RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure

Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3' end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3'-DNA/ds siRNA and includes a 3'-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3'-DNA/ds siRNA, (II) passenger-3'-DNA/ds siRNA, (III) guide-3'-DNA/ss siRNA, and (IV) passenger-3'-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I-III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3'-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3'-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3'-DNA/siRNA constructs are discussed. Molecular Therapy-Nucleic Acids (2014) 2, e141; doi:10.1038/mtna.2013.68; published online 7 January 2014

Honoring Missouri\u27s First People: The Native Partnerships and Programming Alliance

A new initiative began this year in St. Louis; a group of community organizations united together to advance Native partnerships and programming statewide. This collaborative effort is a product of cooperation between educational and cultural institutions in Missouri

Oncogenic viral protein HPV E7 up-regulates the SIRT1 longevity protein in human cervical cancer cells

Senescence is blocked in human cervical keratinocytes infected with high risk human papillomavirus (e.g. HPV type16). Viral oncoproteins HPV E6 and HPV E7 access the cell cycle via cellular p53 and retinoblastoma proteins respectively. Previously we have shown that HPV E7, not HPV E6, is also responsible for cervical cancer cell survival (SiHa cells; HPV type16). We now present evidence that SIRT1, an aging-related NAD-dependent deacetylase, mediates HPV E7 survival function in SiHa cervical cancer cells. Moreover, HPV E7 up-regulates SIRT1 protein when expressed in primary human keratinocytes. Conversely, SIRT1 levels decrease following RNAi-mediated silencing of HPV E7 in SiHa cells. Silencing HPV E6 has no effect on SIRT1 but, as expected, causes marked accumulation of p53 protein accompanied by p53-mediated up-regulation of p21. However, p53 acetylation (K382Ac) was barely detectable. Since p53 is a known SIRT1 substrate we propose that elevated SIRT1 levels (induced by HPV E7) attenuate p53 pro-apoptotic capacity via its de-acetylation. Our discovery that HPV E7 up-regulates SIRT1 links a clinically important oncogenic virus with the multi-functional SIRT1 protein. This link may open the way for a more in-depth understanding of the process of HPV-induced malignant transformation and also of the inter-relationships between aging and cancer

A bi-functional siRNA construct induces RNA interference and also primes PCR amplification for its own quantification

RNA interference (RNAi) is a process of post-transcriptional gene silencing initiated by double-stranded RNAs, including short interfering RNA (siRNA). Silencing is sequence-specific and RNAi has rapidly become central to the study of gene function. RNAi also carries promise for selective silencing of viral and endogenous genes causal for disease. To detect the very low levels of siRNA effective for RNAi we modified the 3′ end of the sense strand of siRNA with a nuclease-resistant DNA hairpin. We show that the modified siRNA-DNA construct (termed ‘crook’ siRNA) functions as a primer for the PCR and describe a novel, yet simple PCR protocol for its quantification (amolar levels/cell). When transfected into mammalian cells, crook siRNA induces selective mRNA knock-down equivalent to its unmodified siRNA counterpart. This new bifunctional siRNA construct will enable future in vivo studies on the uptake, distribution and pharmacokinetics of siRNA, and is particularly important for the development of siRNA-based therapeutics. More generally, PCR-based detection of siRNA carries wide-ranging applications for RNAi reverse genetics

P53 autoantibodies in 1006 patients followed up for breast cancer.

Serial plasma samples from 1006 patients with breast cancer revealed: (i) no correlation of p53 autoantibody status with disease status at the time of sample collection, or with menopausal status at time of primary diagnosis of breast cancer; (ii) 155 out of 1006 (15%) of patients were positive for p53 autoantibodies, and these patients tended to have a persistent autoantibody status throughout follow up, irrespective of disease behaviour; and (iii) where a negative autoantibody status was found at primary diagnosis of breast cancer, this negative status persisted throughout follow up, irrespective of later disease behaviour. We conclude that screening for p53 autoantibody status is not informative on residual tumour activity nor on therapeutic responsiveness.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Network based transcription factor analysis of regenerating axolotl limbs

<p>Abstract</p> <p>Background</p> <p>Studies on amphibian limb regeneration began in the early 1700's but we still do not completely understand the cellular and molecular events of this unique process. Understanding a complex biological process such as limb regeneration is more complicated than the knowledge of the individual genes or proteins involved. Here we followed a systems biology approach in an effort to construct the networks and pathways of protein interactions involved in formation of the accumulation blastema in regenerating axolotl limbs.</p> <p>Results</p> <p>We used the human orthologs of proteins previously identified by our research team as bait to identify the transcription factor (TF) pathways and networks that regulate blastema formation in amputated axolotl limbs. The five most connected factors, c-Myc, SP1, HNF4A, ESR1 and p53 regulate ~50% of the proteins in our data. Among these, c-Myc and SP1 regulate 36.2% of the proteins. c-Myc was the most highly connected TF (71 targets). Network analysis showed that TGF-β1 and fibronectin (FN) lead to the activation of these TFs. We found that other TFs known to be involved in epigenetic reprogramming, such as Klf4, Oct4, and Lin28 are also connected to c-Myc and SP1.</p> <p>Conclusions</p> <p>Our study provides a systems biology approach to how different molecular entities inter-connect with each other during the formation of an accumulation blastema in regenerating axolotl limbs. This approach provides an in silico methodology to identify proteins that are not detected by experimental methods such as proteomics but are potentially important to blastema formation. We found that the TFs, c-Myc and SP1 and their target genes could potentially play a central role in limb regeneration. Systems biology has the potential to map out numerous other pathways that are crucial to blastema formation in regeneration-competent limbs, to compare these to the pathways that characterize regeneration-deficient limbs and finally, to identify stem cell markers in regeneration.</p

‘Nature positive’ must incorporate, not undermine, the mitigation hierarchy

For the concept of nature positive to succeed as the lodestar for international action on biodiversity conservation, it must build upon lessons learned from the application of the mitigation hierarchy — or risk becoming mere greenwash