8 research outputs found
Analysis by siRNA_profile program displays novel thermodynamic characteristics of highly functional siRNA molecules
<p>Abstract</p> <p>Objective</p> <p>Here we report the improved results of a new siRNA design program and analysis tool called <b><it>siRNA_profile </it></b>that reveals an additional criterion for bioinformatic search of highly functional siRNA sequences.</p> <p>Methods</p> <p>We retrospectively analysed over 2400 siRNA sequences from 34 genes and with known efficacies to categorize factors that differentiate highly, moderately and non-functional siRNA sequences in more detail. We tested the biological relevance of <b><it>siRNA_profile </it></b>in CHO cells stably expressing human TRACP.</p> <p>Results</p> <p>The highly functional siRNA molecules exhibited lower overall stabilities than non-functional siRNAs after taking into consideration all the nucleotides from 5'-terminus to the 3'-terminus along the siRNA molecule, in addition to the 5'-section of the antisense strand and the region between 9–14 nucleotides as previously has been acknowledged. Comparison of the <b><it>siRNA_profile </it></b>program to five other programs resulted in a wide range of selected siRNA sequences with diverse gene silencing capacities, even when the target was only 197 nucleotides long. Six siRNA design programs selected 24 different siRNA sequences, and only 6 of them were selected by two or more programs. The other 18 sequences were individually selected by these six programs.</p> <p>Conclusion</p> <p>Low general stability of dsRNA plays a significant role in the RNAi pathway and is a recommended criterion to consider, in addition to 5'-instability, internal instability, nucleotide preferences and target mRNA position, when designing highly efficient siRNAs.</p
Evidence-based national vaccine policy
India has over a century old tradition of development and production of vaccines. The Government rightly adopted self-sufficiency in vaccine production and self-reliance in vaccine technology as its policy objectives in 1986. However, in the absence of a full-fledged vaccine policy, there have been concerns related to demand and supply, manufacture vs. import, role of public and private sectors, choice of vaccines, new and combination vaccines, universal vs. selective vaccination, routine immunization vs. special drives, cost-benefit aspects, regulatory issues, logistics etc. The need for a comprehensive and evidence based vaccine policy that enables informed decisions on all these aspects from the public health point of view brought together doctors, scientists, policy analysts, lawyers and civil society representatives to formulate this policy paper for the consideration of the Government. This paper evolved out of the first ever ICMR-NISTADS national brainstorming workshop on vaccine policy held during 4-5 June, 2009 in New Delhi, and subsequent discussions over email for several weeks, before being adopted unanimously in the present form
Characterisation of ACP5 missense mutations encoding tartrate-resistant acid phosphatase associated with spondyloenchondrodysplasia.
Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism