Effect of constant versus variable small-group facilitators on students' basic science knowledge in an enquiry-based dental curriculum.

Introduction: The role of small-group facilitators is of pivotal importance for the success of curricula based on active learning. Disorganised tutorial processes and superficial study of the problem have been identified as main hindering factors for students' learning. The aim of this study was to evaluate the influence of consistency of facilitation on students' performance in knowledge-based basic science assessments in a hybrid, enquiry-based (EBL) undergraduate dental curriculum.

PrEP-001 prophylactic effect against rhinovirus and influenza virus - RESULTS of 2 randomized trials.

Background: PrEP-001 Nasal Powder, a proprietary formulation of polyriboinosinic and polyribocytidylic acid effectively elicits a cellular innate immune response in nasal epithelium. The aim of these 2 studies was to investigate the safety and efficacy of PrEP-001 prophylaxis against rhinovirus (HRV-A16) and influenza-A (H3N2-IAV).

Methods: Healthy subjects randomly received 2 doses of PrEP-001 or placebo, 48 and 24 h pre-challenge with 10 TCID of HRV-A16 (Study 1) or H3N2-IAV (Study 2).

Mutations in KEOPS-complex genes cause nephrotic syndrome with primary microcephaly.

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality.

Half-life extension of the HIV-fusion inhibitor peptide TRI-1144 using a novel linker technology.

We have previously developed a linker technology for half-life extension of peptides, proteins and small molecule drugs (1). The linkers undergo β-elimination reactions with predictable cleavage rates to release the native drug. Here we utilize this technology for half-life extension of the 38 amino acid HIV-1 fusion inhibitor TRI-1144.

Can virtual nature improve patient experiences and memories of dental treatment? A study protocol for a randomized controlled trial.

Background: Dental anxiety and anxiety-related avoidance of dental care create significant problems for patients and the dental profession. Distraction interventions are used in daily medical practice to help patients cope with unpleasant procedures. There is evidence that exposure to natural scenery is beneficial for patients and that the use of virtual reality (VR) distraction is more effective than other distraction interventions, such as watching television.

Improving dental experiences by using virtual reality distraction: a simulation study.

Dental anxiety creates significant problems for both patients and the dental profession. Some distraction interventions are already used by healthcare professionals to help patients cope with unpleasant procedures. The present study is novel because it a) builds on evidence that natural scenery is beneficial for patients, and b) uses a Virtual Reality (VR) representation of nature to distract participants.

The glycosylation of AGP and its associations with the binding to methadone.

Methadone remains the most common form of pharmacological therapy for opioid dependence; however, there is a lack of explanation for the reports of its relatively low success rate in achieving complete abstinence. One hypothesis is that in vivo binding of methadone to the plasma glycoprotein alpha-1-acid glycoprotein (AGP), to a degree dependent on the molecular structure, may render the drug inactive. This study sought to determine whether alterations present in the glycosylation pattern of AGP in patients undergoing various stages of methadone therapy (titration < two weeks, harm reduction < one year, long-term > one and a half years) could affect the affinity of the glycoprotein to bind methadone.

Screening and rank ordering of reversible mechanism-based inhibitors of hepatitis C virus NS3 protease using electrospray ionization mass spectrometry.

An affinity-selection study using size exclusion chromatography (SEC) combined with off-line electrospray ionization mass spectrometry (ESI-MS) was performed on libraries of peptidic α-ketoamide inhibitors directed against the hepatitis C virus (HCV) NS3 protease. A limiting amount of HCV NS3 protease (25 µM) was incubated with equimolar amounts (100 µM) of 49 reversible mechanism-based ketoamide inhibitors, previously grouped into seven sets to ensure clearly distinguishable mass differences of the enzyme-inhibitor complexes (>10 Da). The unbound compounds were separated rapidly from the protease and the protease-inhibitor complexes by SEC spin columns.

A case of γ-butyrolactone associated with severe withdrawal delirium and acute renal failure.

γ-Butyrolactone (GBL) is a popular drug of abuse which is easily available over the internet. Following a UK classification change to a class C drug in January 2010, internet supply has become difficult. Some of the effects have resulted in sourcing GBL from industrial solvents.

A comparative analysis of the substrate permissiveness of HCV and GBV-B NS3/4A proteases reveals genetic evidence for an interaction with NS4B protein during genome replication.

The hepatitis C virus (HCV) serine protease (NS3/4A) processes the NS3-NS5B segment of the viral polyprotein and also cleaves host proteins involved in interferon signaling, making it an important target for antiviral drug discovery and suggesting a wide breadth of substrate specificity. We compared substrate specificities of the HCV protease with that of the GB virus B (GBV-B), a distantly related nonhuman primate hepacivirus, by exchanging amino acid sequences at the NS4B/5A and/or NS5A/5B cleavage junctions between these viruses within the backbone of subgenomic replicons. This mutagenesis study demonstrated that the GBV-B protease had a broader substrate tolerance, a feature corroborated by structural homology modeling.

Discovery of Narlaprevir (SCH 900518): A Potent, Second Generation HCV NS3 Serine Protease Inhibitor.

Boceprevir (SCH 503034), 1, a novel HCV NS3 serine protease inhibitor discovered in our laboratories, is currently undergoing phase III clinical trials. Detailed investigations toward a second generation protease inhibitor culminated in the discovery of narlaprevir (SCH 900518), 37, with improved potency (∼10-fold over 1), pharmacokinetic profile and physicochemical characteristics, currently in phase II human trials. Exploration of synthetic sequence for preparation of 37 resulted in a route that required no silica gel purification for the entire synthesis.

Key steps in the structure-based optimization of the hepatitis C virus NS3/4A protease inhibitor SCH503034.

The structures of both native and S139A holo-HCV NS3/4A protease domain were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site.

Discovery of the HCV NS3/4A protease inhibitor (1R,5S)-N-[3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]-3- [2(S)-[[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2(S)-carboxamide (Sch 503034) II. Key steps in structure-based optimization.

The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site.

Addressing the efficacy of dihydrocodeine versus methadone as an alternative maintenance treatment for opiate dependence: A randomized controlled trial.

Aim: The aim of this study is to define the efficacy of dihydrocodeine as an alternative to methadone in the maintenance treatment of opiate dependence.

Design: A pragmatic open-label randomized controlled study of patients recommended for opiate maintenance treatment to test equivalence of the two treatment options with follow-up continuing for up to 42 months after recruitment.

Setting: Assessment at either Edinburgh's Community Drug Problem Service or at two general practitioner practices with specialist drug community psychiatric nurses, then with shared care follow-up.

Discovery of (1R,5S)-N-[3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]- 3-[2(S)-[[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2(S)-carboxamide (SCH 503034), a selective, potent, orally bioavailable hepatitis C virus NS3 protease inhibitor: a potential therapeutic agent for the treatment of hepatitis C infection.

Hepatitis C virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 170 million people worldwide. Currently the only therapeutic regimens are subcutaneous interferon-alpha or polyethylene glycol (PEG)-interferon-alpha alone or in combination with oral ribavirin. Although combination therapy is reasonably successful with the majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only about 40% of the patients showing sustained virological response.

Identification and analysis of fitness of resistance mutations against the HCV protease inhibitor SCH 503034.

HCV NS3 protease variants resistant to the protease inhibitor SCH 503034 were selected. Three mutations, T54A, V170A and A156S mutations conferred low to moderate levels of resistance (<20-fold). Longer exposure (>10 passages) or selection with higher levels of compound led to the selection of a more resistant variant, A156T (>100-fold).

Impact of naturally occurring variants of HCV protease on the binding of different classes of protease inhibitors.

HCV drug discovery efforts have largely focused on genotype 1 virus due to its prevalence and relatively poor response to current therapy. However, patients infected with genotype 2 and 3 viruses account for a significant number of cases and would also benefit from new therapies. In vitro studies using two chemically distinct protease inhibitors with clinical potential showed that one, VX-950, was equally active on proteases from all three genotypes, whereas the other, BILN 2061, was significantly less active on genotype 2 and 3 proteases.

Mutations conferring resistance to SCH6, a novel hepatitis C virus NS3/4A protease inhibitor. Reduced RNA replication fitness and partial rescue by second-site mutations.

Drug resistance is a major issue in the development and use of specific antiviral therapies. Here we report the isolation and characterization of hepatitis C virus RNA replicons resistant to a novel ketoamide inhibitor of the NS3/4A protease, SCH6 (originally SCH446211). Resistant replicon RNAs were generated by G418 selection in the presence of SCH6 in a dose-dependent fashion, with the emergence of resistance reduced at higher SCH6 concentrations.

Trans-complementation of HCV replication by non-structural protein 5A.

Although establishment of the subgenomic replicon system has considerably facilitated genetic analysis of HCV replication, many details remain largely unknown. To initially test whether HCV replication could be affected in trans, complementation studies were conducted in which defective replicon RNAs carrying a luciferase reporter were introduced into stable cells bearing functional replicons. The NS3 protease and the NS5B viral polymerase genes on the transfected replicons were rendered null by active site mutations and shown not to be complemented in trans by functional proteins expressed from the endogenous replicons.

Development of a fluorescence polarization bead-based coupled assay to target different activity/conformation states of a protein kinase.

Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states.

A continuous nonradioactive assay for RNA-dependent RNA polymerase activity.

Current assays for the activity of viral RNA-dependent RNA polymerases (RdRps) are inherently end-point measurements, often requiring the use of radiolabeled or chemically modified nucleotides to detect reaction products. In an effort to improve the characterization of polymerases that are essential to the life cycle of RNA viruses and develop antiviral therapies that target these enzymes, a continuous nonradioactive assay was developed to monitor the activity of RdRps by measuring the release of pyrophosphate (PP(i)) generated during nascent strand synthesis. A coupled-enzyme assay method based on the chemiluminescent detection of PP(i), using ATP sulfurylase and firefly luciferase, was adapted to monitor poliovirus 3D polymerase (3D(pol)) and the hepatitis C virus nonstructural protein 5B (NS5B) RdRp reactions.

Conserved C-terminal threonine of hepatitis C virus NS3 regulates autoproteolysis and prevents product inhibition.

Inspection of over 250 hepatitis C virus (HCV) genome sequences shows that a threonine is strictly conserved at the P1 position in the NS3-NS4A (NS3-4A) autoproteolysis junction, while a cysteine is maintained as the P1 residue in all of the putative trans cleavage sites (NS4A-4B, NS4B-5A, and NS5A-5B). To understand why T631 is conserved at the NS3-4A junction of HCV, a series of in vitro transcription-translation studies were carried out using wild-type and mutant (T631C) NS3-4A constructs bearing native, truncated, and mutant NS4A segments. The autocleavage of the wild-type junction was found to be dependent on the presence of the central cofactor domain of NS4A (residues 21 to 34).

The effect of ribavirin and IMPDH inhibitors on hepatitis C virus subgenomic replicon RNA.

The recent development of in vitro hepatitis C virus (HCV) RNA replication systems has provided useful tools for studying the intracellular anti-HCV activity of ribavirin. Ribavirin has been shown to: (1) induce "error catastrophe" in poliovirus, Proc. Natl.

Dosage of treatment to sexual offenders: are we overprescribing?

A sample of 337 offenders who received treatment in a variety of sex offender treatment programs in the Ontario region of Correctional Service Canada between 1993 and 1998 were divided based on the highest intensity sex offender programming that they received (low, moderate, and high). The three groups were compared with reference to a variety of actuarial risk assessment measures, criminogenic factors, and the number and type of treatment programs completed. It was hypothesized that the high-intensity group would have more criminogenic risk factors, higher actuarial scores, and participate in more treatment programs than both the moderate- and low-intensity groups.