Overexpression of Arabidopsis thaliana brassinosteroid-related acyltransferase 1 gene induces brassinosteroid-deficient phenotypes in creeping bentgrass

Brassinosteroids (BRs) are naturally occurring steroidal hormones that play diverse roles in various processes during plant growth and development. Thus, genetic manipulation of endogenous BR levels might offer a way of improving the agronomic traits of crops, including plant architecture and stress tolerance. In this study, we produced transgenic creeping bentgrass (Agrostis stolonifera L.) overexpressing a BR-inactivating enzyme, Arabidopsis thaliana BR-related acyltransferase 1 (AtBAT1), which is known to catalyze the conversion of BR intermediates to inactive acylated conjugates. After putative transgenic plants were selected using herbicide resistance assay, genomic integration of the AtBAT1 gene was confirmed by genomic PCR and Southern blot analysis, and transgene expression was validated by northern blot analysis. The transgenic creeping bentgrass plants exhibited BR-deficient phenotypes, including reduced plant height with shortened internodes (i.e., semi-dwarf), reduced leaf growth rates with short, wide, and thick architecture, high chlorophyll contents, decreased numbers of vascular bundles, and large lamina joint bending angles (i.e., erect leaves). Subsequent analyses showed that the transgenic plants had significantly reduced amounts of endogenous BR intermediates, including typhasterol, 6-deoxocastasterone, and castasterone. Moreover, the AtBAT1 transgenic plants displayed drought tolerance as well as delayed senescence. Therefore, the results of the present study demonstrate that overexpression of an Arabidopsis BR-inactivating enzyme can reduce the endogenous levels of BRs in creeping bentgrass resulting in BR-deficient phenotypes, indicating that the AtBAT1 gene from a dicot plant is also functional in the monocot crop.111Ysciescopu

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Department of Biomedical EngineeringGene therapy has benefited from the use of retroviruses, particularly lentiviruses, as effective gene transfer system to host cells. Lentiviruses have been utilized for treating various genetic diseases and developing cancer immunotherapy strategies. However, the integration of viral genomes into host cells can pose safety concerns because of the unpredictable integration sites. Thus, the detailed profiling of viral genome integration sites is crucial for ensuring safety and quality control in viral applications. Therefore, I developed the unbiased genome-wide detection method, UniQuE-seq. This method is simple and easy to produce the library for sequencing and improves detection sensitivity by using custom transposon loaded Tn5 and lambda exonuclease. I conducted the UniQuE-seq to validate that this method can detect the lentivirus integration sites. A comparison between whole genome sequencing and UniQuE-Seq revealed specific locations where simultaneous outcomes occurred. Interestingly, only 7 integration sites were consistent with whole genome sequencing in the experimental results using a monoclonal stable cell line. Moreover, the number of sites with a significantly low P-value (P-value < 0.0001) was about two times less when lambda exonuclease was treated. Sensitivity analysis demonstrated that UniQuE-Seq is feasible to detect even a single integrated site as well. Furthermore, UniQuE-seq enables to identify the genome-wide integration sites at complex populations. In conclusion, UniQuE-Seq is an unbiased method for detecting viral genome integration sites and improves selective amplification of targeted sequences by using lambda exonuclease and custom-designed transposon-loaded Tn5.clos

Prefoldin 5 and Anti-prefoldin 5 Antibodies as Biomarkers for Uveitis in Ankylosing Spondylitis

Objective: Uveitis is the most common extra-articular manifestation of ankylosing spondylitis (AS), for which no diagnostic biomarkers have been identified. This study was conducted to identify biomarker for uveitis in AS.Methods: To identify autoantibodies associated with uveitis in AS, we performed human protein microarray analysis using sera derived from various autoimmune diseases and ELISA analysis of sera derived from AS and rheumatoid arthritis patients. In the curdlan-induced SKG mice model, ophthalmic examination was performed at week 8 post-immunization and histologic examination of the ocular lesions performed at week 16 post-immunization. Serum levels of target antibodies were assessed at various time-points. To evaluate the functional role of specific autoantibodies, an in vitro apoptosis assay using ARPE-19 cells was performed.Results: Reactivity against prefoldin subunit 5 (PFDN5) was identified in AS with uveitis. Levels of anti-PFDN5 antibodies and PFDN5 in sera from AS with uveitis patients were significantly higher than those in AS without uveitis. At week 8, half of curdlan-treated SKG mice developed anterior uveitis, while all of them developed histologically confirmed uveitis at week 16. The levels of anti-PFDN5 antibodies increased over time in the sera of curdlan-treated SKG mice along with increased expression of PFDN5 and apoptosis in the ocular lesions. Knockdown of PFDN5 in ARPE19 cells resulted in increased apoptosis, suggesting a protective role of PFDN5 against cell death in uveitis.Conclusion: AS patients with uveitis have increased levels of anti-PFDN5 antibodies, and our findings suggest that anti-PFDN5 antibodies could provide a biomarker for uveitis in AS

Decision tree-based approach for online management of PEM fuel cells for residential application

This thesis demonstrates a new intelligent technique for the online optimal management of PEM fuel cells units for onsite energy production to supply residential utilizations. Classical optimization techniques are based on offline calculations and cannot provide the necessary computational speed for online performance. In this research, a Decision Tree (DT) algorithm is employed to obtain the optimal, or quasioptimal, settings of the fuel cell online and in a general framework. The main idea is to employ a classification technique, trained on a sufficient subset of data, to produce an estimate of the optimal setting without repeating the optimization process. A database is extracted from a previously-performed Genetic Algorithm (GA)-based optimization has been used to create a suitable decision tree, which was intended for generalizing the optimization results. The approach provides the flexibility of adjusting the settings of the fuel cell online according to the observed variations in the tariffs and load demands. Results at different operating conditions are presented to confirm the high accuracy of the proposed generalization technique. The accuracy of the decision tree has been tested by evaluating the relative error with respect to the optimized values. Then, the possibility of pruning the tree has been investigated in order to simplify its structure without affecting the accuracy of the results. In addition, the accuracy of the DTs to approximate the optimal performance of the fuel cell is compared to that of the Artificial Neural Networks (ANNs) used for the same purpose. The results show that the DTs can somewhat outperform the ANNs with certain pruning levels

2155-PUB: Diabetic Foot Ulcer Regeneration Platform Based on 4D Bioprinting Technology

Diabetic foot ulcers have a significantly negative impact on patients and are highly prone to contamination, which can lead to amputation. The best possible wound management treatment was essential for diabetic foot ulcers. However, since 4D bio printing is the best way to recapitulate complex and functional human tissue and organ, we have developed the therapeutic 4D bio printer called INVIVO to use in skin regeneration. To find advanced treatment method particularly for wound healing, we tested our own 4D bio printing system with autologous fat tissue for DFU treatment. The extracellular matrix (ECM) derived from Nano-fat consisting of supportive proteins, growth factors and cytokines, has been reported as an important player to provide efficient 3D environments during cell proliferation and differentiation. In the clinical study, we printed autologous ECM (MA-ECM) with bio-inks to apply onto the chronic wound site. Most of DFU patients showed significant reduction of wound size with distinct epithelization only after a week treatment. Therefore, we suggest 4D bio printing system with MA-ECM as an alternative method for dressing materials that successfully promote the mechanism of skin reconstruction for DFU treatment. Reduction in size of wound was measured using ImageJ NIH software and almost all the subjects revealed complete closure of wound maximum of 2∼5 weeks post MA-ECM membrane application. The present study provided a significantly higher healing rate of the subjects and the primary outcome of our study complete epithelization with wound closure was achieved. The autologous fat by nature is loaded with adipocytes and growth factors which could have played a vital role in the healing of non-healing diabetic wounds. Therefore, we suggest 4D bio printing system with MA-ECM as an alternative method for chronic wound healing that successfully promote the mechanism of skin reconstruction for DFU treatment. Disclosure J. Kim: None. </jats:sec