DNA-PK-Dependent RPA2 Hyperphosphorylation Facilitates DNA Repair and Suppresses Sister Chromatid Exchange

Hyperphosphorylation of RPA2 at serine 4 and serine 8 (S4, S8) has been used as a marker for activation of the DNA damage response. What types of DNA lesions cause RPA2 hyperphosphorylation, which kinase(s) are responsible for them, and what is the biological outcome of these phosphorylations, however, have not been fully investigated. In this study we demonstrate that RPA2 hyperphosphorylation occurs primarily in response to genotoxic stresses that cause high levels of DNA double-strand breaks (DSBs) and that the DNA-dependent protein kinase complex (DNA-PK) is responsible for the modifications in vivo. Alteration of S4, S8 of RPA2 to alanines, which prevent phosphorylations at these sites, caused increased mitotic entry with concomitant increases in RAD51 foci and homologous recombination. Taken together, our results demonstrate that RPA2 hyperphosphorylation by DNA-PK in response to DSBs blocks unscheduled homologous recombination and delays mitotic entry. This pathway thus permits cells to repair DNA damage properly and increase cell viability

An Effective Assessment of Simvastatin-Induced Toxicity with NMR-Based Metabonomics Approach

BACKGROUND: Simvastatin, which is used to control elevated cholesterol levels, is one of the most widely prescribed drugs. However, a daily excessive dose can induce drug-toxicity, especially in muscle and liver. Current markers for toxicity reflect mostly the late stages of tissue damage; thus, more efficient methods of toxicity evaluation are desired. METHODOLOGY/PRINCIPAL FINDINGS: As a new way to evaluate toxicity, we performed NMR-based metabonomics analysis of urine samples. Compared to conventional markers, such as AST, ALT, and CK, the urine metabolic profile provided clearer distinction between the pre- and post-treatment groups treated with toxic levels of simvastatin. Through multivariate statistical analysis, we identified marker metabolites associated with the toxicity. Importantly, we observed that the treatment group could be further categorized into two subgroups based on the NMR profiles: weak toxicity (WT) and high toxicity (HT). The distinction between these two groups was confirmed by the enzyme values and histopathological exams. Time-dependent studies showed that the toxicity at 10 days could be reliably predicted from the metabolic profiles at 6 days. CONCLUSIONS/SIGNIFICANCE: This metabonomics approach may provide a non-invasive and effective way to evaluate the simvastatin-induced toxicity in a manner that can complement current measures. The approach is expected to find broader application in other drug-induced toxicity assessments

MEMS Technologies for Energy Harvesting

The objective of this chapter is to introduce the technology of Microelectromechanical Systems, MEMS, and their application to emerging energy harvesting devices. The chapter begins with a general introduction to the most common MEMS fabrication processes. This is followed with a survey of design mechanisms implemented in MEMS energy harvesters to provide nonlinear mechanical actuations. Mechanisms to produce bistable potential will be studied, such as introducing fixed magnets, buckling of beams or using slightly slanted clamped-clamped beams. Other nonlinear mechanisms are studied such as impact energy transfer, or the design of nonlinear springs. Finally, due to their importance in the field of MEMS and their application to energy harvesters, an introduction to actuation using piezoelectric materials is given. Examples of energy harvesters found in the literature using this actuation principle are also presented

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Hybrid tin oxide-SWNT nanostructures based gas sensor

A facile electrochemical functionalization method was utilized to decorate single-walled carbon nanotubes (SWNTs) with tin oxide and their gas sensing performance toward various analytes (NH3, NO2, H-2, H2S, acetone, and water vapor) was evaluated at room temperature. Tin oxy-hydroxide was sitespecifically precipitated on the surface of SWNTs because of an increase in local pH during electrochemical reduction of nitrate to nitrite ions. By adjusting the amount of charge passed during deposition, the amount of tin oxide deposited on SWNTs was controlled, which altered the electronic and gas sensing properties of the nanostructures. The resulting hybrid nanostructures showed excellent sensitivities upon exposure to trace amounts of both oxidizing gases (limit of detection (LOD) of 25 ppbv for NO2) and reducing gases (LOD of 10 ppmv for H-2) at room temperature. The enhanced sensing performance was due to the charge transfer between the surface active tin oxide nanoparticles and SWNTs, with the direction of charge transfer depending on the analyte gas. This approach can be applied to fabricate other hybrid metal oxide-SWNTs nanostructures to create highly sensitive gas sensor arrays. (C) 2013 Elsevier Ltd. All rights reserved