Application and Optimization of Bioluminescence Resonance Energy Transfer (BRET) for Real Time Detection of Protein-Protein Interactions in Transgenic \u3cem\u3eArabidopsis\u3c/em\u3e as well as Structure-Based Functional Studies on the Active Site of Coelenterazine-dependent Luciferase from \u3cem\u3eRenilla\u3c/em\u3e and its Improvement by Protein Engineering

Bioluminescence resonance energy transfer (BRET) is a biological phenomenon in some marine organisms such as Renilla reniformis and Aequorea victoria. In BRET, resonance energy from decarboxylation of coelenterazine, a substrate of Renilla luciferase (RLUC), is transferred to its acceptor such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP), dependent on a distance of around 5 nm between the energy donor (RLUC) and its acceptor. The activation of the energy acceptor results in a spectral change in luminescence emission. The BRET system allows investigation of in vivo protein-protein interactions in real time. This was demonstrated with two heterodimeric interactions in transgenic Arabidopsis. In an attempt to optimize the activity and to address the reaction mechanism of the RLUC enzyme, a homology model of RLUC was obtained using a haloalkane dehalogenase, LinB, as a template. Furthermore, the homology model and the crystal structures of RLUC were docked with coelenterazine. The computational analyses suggested potential roles of catalytic triad residues (Asp120, Glu144, and His285) and substrate binding residues (N53, W121, and P220) in the active site. Mutagenesis, spectroscopy, and expression in E. coli were carried out to elucidate the reaction mechanism of RLUC and the possible roles of the residues. Moreover, the catalytic triad was probed using pharmacological tests. Using random mutagenesis, a new triple mutant was isolated, which showed increased kcat, increased half-life, and higher resistance to substrate inhibition. These results establish enzymatic characteristics of RLUC and, furthermore, suggest that the triple mutant may result in potentially advantageous properties for BRET assays, including imaging routines in Arabidopsis

Mutational optimization of the coelenterazine-dependent luciferase from Renilla

Renilla luciferase (RLUC) is a popular reporter enzyme for gene expression and biosensor applications, but it is an unstable enzyme whose catalytic mechanism remains to be elucidated. We titrated that one RLUC molecule can turn over about one hundred molecules of coelenterazine substrate. Mutagenesis of active site residue Pro220 extended the half-life of photon emission, yielding brighter luminescence in E. coli. Random mutagenesis uncovered two new mutations that stabilized and increased photon emission in vivo and in vitro, while ameliorating substrate inhibition. Further amended with a previously identified mutation, a new triple mutant showed a threefold improved kcat, as well as elevated luminescence in Arabidopsis. This advances the utility of RLUC as a reporter protein, biosensor, or resonance energy donor

Clinical outcomes of FOLFIRINOX in locally advanced pancreatic cancer: A single center experience

Systemic chemotherapy or chemoradiotherapy is the initial primary option for patients with locally advanced pancreatic cancer (LAPC). This study analyzed the effect of FOLFIRINOX and assessed the factors influencing conversion to surgical resectability for LAPC.Sixty-four patients with LAPC who received FOLFIRINOX as initial chemotherapy were enrolled retrospectively. Demographic characteristics, tumor status, interval/dosage/cumulative relative dose intensity (cRDI) of FOLFIRINOX, conversion to resection, and clinical outcomes were reviewed and factors associated with conversion to resectability after FOLFIRINOX were analyzed.After administration of FOLFIRINOX (median 9 cycles, 70% of cRDI), the median patient overall survival (OS) was 17.0 months. Fifteen of 64 patients underwent surgery and R0 resection was achieved in 11 patients. During a median follow-up time of 9.4 months after resection, cumulative recurrence rate was 28.5% at 18 months after resection. The estimated median OS was significantly longer for the resected group (>40 months vs 13 months). There were no statistical differences between the resected and non-resected groups in terms of baseline characteristics, tumor status and hematologic adverse effects. The patients who received standard dose of FOLFIRINOX had higher probability of subsequent resection compared with patients who received reduced dose, although cRDIs did not differ between groups.FOLFIRINOX is an active regimen in patients with LAPC, given acceptable resection rates and promising R0 resection rates. Additionally, our data demonstrate it is advantageous for obtaining resectability to administer FOLFIRINOX without dose reduction

CRYPTO-MINE: Cryptanalysis via Mutual Information Neural Estimation

The use of Mutual Information (MI) as a measure to evaluate the efficiency of cryptosystems has an extensive history. However, estimating MI between unknown random variables in a high-dimensional space is challenging. Recent advances in machine learning have enabled progress in estimating MI using neural networks. This work presents a novel application of MI estimation in the field of cryptography. We propose applying this methodology directly to estimate the MI between plaintext and ciphertext in a chosen plaintext attack. The leaked information, if any, from the encryption could potentially be exploited by adversaries to compromise the computational security of the cryptosystem. We evaluate the efficiency of our approach by empirically analyzing multiple encryption schemes and baseline approaches. Furthermore, we extend the analysis to novel network coding-based cryptosystems that provide individual secrecy and study the relationship between information leakage and input distribution

Discovery of Novel Human Breast Cancer MicroRNAs from Deep Sequencing Data by Analysis of Pri-MicroRNA Secondary Structures

MicroRNAs (miRNAs) are key regulators of gene expression and contribute to a variety of biological processes. Abnormal miRNA expression has been reported in various diseases including pathophysiology of breast cancer, where they regulate protumorigenic processes including vascular invasiveness, estrogen receptor status, chemotherapy resistance, invasion and metastasis. The miRBase sequence database, a public repository for newly discovered miRNAs, has grown rapidly with approximately >10,000 entries to date. Despite this rapid growth, many miRNAs have not yet been validated, and several others are yet to be identified. A lack of a full complement of miRNAs has imposed limitations on recognizing their important roles in cancer, including breast cancer. Using deep sequencing technology, we have identified 189 candidate novel microRNAs in human breast cancer cell lines with diverse tumorigenic potential. We further show that analysis of 500-nucleotide pri-microRNA secondary structure constitutes a reliable method to predict bona fide miRNAs as judged by experimental validation. Candidate novel breast cancer miRNAs with stem lengths of greater than 30 bp resulted in the generation of precursor and mature sequences in vivo. On the other hand, candidates with stem length less than 30 bp were less efficient in producing mature miRNA. This approach may be used to predict which candidate novel miRNA would qualify as bona fide miRNAs from deep sequencing data with approximately 90% accuracy

Physical-Security for Wireless with Orbital Angular Momentum Wave

As technology progresses and an ever-increasing number of digital data are transmitted day to day, securing data has emerged as a major field of research. Conventional cryptography in higher layers of protocol stack has been studied as a data protection technique from an unauthorized party by converting secret data into a non-readable binary form. In this work, we leverage an OAM-wave-based transmission as an additional layer of physical security to be used with data encryption. A trustworthy key distribution mechanism for symmetric cryptography protocol is proposed by exploiting randomly hopping among the orthogonal OAM-wave modes and phases. Keccak block generates randomness for OAM modes, and AES is employed for encryption. This work provides physical-layer security, which is compatible with any higher layer encryption techniques. The hardware is implemented in 65nm CMOS technology, and post place-and-route simulation results are presented.S.M