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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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. 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. 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

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Transglutaminase 2 (TGase2) is a ubiquitously expressed enzyme that catalyzes irreversible post-translational modification of protein, forming cross-linked protein aggregates. We previously reported that intracellular TGase2 is activated by oxidative stress. To elucidate the functional role of TGase2 activation in cells under the oxidatively stressed condition, we identified the mediator that activates TGase2. In this study, we showed that low levels of oxidative stress trigger the release of TGFbeta, which subsequently activates TGase2 through the nuclear translocation of Smad3. Analysis of substrate proteins reveals that TGase2-mediated protein modification results in a decrease of protein solubility and a collapse of intermediate filament network, which leads to aggregation of proteins. We confirm these results using lens tissues from TGase2-deficient mice. Among several antioxidants tried, only N-acetylcysteine effectively inhibits TGFbeta-mediated activation of TGase2. These results indicate that TGFbeta mediates oxidative stress-induced protein aggregation through activation of TGase2 and suggest that the formation of protein aggregation may not be a passive process of self-assembly of oxidatively damaged proteins but may be an active cellular response to oxidative stress. Therefore, TGFbeta-TGase2 pathway may have implications for both the pathogenesis of age-related degenerative diseases and the development of pharmaceutics.

Multifunctional carbon nanotube (CNT) composite fibers are currently of considerable interest in applications where actuation and energy-storage functions are highly desirable, such as electronic textiles. CNT fibers have been shown to function as excellent electrochemical supercapacitors giving specific capacitances of 100 F g -1 [1] . CNT assemblies can also produce useful actuation strains when electrochemically charged [2] and can potentially operate to high stresses because of the excellent mechanical properties of individual CNTs. The development of CNT fibers that simultaneously produce a high capacitance and useful actuation performance remains a challenge however, because the high surface area needed for high capacitance significantly reduces the strength and compromises actuation performance. To date, it has not been possible to develop an ion-conducting binder that mechanically stabilizes the CNT assembly, maintains electrical connectivity between nanotubes, and allows free transport of ions between the nanotubes and an external electrolyte. Pioneering work by Poulin [3] and Baughman [4] have established processing methods for preparing continuous fibers of CNTs and CNT composites that are ideal for electronic textiles. Various studies on these fibers and other CNT assemblies have highlighted the difficulties involved in producing mechanically robust, high-conductivity and high-surface-area electrodes. While single wall carbon nanotube (SWNT) fibers containing ∼40 % poly(vinyl alcohol) (PVA) binder give exceptional mechanical properties, their conductivity is very low at 0.2 S cm -1 .[1] The binder can be removed by pyrolysis to improve conductivity so that the fibers can be operated as electromechancial actuators. While quite high actuation stresses were obtained in these thermally annealed CNT fibers, their low flexibility and high creep during charge and discharge were noted as significant problems.[5] Similarly, fibers spun without the aid of a polymer binder [1] produce high conductivities (140 S cm -1 after thermal annealing) and capacitances (100 F g -1 ) but are mechanically fragile. To resolve these problems, crosslinked DNA has been chosen as a binder for CNT fibers. DNA is a good candidate for improved electrical conductivity for electrochemical devices with CNTs, as DNA has electrical characteristics similar to those of semiconducting diodes in that current flows in one direction only. [6][7][8] In addition, DNA more effectively coats, separates, and solubilizes CNTs than other surfactants because of the large surface area of its phosphate backbone, which interacts with water, and there are many bases in DNA that can bind to CNTs.[9] Therefore, DNA wrapping can debundle CNTs in high concentration CNT dispersions. Consequently, DNA wrapping may improve electrochemical actuation and capacitance of nanotubes in composite fibers by its improved electrical conductivity, high CNT surface area and enhanced mechanical stability due to the p-p interaction between the DNA and the CNT sidewall. We rep...

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SummaryDendritic cells (DCs) are promising therapeutic agents in the field of cancer immunotherapy due to their intrinsic immune-priming capacity. The potency of DCs, however, is readily attenuated immediately after their administration in patients as tumours and various immune cells, including DCs, produce various immunosuppressive factors such as interleukin (IL)-10 and transforming growth factor (TGF)-b that hamper the function of DCs. In this study, we used small interfering RNA (siRNA) to silence the expression of endogenous molecules in DCs, which can sense immunosuppressive factors. Among the siRNAs targeting various immunosuppressive molecules, we observed that DCs transfected with siRNA targeting IL-10 receptor alpha (siIL-10RA) initiated the strongest antigen-specific CD8 1 T cell immune responses. The potency of siIL-10RA was enhanced further by combining it with siRNA targeting TGF-b receptor (siTGF-bR), which was the next best option during the screening of this study, or the previously selected immunoadjuvant siRNA targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or Bcl-2-like protein 11 (BIM). In the midst of sorting out the siRNA cocktails, the cocktail of siIL-10RA and siTGF-bR generated the strongest antigen-specific CD8 1 T cell immunity. Concordantly, the knock-down of both IL-10RA and TGF-bR in DCs induced the strongest anti-tumour effects in the TC-1 P0 tumour model, a cervical cancer model expressing the human papillomavirus (HPV)-16 E7 antigen, and even in the immune-resistant TC-1 (P3) tumour model that secretes more IL-10 and TGF-b than the parental tumour cells (TC-1 P0). These results provide the groundwork for future clinical development of the siRNA cocktail-mediated strategy by co-targeting immunosuppressive molecules to enhance the potency of DC-based vaccines.

AIM:To investigate the characteristics of slow electrical waves and the presence of transient receptor potential melastatin-type 7 (TRPM7) in the human gastrointestinal (GI) tract. METHODS:Conventional microelectrode techniques were used to record intracellular electrical responses from human GI smooth muscle tissue. Immunohistochemistry was used to identify TRPM7 channels in interstitial cells of Cajal (ICCs). RESULTS:The human GI tract generated slow electrical waves and had ICCs which functioned as pacemaker cells. Flufenamic acid, a nonselective cation channel blocker, and 2-APB (2-aminoethoxydiphenyl borate) and La 3+, TRPM7 channel blockers, inhibited the slow waves. Also, TRPM7 channels were expressed in ICCs in human tissue. CONCLUSION:These results suggest that the human GI tract generates slow waves and that TRPM7 channels expressed in the ICCs may be involved in the generation of the slow waves.