Search for α3β2/3γ2 subtype selective ligands that are stable on human liver microsomes

Selective modulation of specific benzodiazepine receptor (BzR) gamma amino butyric acid-A (GABA_A) receptor ion channels has been identified as an important method for separating out the variety of pharmacological effects elicited by BzR-related drugs. Importantly, it has been demonstrated that both α2β_(2/3)γ2 (α2BzR) and α3BzR (and/or α2/α3) BzR subtype selective ligands exhibit anxiolytic effects with little or no sedation. Previously we have identified several such ligands; however, three of our parent ligands exhibited significant metabolic liability in rodents in the form of a labile ester group. Here eight analogs are reported which were designed to circumvent this liability by utilizing a rational replacement of the ester moiety based on medicinal chemistry precedents. In a metabolic stability study using human liver microsomes, four compounds were found to undergo slower metabolic transformation, as compared to their corresponding ester analogs. These compounds were also evaluated in in vitro efficacy assays. Additionally, bioisostere 11 was evaluated in a rodent model of anxiety. It exhibited anxiolytic activity at doses of 10 and 100 mg/kg and was devoid of sedative properties.     

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative,nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes

In the present study, one has determined the relative role of plasma membrane equilibrative (Na⁺-independent) ENT nucleoside transport proteins (particularly ENT2) in the uptake of antiviral nucleoside analogues for comparison with the previously reported drug transport properties of concentrative (Na⁺-dependent) CNT nucleoside transport proteins. The human and rat nucleoside transport proteins hENT1, rENT1, hENT2 and rENT2 were produced in Xenopus oocytes and investigated for their ability to transport three 3'-deoxy-nucleoside analogues, ddC (2' 3'-dideoxycytidine), AZT (3'-azido-3'-deoxythymidine)and ddI (2' 3'-dideoxyinosine), used in human immunodeficiency virus (HIV) therapy. The results show, for the first time, that the ENT2 transporter isoform represents a mechanism for cellular uptake of these clinically important nucleoside drugs. Recombinant h/rENT2 transported ddC, ddI and AZT, whilst h/rENT1 transported only ddC and ddI. Relative to uridine, h/rENT2 mediated substantially larger fluxes of ddC and ddI than h/rENT1. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 transport-positive for AZT and enhanced the uptake of both ddC and ddI, identifying this region as a major site of 3'-deoxy-nucleoside drug interaction.     

Isolation and identification of gastrointestinal microbiota from the short-nosed fruit bat Cynopterus brachyotis brachyotis

Studies on the microbial ecology of gut microbiota in bats are limited and such information is necessary in determining the ecological significance of these hosts. Short-nosed fruit bats (Cynopterus brachyotis brachyotis) are good candidates for microbiota studies given their close association with humans in urban areas. Thus, this study explores the gut microbiota of this species from Peninsular Malaysia by means of biochemical tests and 16S rRNA gene sequences analysis. The estimation of viable bacteria present in the stomach and intestine of C. b. brachyotis ranged from 3.06 × 10¹⁰ to 1.36 × 10¹⁵ CFU/ml for stomach fluid and 1.92 × 10¹⁰ to 6.10 × 10¹⁵ CFU/ml for intestinal fluid. A total of 34 isolates from the stomach and intestine of seven C. b. brachyotis were retrieved. A total of 16 species of bacteria from eight genera (Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Pantoea, Pseudomonas and Serratia) were identified, Enterobacteriaceae being the most prevalent, contributing 12 out of 16 species isolated. Most isolates from the Family Enterobacteriaceae have been reported as pathogens to humans and wildlife. With the possibility of human wildlife transmission, the findings of this study focus on the importance of bats as reservoirs of potential bacterial pathogens.     

Structure and secretion of CofJ,a putative colonization factor of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) colonize the human gut, causing severe cholera‐like diarrhoea. ETEC utilize a diverse array of pili and fimbriae for host colonization, including the Type IVb pilus CFA/III. The CFA/III pilus machinery is encoded on the cof operon, which is similar in gene sequence and synteny to the tcp operon that encodes another Type IVb pilus, the Vibrio cholerae toxin co‐regulated pilus (TCP). Both pilus operons possess a syntenic gene encoding a protein of unknown function. In V. cholerae, this protein, TcpF, is a critical colonization factor secreted by the TCP apparatus. Here we show that the corresponding ETEC protein, CofJ, is a soluble protein secreted via the CFA/III apparatus. We present a 2.6 Å resolution crystal structure of CofJ, revealing a large β‐sandwich protein that bears no sequence or structural homology to TcpF. CofJ has a cluster of exposed hydrophobic side‐chains at one end and structural homology to the pore‐forming proteins perfringolysin O and α‐haemolysin. CofJ binds to lipid vesicles and epithelial cells, suggesting a role in membrane attachment during ETEC colonization.     

Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.     

Evolutionary History of HIV-1 Subtype B and CRF01_AE Transmission Clusters among Men Who Have Sex with Men (MSM) in Kuala Lumpur,Malaysia

HIV-1 epidemics among men who have sex with men (MSM) continue to expand in developed and developing countries. Although HIV infection in MSM is amongst the highest of the key affected populations in many countries in Southeast Asia, comprehensive molecular epidemiological study of HIV-1 among MSM remains inadequate in the region including in Malaysia. Here, we reported the phylodynamic profiles of HIV-1 genotypes circulating among MSM population in Kuala Lumpur, Malaysia. A total of n = 459 newly-diagnosed treatment-naïve consenting subjects were recruited between March 2006 and August 2012, of whom 87 (18.9%) were self-reported MSM. Transmitted drug resistance mutations were absent in these isolates. Cumulatively, phylogenetic reconstructions of the pro-rt gene (HXB2∶2253–3275) showed that HIV-1 subtype B and CRF01_AE were predominant and contributed to approximately 80% of the total HIV-1 infection among MSM. In addition to numerous unique transmission lineages within these genotypes, twelve monophyletic transmission clusters of different sizes (2–7 MSM sequences, supported by posterior probability value of 1) were identified in Malaysia. Bayesian coalescent analysis estimated that the divergence times for these clusters were mainly dated between 1995 and 2005 with four major transmission clusters radiating at least 12 years ago suggesting that active spread of multiple sub-epidemic clusters occurred during this period. The changes in effective population size of subtype B showed an exponential growth within 5 years between 1988 and 1993, while CRF01_AE lineage exhibited similar expansion between 1993 and 2003. Our study provides the first insight of the phylodynamic profile of HIV-1 subtype B and CRF01_AE circulating among MSM population in Kuala Lumpur, Malaysia, unravelling the importance of understanding transmission behaviours as well as evolutionary history of HIV-1 in assessing the risk of outbreak or epidemic expansion.     

Predicting National Suicide Numbers with Social Media Data

Suicide is not only an individual phenomenon, but it is also influenced by social and environmental factors. With the high suicide rate and the abundance of social media data in South Korea, we have studied the potential of this new medium for predicting completed suicide at the population level. We tested two social media variables (suicide-related and dysphoria-related weblog entries) along with classical social, economic and meteorological variables as predictors of suicide over 3 years (2008 through 2010). Both social media variables were powerfully associated with suicide frequency. The suicide variable displayed high variability and was reactive to celebrity suicide events, while the dysphoria variable showed longer secular trends, with lower variability. We interpret these as reflections of social affect and social mood, respectively. In the final multivariate model, the two social media variables, especially the dysphoria variable, displaced two classical economic predictors – consumer price index and unemployment rate. The prediction model developed with the 2-year training data set (2008 through 2009) was validated in the data for 2010 and was robust in a sensitivity analysis controlling for celebrity suicide effects. These results indicate that social media data may be of value in national suicide forecasting and prevention.     

A reliability and validity study for Scolioscan: a radiation-free scoliosis assessment system using 3D ultrasound imaging

Background

Radiographic evaluation for patients with scoliosis using Cobb method is the current gold standard, but radiography has radiation hazards. Several groups have recently demonstrated the feasibility of using 3D ultrasound for the evaluation of scoliosis. Ultrasound imaging is radiation-free, comparatively more accessible, and inexpensive. However, a reliable and valid 3D ultrasound system ready for clinical scoliosis assessment has not yet been reported. Scolioscan is a newly developed system targeted for scoliosis assessment in clinics by using coronal images of spine generated by a 3D ultrasound volume projection imaging method. The aim of this study is to test the reliability of spine deformity measurement of Scolioscan and its validity compared to the gold standard Cobb angle measurements from radiography in adolescent idiopathic scoliosis (AIS) patients.

Methods

Prospective study divided into two stages: 1) Investigation of intra- and inter- reliability between two operators for acquiring images using Scolioscan and among three raters for measuring spinal curves from those images; 2) Correlation between the Cobb angle obtained from radiography by a medical doctor and the spine curve angle obtained using Scolioscan (Scolioscan angle). The raters for ultrasound images and the doctors for evaluating radiographic images were mutually blinded. The two stages of tests involved 20 (80 % females, total of 26 angles, age of 16.4?±?2.7 years, and Cobb angle of 27.6?±?11.8°) and 49 (69 % female, 73 angles, 15.8?±?2.7 years and 24.8?±?9.7°) AIS patients, respectively. Intra-class correlation coefficients (ICC) and Bland-Altman plots and root-mean-square differences (RMS) were employed to determine correlations, which interpreted based on defined criteria.

Results

We demonstrated a very good intra-rater and intra-operator reliability for Scolioscan angle measurement with ICC larger than 0.94 and 0.88, respectively. Very good inter-rater and inter-operator reliability was also demonstrated, with both ICC larger than 0.87. For the thoracic deformity measurement, the RMS were 2.5 and 3.3° in the intra- and inter-operator tests, and 1.5 and 3.6° in the intra- and inter-rater tests, respectively. The RMS differences were 3.1, 3.1, 1.6, 3.7° in the intra- and inter-operator and intra- and inter-rater tests, respectively, for the lumbar angle measurement. Moderate to strong correlations (R²?>?0.72) were observed between the Scolioscan angles and Cobb angles for both the thoracic and lumbar regions. It was noted that the Scolioscan angle slightly underestimated the spinal deformity in comparison with Cobb angle, and an overall regression equation y?=?1.1797x (R²?=?0.76) could be used to translate the Scolioscan angle (x) to Cobb angle (y) for this group of patients. The RMS difference between Scolioscan angle and Cobb angle was 4.7 and 6.2°, with and without the correlation using the overall regression equation.

Conclusions

We showed that Scolioscan is reliable for measuring coronal deformity for patients with AIS and appears promising in screening large numbers of patients, for progress monitoring, and evaluation of treatment outcomes. Due to it being radiation-free and relatively low-cost, Scolioscan has potential to be widely implemented and may contribute to reducing radiation dose during serial monitoring.

     

Production of Mucosally Transmissible SHIV Challenge Stocks from HIV-1 Circulating Recombinant Form 01_AE env Sequences

Simian-human immunodeficiency virus (SHIV) challenge stocks are critical for preclinical testing of vaccines, antibodies, and other interventions aimed to prevent HIV-1. A major unmet need for the field has been the lack of a SHIV challenge stock expressing circulating recombinant form 01_AE (CRF01_AE) env sequences. We therefore sought to develop mucosally transmissible SHIV challenge stocks containing HIV-1 CRF01_AE env derived from acutely HIV-1 infected individuals from Thailand. SHIV-AE6, SHIV-AE6RM, and SHIV-AE16 contained env sequences that were >99% identical to the original HIV-1 isolate and did not require in vivo passaging. These viruses exhibited CCR5 tropism and displayed a tier 2 neutralization phenotype. These challenge stocks efficiently infected rhesus monkeys by the intrarectal route, replicated to high levels during acute infection, and established chronic viremia in a subset of animals. SHIV-AE16 was titrated for use in single, high dose as well as repetitive, low dose intrarectal challenge studies. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines, monoclonal antibodies, and other interventions targeted at preventing HIV-1 CRF01_AE infection.