Nucleic acid extraction (NAE) plays a vital role in molecular biology as the primary step for many downstream applications. Many modifications have been introduced to the original 1869 method. Modern processes are categorized into chemical or mechanical, each with peculiarities that influence their use, especially in point-of-care diagnostics (POC-Dx). POC-Dx is a new approach aiming to replace sophisticated analytical machinery with microanalytical systems, able to be used near the patient, at the point of care or point of need. Although notable efforts have been made, a simple and effective extraction method is still a major challenge for widespread use of POC-Dx. In this review, we dissected the working principle of each of the most common NAE methods, overviewing their advantages and disadvantages, as well their potential for integration in POC-Dx systems. At present, it seems difficult, if not impossible, to establish a procedure which can be universally applied to POC-Dx. We also discuss the effects of the NAE chemicals upon the main plastic polymers used to mass produce POC-Dx systems. We end our review discussing the limitations and challenges that should guide the quest for an efficient extraction method that can be integrated in a POC-Dx system.

Abstract-Ischemic and pharmacological preconditioning can be triggered by an intracellular signaling pathway in which G i -coupled surface receptors activate a cascade including phosphatidylinositol 3-kinase, endothelial nitric oxide synthase, guanylyl cyclase, and protein kinase G (PKG). Activated PKG opens mitochondrial K ATP channels (mitoK ATP ) which increase production of reactive oxygen species. Steps between PKG and mitoK ATP opening are unknown. We describe effects of adding purified PKG and cGMP on K ϩ transport in isolated mitochondria. Light scattering and respiration measurements indicate PKG induces opening of mitoK ATP similar to K ATP channel openers like diazoxide and cromakalim in heart, liver, and brain mitochondria. This effect was blocked by mitoK ATP inhibitors 5-hydroxydecanoate, tetraphenylphosphonium, and glibenclamide, PKG-selective inhibitor KT5823, and protein kinase C (PKC) inhibitors chelerythrine, Ro318220, and PKC-⑀ peptide antagonist ⑀V 1-2 . MitoK ATP are opened by the PKC activator 12-phorbol 13-myristate acetate. We conclude PKG is the terminal cytosolic component of the trigger pathway; it transmits the cardioprotective signal from cytosol to inner mitochondrial membrane by a pathway that includes PKC-⑀. (Circ Res. 2005;97:329-336.)Key Words: ATP-sensitive K ϩ channel Ⅲ cGMP Ⅲ preconditioning Ⅲ protein kinase C Ⅲ protein kinase G I schemic (IPC) and pharmacological preconditioning by ligands such as acetylcholine and bradykinin initiates a signaling cascade that opens mitochondrial ATP-sensitive K ϩ channels (mitoK ATP ). Many components of this signaling pathway have been identified. 1 Surface receptors activate phosphatidylinositol 3-(PI3-) kinase by transactivation of epidermal growth factor receptors (EGFRs). A signaling complex composed of transactivated EGFR, Src kinase, and PI3-kinase causes phosphorylation of phosphatidylinositol bisphosphate, which in turn activates the phosphatidylinositol-dependent kinases (PDKs). 2 The PDKs then phosphorylate Akt 3 which activates the remainder of the cytosolic signaling pathway (Figure 1) including phosphorylation of endothelial nitric oxide synthase (eNOS), production of NO, stimulation of guanylyl cyclase, generation of cGMP, and activation of protein kinase G (PKG).cGMP and presumably PKG activation are important during preconditioning. Delayed preconditioning by diazoxide involves NO, 4 and cGMP accumulation after inhibition of cGMP-specific phosphodiesterase by sildenafil induces acute and delayed preconditioning. 5 Direct activation of PKG increases generation of reactive oxygen species (ROS) in cardiomyocytes, and this effect depends on mitoK ATP opening. 3 Whereas PKG antagonists abort ROS generation by PKG activators, they have no effect on ROS triggered by diazoxide, a direct opener of mitoK ATP , indicating PKG is upstream of mitoK ATP . A direct activator of PKG mimics IPC in intact hearts. 6 We hypothesized that PKG opens mitoK ATP by causing its phosphorylation, 7 and that the open channel increases generation of mi...

Coronary artery disease and its sequelae-ischemia, myocardial infarction, and heart failure-are leading causes of morbidity and mortality in man. Considerable effort has been devoted toward improving functional recovery and reducing the extent of infarction after ischemic episodes. As a step in this direction, it was found that the heart was significantly protected against ischemia-reperfusion injury if it was first preconditioned by brief ischemia or by administering a potassium channel opener. Both of these preconditioning strategies were found to require opening of a K(ATP) channel, and in 1997 we showed that this pivotal role was mediated by the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). This paper will review the evidence showing that opening mitoK(ATP) is cardioprotective against ischemia-reperfusion injury and, moreover, that mitoK(ATP) plays this role during all three phases of the natural history of ischemia-reperfusion injury preconditioning, ischemia, and reperfusion. We discuss two distinct mechanisms by which mitoK(ATP) opening protects the heart-increased mitochondrial production of reactive oxygen species (ROS) during the preconditioning phase and regulation of intermembrane space (IMS) volume during the ischemic and reperfusion phases. It is likely that cardioprotection by ischemic preconditioning (IPC) and K(ATP) channel openers (KCOs) arises from utilization of normal physiological processes. Accordingly, we summarize the results of new studies that focus on the role of mitoK(ATP) in normal cardiomyocyte physiology. Here, we observe the same two mechanisms at work. In low-energy states, mitoK(ATP) opening triggers increased mitochondrial ROS production, thereby amplifying a cell signaling pathway leading to gene transcription and cell growth. In high-energy states, mitoK(ATP) opening prevents the matrix contraction that would otherwise occur during high rates of electron transport. MitoK(ATP)-mediated volume regulation, in turn, prevents disruption of the structure-function of the IMS and facilitates efficient energy transfers between mitochondria and myofibrillar ATPases.