Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection

Delivering gene constructs into the dorsal root ganglia (DRG) is a powerful but challenging therapeutic strategy for sensory disorders affecting the DRG and their peripheral processes. The current delivery methods of direct intra-DRG injection and intrathecal injection have several disadvantages, including potential injury to DRG neurons and low transfection efficiency, respectively. This study aimed to develop a spinal nerve injection strategy to deliver polyethylenimine mixed with plasmid (PEI/DNA polyplexes) containing green fluorescent protein (GFP). Using this spinal nerve injection approach, PEI/DNA polyplexes were delivered to DRG neurons without nerve injury. Within one week of the delivery, GFP expression was detected in 82.8% ± 1.70% of DRG neurons, comparable to the levels obtained by intra-DRG injection (81.3% ± 5.1%, p = 0.82) but much higher than those obtained by intrathecal injection. The degree of GFP expression by neurofilament(+) and peripherin(+) DRG neurons was similar. The safety of this approach was documented by the absence of injury marker expression, including activation transcription factor 3 and ionized calcium binding adaptor molecule 1 for neurons and glia, respectively, as well as the absence of behavioral changes. These results demonstrated the efficacy and safety of delivering PEI/DNA polyplexes to DRG neurons via spinal nerve injection.National Science Council of Taiwan (100-2321-B-002-007)National Science Council of Taiwan (100-2320-B-002-083-MY3)Taiwan. Ministry of Science and Technology (104-2300-B-002-019-MY3)National Taiwan University. College of Medicine (Translational Medicine Project)National Taiwan University Hospital (101C101-201

SUMOylation attenuates c-Maf-dependent IL-4 and IL-21 expression

轉錄因子的功能是藉由控制其生成、活性與降解來嚴格調控的，而SUMO化修飾是在轉錄後層級調控蛋白質的活性。c-Maf蛋白是屬於大Maf家族的鹼性白氨酸拉鍊蛋白，在第二或第十七型輔助T細胞上分別為介白素四與介白素二十一的專一性轉錄因子。我們藉由酵母雙雜交系統來尋找c-Maf的交互作用蛋白。我們發現兩個SUMO化修飾的關鍵酵素：Ubc9與PIAS1，可以與c-Maf蛋白交互作用。在T細胞中，PIAS1可以與c-Maf蛋白交互作用；這兩個SUMO化修飾的關鍵酵素也與c-Maf蛋白共同位於細胞核中。我們也確認c-Maf蛋白在活體外與活體內都可被SUMO修飾。c-Maf蛋白N端第33個離胺酸是SUMO蛋白的修飾位。SUMO化修飾會降低c-Maf蛋白對介白素四的轉錄活性，而無法被SUMO化修飾的c-Maf蛋白卻有較強的轉錄活性。同時，我們也發現c-Maf是介白素二十一的專一性轉錄因子。SUMO化修飾也會影響c-Maf蛋白相關之介白素二十一的產生。SUMO化修飾不會影響c-Maf蛋白的穩定性，但無法SUMO化修飾的c-Maf蛋白匯集到啟動子的能力卻較強。我們的結論是c-Maf蛋白的SUMO化修飾對其在輔助T細胞中的活性十分重要。The function of transcription factor is tightly regulated by controlling their synthesis, activity and degradation. SUMOylation modulates target protein activity on post-translational level. c-Maf, the cellular homologue of v-Maf, is a basic-leucine zipper protein and belongs to the large Maf family. In helper T cells, c-Maf is the specific transcription factor of the IL-4 and IL-21 genes in type 2 T helper (Th2) and type 17 T helper (Th17) cells, respectively. In our study, we performed the yeast two-hybrid assay to identify the c-Maf interacting proteins. We found that c-Maf can interact with Ubc9 and PIAS1, the key enzymes of SUMOylation system. In T cells, c-Maf interacts with PIAS1 in primary T cells and also co-localizes with these two SUMO ligases in the nucleus. We also demonstrated that c-Maf can be SUMOylated in vitro and also in vivo. We identified the c-Maf SUMO acceptor site(s) by mutated the putative conjugating lysine residues. We demonstrated that N-terminal lysine-33 within the activation domain is the SUMO acceptor site for c-Maf. SUMO modification attenuates Wt-c-Maf transcriptional activity. Conversely, c-Maf SUMO deficient mutant is more potent to drive IL-4 production in Th2 cells. Furthermore, we showed that c-Maf, but not other transcription factor, transactivates IL-21 gene expression. SUMOylation also affects c-Maf dependent IL-21 production. In addition, SUMO deficient c-Maf does not alter the localization and the protein stability, but further enhances its recruitment to the Il4-promoter. We conclude that post-translational lysine-33 SUMOylation is critical for c-Maf activity in helper T cells

SUMOYLATION ATTENUATES C-MAF-DEPENDENT IL-4 EXPRESSION

The function of transcription factors can be critically regulated by SUMOylation. c-Maf, the cellular counterpart of v-maf oncogene, is a potent transactivator of the IL-4 gene in Th2 cells. We found in a yeast two-hybrid screen that c- Maf can interact with Ubc9 and PIAS1, two key enzymes of the SUMOylation pathway. In this study, we report that c-Maf co-localized with these two SUMO (small ubiquitin-like modifier) ligases in the nucleus and that c-Maf can be SUMOylated in vitro and also in primary Th2 cells. We also demonstrated that lysine-33 is the dominant, if not the only , SUMO acceptor site of c-Maf. SUMOylation of c-Maf attenuated its transcriptional activity. Reciprocally, a SUMOylation resistant c-Maf was more potent than WT-c-Maf in driving IL-4 production in c-Maf-deficient Th2 cells. Furthermore, we showed that ablation of the SUMO site did not alter the subcellular localization or the stability of c -Maf protein but instead enhanced its recruitment to the I14 -promoter. We conclude that SUMOylation at lysine-33 is a functionally critical post-translational modification event of c-Maf in Th cells

Spatiotemporal integration of visual stimuli and its relevance to the use of a divisional power supply scheme for retinal prosthesis.

A wireless photovoltaic retinal prosthesis is currently being studied with the aim of providing prosthetic vision to patients with retinitis pigmentosa (RP) and age-related macular degeneration (AMD). The major challenge of a photovoltaic device is its limited power efficiency. Our retinal prosthetic design implements a unique divisional power supply scheme (DPSS) system that provides the electrical power generated by all of the solar cells to only a subset of electrodes at any moment in time. The aim of the present study was to systematically characterize the spatiotemporal integration performance of the system under various DPSS conditions using human subjects and a psychophysical approach. A 16x16 pixels LED array controlled by Arduino was used to simulate the output signal of the DPSS design, and human performance under different visual stimulations at various update frequencies was then used to assess the spatiotemporal capability of retinal prostheses. The results showed that the contrast polarity of the image, image brightness, and division number influenced the lower limit of the update frequency of the DPSS system, while, on the other hand, visual angle, ambient light level, and stimulation order did not affect performance significantly. Pattern recognition by visual persistence with spatiotemporal integration of multiple frames of sparse dots is a feasible approach in retinal prosthesis design. These findings provide an insight into how to optimize a photovoltaic retinal prosthesis using a DPSS design with an appropriate update frequency for reliable pattern recognition. This will help the development of a wireless device able to restore vision to RP and AMD patients in the future

sj-docx-1-npx-10.1177_1934578X221142797 - Supplemental material for Comparison of the Immunomodulatory Effect of TCM Formulas Containing Either Astragali Radix or With This Replaced by Hedysari Radix

Supplemental material, sj-docx-1-npx-10.1177_1934578X221142797 for Comparison of the Immunomodulatory Effect of TCM Formulas Containing Either Astragali Radix or With This Replaced by Hedysari Radix by Yu-Chi Tsai, Ming-Kuem Lin, Wen-Huang Peng, Chih-Kai Tseng, Meng-Shiou Lee, Bo-Cheng Yang and Wen-Te Chang in Natural Product Communications</p

A Pilot Plant Study on the Autoclaving of Food Wastes for Resource Recovery and Reutilization

Autoclaving of food wastes (FW) for the resource recovery and reutilization was studied using the pilot plant scale. Experiments were conducted at various temperatures of 408, 428, and 438 K and times of 15 and 60 min. The in-filled steam to the autoclave was supplied by the incineration plant with a gauge pressure of 7 kg/cm2 and a temperature of 443 K or above. The results obtained from the experiments show that the less energy- and time-consuming autoclaving conditions (408 K and 15 min, denoted as Case A408-15) are effective. Comparisons of the properties and characteristics of autoclaved FW (FWA) of Case A408-15 with those of FW are made. The wet bulk volume and wet bulk density of FW A are dramatically reduced to 15.64% and increased to 313.37% relative to those of FW, respectively. This makes the subsequent processing and reuse for FWA more convenient than FW. The autoclaving results in an increase of carbon content and a decrease of nitrogen content, and thus an increase of the C/N ratio of FWA. The contents of sulfur, hemi-cellulose, and cellulose of FWA are also reduced. All these fluctuations are beneficial for making compost or other usages from FWA than FW. The autoclaved liquid product (LA) separated from FWA and liquid condensate (LC) from the released gas possess high COD and TOC. These two liquids can be mixed for use as liquid fertilizers with proper conditioning. Alternatively, further anaerobic digestion of the mixture of FWA, LA, and LC can offer enhanced biogas production for power generation. All these thus match the appeal of sustainable materials management and circular economy. The emitted gas from autoclaving contains no CO and some hydrocarbons. Suitable air pollution control is needed. The results and information obtained are useful for the proper recovery and reuse of abundant food wastes from domestic households and food industries

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field