration rate and basal urinary osmolality also were prominent. These results indicate that neonatal sympathectomy has an adverse effect on the biochemical and functional development of the kidney.

Do sympathetic neurons coordinate cellular development in the heart and kidney? Effects of neonatal central and peripheral catecholaminergic lesions on cardiac and renal nucleic acids and proteins.

Sympathetic nerve activity has been hypothesized to set the timing of cellular maturational events in target tissues. In the current study, this hypothesis was tested by lesioning catecholamine pathways in the periphery and central nervous system through the use of subcutaneous or intracisternal injections of 6-hydroxydopamine. Systemically administered 6-hydroxydopamine completely depleted peripheral norepinephrine. The central treatment completely ablated the developmental rise in brain norepinephrine and dopamine and had little effect on peripheral norepinephrine levels, but has been shown to reduce sympathetic tone. In both the heart and the kidney, either type of lesion resulted in deficits in cell acquisition (DNA) with some evidence of compensatory increases in other macromolecules involved in cell enlargement (particularly RNA), thus maintaining the tissue growth rate at only slightly subnormal levels. The peak effect was always seen during the stages at which sympathetic neuronal synaptogenesis and impulse activity ordinarily undergo their most rapid development. Most of the 6-hydroxydopamine-induced differences in nucleic acids lessened or disappeared toward weaning, and thus these data support the view that sympathetic neuronal input influences the timing of maturational control of macromolecules, but not their final set-point. In combination with earlier studies showing termination of DNA synthesis caused by exposure of heart and kidney acutely to high levels of catecholamines, the results suggest that neuronal activity provides a biphasic signal, with positive trophic effects predominating during early development when sympathetic tone is low, and negative effects appearing when sympathetic tone is elevated during the late preweanling stage.

Biochemical determinants of growth sparing during neonatal nutritional deprivation or enhancement: ornithine decarboxylase, polyamines, and macromolecules in brain regions and heart.

In order to elucidate the biochemical mechanisms operating to protect the brain from growth retardation in response to nutritional deprivation, comparisons were made of markers of cellular development in brain regions (cerebellum, cerebral cortex, midbrain + brainstem) and in a tissue which is not spared (heart). Nutritional status of neonatal rats was manipulated by increasing or decreasing the litter size beginning at birth, and development of DNA, RNA, and proteins followed throughout the neonatal period. In addition, we assessed the activity and levels of ornithine decarboxylase and its metabolic products, the polyamines, which are known to coordinate macromolecule synthesis in immature tissue and to provide an early index of perturbed development. Cardiac ornithine decarboxylase and polyamines were altered within 48 h of initiating the changes in litter size, and the direction and magnitude of these biochemical effects were predictive of subsequent impairment or enhancement of organ growth and of cellular development. All three brain regions were buffered from growth alterations relative to the heart, but the cerebellum, which undergoes major phases of cell replication later than the other two regions, was somewhat less protected. The spared brain regions also showed evidence of compensatory hypertrophy in nutritional deprivation (increased protein/DNA ratio) which accounts for maintenance of growth in the presence of reduced cell numbers. Thus, brain growth sparing involves specific cellular responses which are dependent on the maturational profile of each brain region.