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1.  Design, Synthesis, Biochemical, and Antiviral Evaluations of C6 Benzyl and C6 Biarylmethyl Substituted 2-Hydroxylisoquinoline-1,3-diones: Dual Inhibition against HIV Reverse Transcriptase-Associated RNase H and Polymerase with Antiviral Activities 
Journal of Medicinal Chemistry  2014;58(2):651-664.
Reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not targeted by current chemotherapy against human immunodeficiency virus (HIV). Although numerous chemotypes have been reported to inhibit HIV RNase H biochemically, few show significant antiviral activity against HIV. We report herein the design, synthesis, and biological evaluations of a novel variant of 2-hydroxyisoquinoline-1,3-dione (HID) scaffold featuring a crucial C-6 benzyl or biarylmethyl moiety. The synthesis involved a recently reported metal-free direct benzylation between tosylhydrazone and boronic acid, which allowed the generation of structural diversity for the hydrophobic aromatic region. Biochemical studies showed that the C-6 benzyl and biarylmethyl HID analogues, previously unknown chemotypes, consistently inhibited HIV RT-associated RNase H and polymerase with IC50s in low to submicromolar range. The observed dual inhibitory activity remained uncompromised against RT mutants resistant to non-nucleoside RT inhibitors (NNRTIs), suggesting the involvement of binding site(s) other than the NNRTI binding pocket. Intriguingly, these same compounds inhibited the polymerase, but not the RNase H function of Moloney Murine Leukemia Virus (MoMLV) RT and also inhibited Escherichia coli RNase H. Additional biochemical testing revealed a substantially reduced level of inhibition against HIV integrase. Molecular docking corroborates favorable binding of these analogues to the active site of HIV RNase H. Finally, a number of these analogues also demonstrated antiviral activity at low micromolar concentrations.
PMCID: PMC4306517  PMID: 25522204
2.  In Ovo Delivery of CpG DNA Reduces Avian Infectious Laryngotracheitis Virus Induced Mortality and Morbidity 
Viruses  2015;7(4):1832-1852.
Endosomal toll-like receptor-21 and -9 sense CpG DNA activating production of pro-inflammatory mediators with antimicrobial effects. Here, we investigated the induction of antiviral response of in ovo delivered CpG DNA against infectious laryngotracheitis virus (ILTV) infection. We found that in ovo delivered CpG DNA significantly reduces ILTV infection pre-hatch correlating with the expression of IL-1β and increase of macrophages in lungs. As assessed in vitro, CpG DNA stimulated avian macrophages could be a potential source of IL-1β and other pro-inflammatory mediators. Since we also found that in ovo CpG DNA delivery maintains increased macrophages in the lungs post-hatch, we infected the chickens on the day of hatch with ILTV. We found that in ovo delivered CpG DNA significantly reduces mortality and morbidity resulting from ILTV infection encountered post-hatch. Thus, CpG DNA can be a candidate innate immune stimulant worthy of further investigation for the control of ILTV infection in chickens.
PMCID: PMC4411679  PMID: 25856635
In ovo; toll-like receptor 21; CpG-motif containing synthetic DNA; infectious laryngotracheitis virus; lung; chicken
3.  Enhanced Bovine Herpesvirus Type 1 Neutralization by Multimerized Single-Chain Variable Antibody Fragments Regardless of Differential Glycosylation 
Single-chain variable antibody fragments (scFvs) with a 2-amino-acid linker capable of multimerization as di-, tri-, or tetrabodies that neutralize bovine herpesvirus type 1 (BoHV-1) in vitro were constructed and expressed in Pichia pastoris. In contrast to the monomeric form, multimeric scFvs had a higher virus neutralization potency, as evidenced by a 2-fold increase in their ability to neutralize BoHV-1 due to avidity effects. Mass spectrum (quadrupole time of flight [Q-TOF]) analyses of multimeric scFv demonstrated extensive heterogeneity due to differential cleavage, variable glycosylation (1 to 9 mannose residues), and the incorporation of minor unidentified adducts. Regardless of the differential glycosylation patterns, the scFvs recognized non-gB or -gE target viral epitopes in the BoHV-1 envelope fraction in a Western blot and also neutralized BoHV-1 in infected Madin-Darby kidney (MDBK) cells in vitro. Indirect evidence for the noncovalent multimerization of scFv was the presence of a major peak of multimerized scFv without a His tag (due to differential cleavage) in the Q-TOF profile, unlike monomeric scFv, which copurified with normally His-tagged scFv and recognized the target antigen. Overall, differentially glycosylated recombinant scFvs against BoHV-1 with a short linker (2 amino acids) are capable of assembly into functional multimers that confer high avidity, resulting in increased virus neutralization in vitro compared to that of monovalent scFv with a long (18-amino-acid) flexible linker. Overall, recombinant multimerized scFv5-2L potentially provides a high-potency therapeutic and immunodiagnostic reagent against BoHV-1, which is suitable for passive immunization and topical application.
PMCID: PMC3416088  PMID: 22695166
4.  Effectiveness of an e-learning course in evidence-based medicine for foundation (internship) training 
To evaluate the educational effectiveness of a clinically integrated e-learning course for teaching basic evidence-based medicine (EBM) among postgraduate medical trainees compared to a traditional lecture-based course of equivalent content.
We conducted a cluster randomized controlled trial to compare a clinically integrated e-learning EBM course (intervention) to a lecture-based course (control) among postgraduate trainees at foundation or internship level in seven teaching hospitals in the UK West Midlands region. Knowledge gain among participants was measured with a validated instrument using multiple choice questions. Change in knowledge was compared between groups taking into account the cluster design and adjusted for covariates at baseline using generalized estimating equations (GEE) model.
There were seven clusters involving teaching of 237 trainees (122 in the intervention and 115 in the control group). The total number of postgraduate trainees who completed the course was 88 in the intervention group and 72 in the control group. After adjusting for baseline knowledge, there was no difference in the amount of improvement in knowledge of EBM between the two groups. The adjusted post course difference between the intervention group and the control group was only 0.1 scoring points (95% CI −1.2–1.4).
An e-learning course in EBM was as effective in improving knowledge as a standard lecture-based course. The benefits of an e-learning approach need to be considered when planning EBM curricula as it allows standardization of teaching materials and is a potential cost-effective alternative to standard lecture-based teaching.
PMCID: PMC2895523  PMID: 20522698
5.  Bisphosphonate inhibitors of ATP-mediated HIV-1 reverse transcriptase catalyzed excision of chain-terminating 3′-azido, 3′-deoxythymidine: A QSAR investigation 
Bioorganic & medicinal chemistry  2008;16(19):8959-8967.
We report the results of an investigation of the inhibition of the ATP-mediated HIV-1 reverse transcriptase catalyzed phosphorolysis in vitro of AZT from AZT-terminated DNA primers by a series of 42 bisphosphonates. The four most active compounds possess neutral, halogen-substituted phenyl or biphenyl sidechains and have IC50 values < 1 μM in excision inhibition assays. Use of two comparative molecular similarity analysis methods to analyze these inhibition results yielded a classification model with an overall accuracy of 94%, and a regression model having good accord with experiment (q2 = 0.63, r2 = 0.91), with the experimental activities being predicted within, on average, a factor of 2. The most active species had little or no toxicity against three human cell lines (IC50avg >200 μM). These results are of general interest since they suggest that it may be possible to develop potent bisphosphonate-based AZT excision inhibitors with little cellular toxicity, opening up a new route to restoring AZT sensitivity in otherwise resistant HIV-1 strains.
PMCID: PMC2586422  PMID: 18789701
Reverse transcriptase; Nucleoside reverse transcriptase inhibitor (NRTI); Bisphosphonate; Quantitative structure activity relationship (QSAR)
6.  The effectiveness of a clinically integrated e-learning course in evidence-based medicine: A cluster randomised controlled trial 
To evaluate the educational effects of a clinically integrated e-learning course for teaching basic evidence-based medicine (EBM) among postgraduates compared to a traditional lecture-based course of equivalent content.
We conducted a cluster randomised controlled trial in the Netherlands and the UK involving postgraduate trainees in six obstetrics and gynaecology departments. Outcomes (knowledge gain and change in attitude towards EBM) were compared between the clinically integrated e-learning course (intervention) and the traditional lecture based course (control). We measured change from pre- to post-intervention scores using a validated questionnaire assessing knowledge (primary outcome) and attitudes (secondary outcome).
There were six clusters involving teaching of 61 postgraduate trainees (28 in the intervention and 33 in the control group). The intervention group achieved slightly higher scores for knowledge gain compared to the control, but these results were not statistically significant (difference in knowledge gain: 3.5 points, 95% CI -2.7 to 9.8, p = 0.27). The attitudinal changes were similar for both groups.
A clinically integrated e-learning course was at least as effective as a traditional lecture based course and was well accepted. Being less costly than traditional teaching and allowing for more independent learning through materials that can be easily updated, there is a place for incorporating e-learning into postgraduate EBM curricula that offer on-the-job training for just-in-time learning.
Trial registration
Trial registration number: ACTRN12609000022268.
PMCID: PMC2688004  PMID: 19435520
7.  Harmonising Evidence-based medicine teaching: a study of the outcomes of e-learning in five European countries 
We developed and evaluated the outcomes of an e-learning course for evidence based medicine (EBM) training in postgraduate medical education in different languages and settings across five European countries.
We measured changes in knowledge and attitudes with well-developed assessment tools before and after administration of the course. The course consisted of five e-learning modules covering acquisition (formulating a question and search of the literature), appraisal, application and implementation of findings from systematic reviews of therapeutic interventions, each with interactive audio-visual learning materials of 15 to 20 minutes duration. The modules were prepared in English, Spanish, German and Hungarian. The course was delivered to 101 students from different specialties in Germany (psychiatrists), Hungary (mixture of specialties), Spain (general medical practitioners), Switzerland (obstetricians-gynaecologists) and the UK (obstetricians-gynaecologists). We analysed changes in scores across modules and countries.
On average across all countries, knowledge scores significantly improved from pre- to post-course for all five modules (p < 0.001). The improvements in scores were on average 1.87 points (14% of total score) for module 1, 1.81 points (26% of total score) for module 2, 1.9 points (11% of total score) for module 3, 1.9 points (12% of total score) for module 4 and 1.14 points (14% of total score) for module 5. In the country specific analysis, knowledge gain was not significant for module 4 in Spain, Switzerland and the UK, for module 3 in Spain and Switzerland and for module 2 in Spain. Compared to pre-course assessment, after completing the course participants felt more confident that they can assess research evidence and that the healthcare system in their country should have its own programme of research about clinical effectiveness.
E-learning in EBM can be harmonised for effective teaching and learning in different languages, educational settings and clinical specialties, paving the way for development of an international e-EBM course.
PMCID: PMC2386125  PMID: 18442424

Results 1-8 (8)