COLD-PCR enhanced melting curve analysis improves diagnostic accuracy for KRAS mutations in colorectal carcinoma

<p>Abstract</p> <p>Background</p> <p><it>KRAS </it>mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (<it>EGFR</it>) in patients with metastatic colorectal cancer. Sensitive methods are required to reliably detect <it>KRAS </it>mutations in tumor samples due to admixture with non-mutated cells. Many laboratories have implemented sensitive tests for <it>KRAS </it>mutations, but the methods often require expensive instrumentation and reagents, parallel reactions, multiple steps, or opening PCR tubes.</p> <p>Methods</p> <p>We developed a highly sensitive, single-reaction, closed-tube strategy to detect all clinically significant mutations in <it>KRAS </it>codons 12 and 13 using the Roche LightCycler^®instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (<it>T_c</it>) of 81°C increased the sensitivity of the assay >10-fold for the majority of <it>KRAS </it>mutations.</p> <p>Results</p> <p>We compared the COLD-PCR enhanced melting curve method to melting curve analysis without COLD-PCR and to traditional Sanger sequencing. In a cohort of 61 formalin-fixed paraffin-embedded colorectal cancer specimens, 29/61 were classified as mutant and 28/61 as wild type across all methods. Importantly, 4/61 (6%) were re-classified from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significant <it>KRAS </it>mutations by COLD-PCR DNA sequencing. Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background.</p> <p>Conclusions</p> <p>We have developed and validated an inexpensive, rapid, and highly sensitive clinical assay for <it>KRAS </it>mutations that is the first report of COLD-PCR combined with probe-based melting curve analysis. This assay significantly improved diagnostic accuracy compared to traditional PCR and direct sequencing.</p

Internet of Things in Sustainable Energy Systems

Our planet has abundant renewable and conventional energy resources but technological capability and capacity gaps coupled with water-energy needs limit the benefits of these resources to citizens. Through IoT technology solutions and state-of-the-art IoT sensing and communications approaches, the sustainable energy-related research and innovation can bring a revolution in this area. Moreover, by the leveraging current infrastructure, including renewable energy technologies, microgrids, and power-to-gas (P2G) hydrogen systems, the Internet of Things in sustainable energy systems can address challenges in energy security to the community, with a minimal trade-off to environment and culture. In this chapter, the IoT in sustainable energy systems approaches, methodologies, scenarios, and tools is presented with a detailed discussion of different sensing and communications techniques. This IoT approach in energy systems is envisioned to enhance the bidirectional interchange of network services in grid by using Internet of Things in grid that will result in enhanced system resilience, reliable data flow, and connectivity optimization. Moreover, the sustainable energy IoT research challenges and innovation opportunities are also discussed to address the complex energy needs of our community and promote a strong energy sector economy

Quantitative estimates on localized finite differences for the fractional Poisson problem, and applications to regularity and spectral stability

We establish new quantitative estimates for localized finite differences of solutions to the Poisson problem for the fractional Laplace operator with homogeneous Dirichlet conditions of solid type settled in bounded domains satisfying the Lipschitz cone regularity condition. We then apply these estimates to obtain (i) regularity results for solutions of fractional Poisson problems in Besov spaces; (ii) quantitative stability estimates for solutions of fractional Poisson problems with respect to domain perturbations; (iii) quantitative stability estimates for eigenvalues and eigenfunctions of fractional Laplace operators with respect to domain perturbations

Regeneration of the entire human epidermis using transgenic stem cells

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, lifethreatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies

Acute Porphyrias in the USA: Features of 108 Subjects from Porphyrias Consortium

BackgroundRecent descriptions of the clinical and laboratory features of subjects with acute porphyrias in the US are lacking. Our aim was to describe clinical, biochemical, and genetic features of 108 subjects.MethodsBetween September 2010 and December 2012, 108 subjects with acute porphyrias (90 acute intermittent porphyrias, 9 hereditary coproporphyrias, 9 variegate porphyrias) were enrolled into an observational study. Genetic testing was performed at a central genetic testing laboratory and clinical information entered into a central database. Selected features were compared with data for adults in the US.ResultsMost subjects (88/108, 81%) were female, with self-reported onset of symptoms in the second through fourth decades of life. The most common symptom was abdominal pain. Appendectomies and cholecystectomies were common before a diagnosis of porphyria. The diagnosis was delayed by a mean of 15 years. Anxiety and depression were common, and 18% complained of chronic symptoms, especially neuropathic and other pains. The incidences of systemic arterial hypertension, chronic kidney disease, seizure disorders, and psychiatric conditions were markedly increased. Mutations of the known causative genes were found in 102/105 of those tested, with novel mutations being found in 37, including in 7/8 subjects with hereditary coproporphyria. Therapy with intravenous hematin was the most effective therapy both for treatment of acute attacks and for prevention of recurrent attacks.ConclusionsAcute porphyrias often remain undiagnosed for more than a decade after first symptoms develop. Intravenous hematin is the treatment of choice, both for treatment of acute attacks and for prevention of recurrent attacks