sIR: siRNA Information Resource, a web-based tool for siRNA sequence design and analysis and an open access siRNA database

<p>Abstract</p> <p>Background</p> <p>RNA interference has revolutionized our ability to study the effects of altering the expression of single genes in mammalian (and other) cells through targeted knockdown of gene expression. In this report we describe a web-based computational tool, siRNA Information Resource (sIR), which consists of a new open source database that contains validation information about published siRNA sequences and also provides a user-friendly interface to design and analyze siRNA sequences against a chosen target sequence.</p> <p>Results</p> <p>The siRNA design tool described in this paper employs empirically determined rules derived from a meta-analysis of the published data; it uses a weighted scoring system that determines the optimal sequence within a target mRNA and thus aids in the rational selection of siRNA sequences. This scoring system shows a non-linear correlation with the knockdown efficiency of siRNAs. sIR provides a fast, customized BLAST output for all selected siRNA sequences against a variety of databases so that the user can verify the uniqueness of the design. We have pre-designed siRNAs for all the known human genes (24,502) in the Refseq database. These siRNAs were pre-BLASTed against the human Unigene database to estimate the target specificity and all results are available online.</p> <p>Conclusion</p> <p>Although most of the rules for this scoring system were influenced by previously published rules, the weighted scoring system provides better flexibility in designing an appropriate siRNA when compared to the un-weighted scoring system. sIR is not only a comprehensive tool used to design siRNA sequences and lookup pre-designed siRNAs, but it is also a platform where researchers can share information on siRNA design and use.</p

Exon and junction microarrays detect widespread mouse strain- and sex-bias expression differences

Background: Studies have shown that genetic and sex differences strongly influence gene expression in mice. Given the diversity and complexity of transcripts produced by alternative splicing, we sought to use microarrays to establish the extent of variation found in mouse strains and genders. Here, we surveyed the effect of strain and sex on liver gene and exon expression using male and female mice from three different inbred strains. Results: 71 liver RNA samples from three mouse strains - DBA/2J, C57BL/6J and C3H/HeJ - were profiled using a custom-designed microarray monitoring exon and exon-junction expression of 1,020 genes representing 9,406 exons. Gene expression was calculated via two different methods, using the 3'-most exon probe ("3' gene expression profiling") and using all probes associated with the gene ("whole-transcript gene expression profiling"), while exon expression was determined using exon probes and flanking junction probes that spanned across the neighboring exons ("exon expression profiling"). Widespread strain and sex influences were detected using a two-way Analysis of Variance (ANOVA) regardless of the profiling method used. However, over 90% of the genes identified in 3' gene expression profiling or whole transcript profiling were identified in exon profiling, along with 75% and 38% more genes, respectively, showing evidence of differential isoform expression. Overall, 55% and 32% of genes, respectively, exhibited strain- and sex-bias differential gene or exon expression. Conclusion: Exon expression profiling identifies significantly more variation than both 3' gene expression profiling and whole-transcript gene expression profiling. A large percentage of genes that are not differentially expressed at the gene level demonstrate exon expression variation suggesting an influence of strain and sex on alternative splicing and a need to profile expression changes at sub-gene resolution

Reaching the poor with health interventions: Programme-incidence analysis of seven randomised trials of women's groups to reduce newborn mortality in Asia and Africa

Background Efforts to end preventable newborn deaths will fail if the poor are not reached with effective interventions. To understand what works to reach vulnerable groups, we describe and explain the uptake of a highly effective community-based newborn health intervention across social strata in Asia and Africa. Methods We conducted a secondary analysis of seven randomised trials of participatory women's groups to reduce newborn mortality in India, Bangladesh, Nepal and Malawi. We analysed data on 70 574 pregnancies. Socioeconomic and sociodemographic differences in group attendance were tested using logistic regression. Qualitative data were collected at each trial site (225 focus groups, 20 interviews) to understand our results. Results Socioeconomic differences in women's group attendance were small, except for occasional lower attendance by elites. Sociodemographic differences were large, with lower attendance by young primigravid women in African as well as in South Asian sites. The intervention was considered relevant and interesting to all socioeconomic groups. Local facilitators ensured inclusion of poorer women. Embarrassment and family constraints on movement outside the home restricted attendance among primigravid women. Reproductive health discussions were perceived as inappropriate for them. Conclusions Community-based women's groups can help to reach every newborn with effective interventions. Equitable intervention uptake is enhanced when facilitators actively encourage all women to attend, organise meetings at the participants' convenience and use approaches that are easily understandable for the less educated. Focused efforts to include primigravid women are necessary, working with families and communities to decrease social taboos

Stereoselective, competitive, and nonlinear plasma protein binding of ibuprofen enantiomers as determined in vivo in healthy subjects

The plasma protein binding and competitive inhibition parameters of R(−)- and S(+)-ibuprofen were determined in vivo in 12 healthy subjects. Subjects participated in a 4×4 Latin square design in which oral solutions of drug were administered as 300 mg R (−)-ibuprofen, 300 mg S (+)-ibuprofen, 300 mg R (−)-+300 mg S (+)-ibuprofen, and 300 mg R(−)-+600 mg S (+)-ibuprofen. Unlabeled ibuprofen enantiomers were quantitated using a stereospecific reversed-phase HPLC assay, and plasma protein binding experiments were performed using radiolabeled 14 C-enantiomers and an ultrafiltration method at 37C. At therapeutic drug concentrations, the protein binding of each enantiomer was greater than 99%. Furthermore, the binding of ibuprofen enantiomers was Stereoselective and mutually competitive, as well as nonlinear. The bound-free data were fitted to a model in which the non-linearity of plasma protein binding and competition between enantiomers for binding sites could be accommodated. There were substantial differences in the affinity of ibuprofen enantiomers for protein binding sites (RP2=0.358±0.185 vs. SP2=0.979 ±0.501 μg/ml; X±SD) but no differences in their binding capacity (RP1=160±86 vs. SP1=161 ±63 μg/ml). Although statistically significant, the differences in competitive inhibition parameters were more modest (SKI=0.661 ±0.363 vs. RKI=0.436 ±0.210 μg/ml). As a result, the intrinsic binding (i.e.), P1/P2J of R(−)-ibuprofen was greater than S(±)-ibuprofen, and the unbound fraction was significantly greater for S-enantiomer vs. R-enantiomer after a given dose of R-ibuprofen or racemate.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45048/1/10928_2005_Article_BF01059767.pd

Piloting Upfront Xpert MTB/RIF Testing on Various Specimens under Programmatic Conditions for Diagnosis of TB & DR-TB in Paediatric Population

India accounts for one-fifth of the global TB incidence. While the exact burden of childhood TB is not known, TB remains one of the leading causes of childhood mortality in India. Bacteriological confirmation of TB in children is challenging due to difficulty in obtaining quality specimens, in the absence of which diagnosis is largely based on clinical judgement. While testing multiple specimens can potentially contribute to higher proportion of laboratory confirmed paediatric TB cases, lack of high sensitivity tests adds to the diagnostic challenge. We describe here our experiences in piloting upfront Xpert MTB/RIF testing, for diagnosis of TB in paediatric population in respiratory and extra pulmonary specimens, as recently recommended by WHO.Xpert MTB/RIF testing was offered to all paediatric (0-14 years) presumptive TB cases (both pulmonary and extra-pulmonary) seeking care at public and private health facilities in the project areas covering 4 cities of India.Under this pilot project, 8,370 paediatric presumptive TB & presumptive DR-TB cases were tested between April and-November 2014. Overall, 9,149 specimens were tested, of which 4,445 (48.6%) were non-sputum specimens. Xpert MTB/RIF gave 9,083 (99.2%, CI 99.0-99.4) valid results. Of the 8,143 presumptive TB cases enrolled, 517 (6.3%, CI 5.8-6.9) were bacteriologically confirmed. TB detection rates were two fold higher with Xpert MTB/RIF as compared to smear microscopy. Further, a total of 60 rifampicin resistant TB cases were detected, of which 38 were detected among 512 presumptive TB cases while 22 were detected amongst 227 presumptive DR-TB cases tested under the project.Xpert MTB/RIF with advantages of quick turnaround testing-time, high proportion of interpretable results and feasibility of rapid rollout, substantially improved the diagnosis of bacteriologically confirmed TB in children, while simultaneously detecting rifampicin resistance

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Magnetoelectric coupling of multiferroic chromium doped barium titanate thin film probed by magneto-impedance spectroscopy

Thin film of BaTiO3 doped with 0.1 at.% Cr (Cr:BTO) has been prepared by pulsed laser deposition technique. Film was deposited on Pt/SrTiO3 substrate at 500 degrees C in 50 mTorr Oxygen gas pressure using KrF (298 nm) laser. Polycrystalline growth of single phase Cr: BTO thin film has been confirmed by grazing angle X-ray diffraction. Cr: BTO film exhibited remnant polarization 6.4 mu C/cm(2) and 0.79 MV/cm coercivity. Magnetization measurement of Cr: BTO film showed magnetic moment 12 emu/cc. Formation of weakly magnetic domains has been captured by magnetic force microscopy. Theoretical impedance equation fitted to experimental data in Cole-Cole plot for thin film in presence of transverse magnetic field resolved the increase in grain capacitance from 4.58 x 10(-12) to 5.4 x 10(-11) F. Film exhibited high value 137 mV/cm-Oe magneto-electric (ME) coupling coefficient at room temperature. The high value of ME coupling obtained can reduce the typical processing steps involved in multilayer deposition to obtain multiferrocity in thin film. Barium titanate being best ferroelectric material has been tailored to be multiferroic by non ferromagnetic element, Cr, doping in thin film form opens an avenue for more stable and reliable spintronic material for low power magnetoelectric random excess memory applications