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As part of an ongoing program to identify genes involved in maintaining circadian rhythms of zebrafish, 6,500 mutagenized genomes were screened for dominant mutants affecting circadian locomotor activity. Molecular analysis of one of these mutant lines, Clk1dg3, revealed an I254N mutation in the PAS domain of the Clock1 protein. This isoleucine is tightly conserved in the Clock genes of several different species, and the I254N was not seen in any of the wild-type zebrafish population tested. Analysis of circadian activity rhythms as well as melatonin rhythms in homozygotes revealed the biological clock runs with a shortened period. The effect of this Clock1 mutation was characterized in vitro using a cell culture system where it appears to enhance the transactivation ability of the I254N Clock1 protein compared with that of the normal gene product.
// Xiang Zhang 1, 2 , Jiangang Hu 1, 2 , Guanjian Zhao 1, 2 , Ning Huang 1 , Ying Tan 1 , Li Pi 1 , Qing Huang 1 , Feng Wang 1 , Zhigang Wang 2 , Zhibiao Wang 3 , Yuan Cheng 1 1 Department of Neurosurgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China 2 Chongqing Key Laboratory of Ultrasound Molecular Imaging, Institute of Ultrasound Imaging of Chongqing Medical University, Chongqing, 400016, China 3 Chongqing Key Laboratory of Biomedical Engineering, College of Biomedical Engineering of Chongqing Medical University, Chongqing, 400016, China Correspondence to: Yuan Cheng, email: chengyuan023@aliyun.com Keywords: blood brain barrier, focused ultrasound, acoustic droplet vaporization, perfluoropentane, PLGA Received: January 17, 2017 Accepted: April 05, 2017 Published: April 17, 2017 ABSTRACT Previous studies have shown that focused ultrasound (FUS) combined with systematic administration of microbubbles (MBs) can open the blood brain barrier (BBB) locally, transiently and reversibly. However, because of the micro size diameters, MBs are restricted in the intravascular space and cannot extravasate into diseased sites through the opened BBB. In this study, we fabricated one kind of nanoscale droplets which consisted of encapsulated liquid perfluoropentane cores and poly (ethyleneglycol) - poly (lactide-co-glycolic acid) shells. The nanodroplets had the capacity to realize liquid to gas phase shift under FUS. Significant extravasation of Evan's blue appeared when acoustic pressure reached 1.0 MPa. Intracerebral hemorrhages and erythrocyte extravasations were observed when the pressure was increased to 1.5 MPa. Prolonged sonication duration could enhance the level of BBB opening and broaden the time window simultaneously. Furthermore, compared with MBs, the distribution of EB extravasation was firmly confined within narrow region in the center of focal zone, suggesting the site of FUS induced BBB opening could be controlled with high precision by this procedure. Our results show the feasibility of serving PEGylated PLGA-based phase shift nanodroplet as an effective alternative mediating agent for FUS induced BBB opening.
Planar-chiral cycloalkenes are challenging synthetic targets due to the medium-sized ring and the trans-configured olefin. Asymmetric resolution of racemic E-cycloalkenes, UV-induced asymmetric isomerization of Z-cycloalkenes, and asymmetric intramolecular cyclization of olefinic substrates are known as the limited approaches to these scaffolds. However, these methods usually suffer from tedious synthesis, harsh conditions, and insufficient structural diversity. Here, we present a novel palladium-catalyzed asymmetric [7+2] cycloaddition to facilitate the modular synthesis of planar-chiral 9-membered cycloalkenes from readily available chemicals. Excellent selectivity for planar chirality was achieved by retaining the trans-2H configuration of the π-allyl-Pd species throughout the whole process. Mechanistic studies were performed to elucidate the pivotal role of hydrogen bonding from the chiral ligand in enhancing both reaction efficiency and stereocontrol. In addition, synthetic transformations of planar chiral cycloalkene products and their applications in the identification of anticancer agents and selective bioimaging of cancer cells demonstrated the synthetic value of this new methodology. This research provides a promising way to access synthetically valued planar-chiral cycloalkenes.
The detection of proteins is of great biological significance as disease biomarkers in early diagnosis, prognosis tracking and therapeutic evaluation. Thus, we developed a simple, sensitive and universal protein-sensing platform based on peptide and graphene oxide (GO). The design consists of a fluorophore (TAMRA, TAM), a peptide containing eight arginines and peptide ligand that could recognize the target protein, and GO used as a quencher. To demonstrate the feasible use of the sensor for target detection, Bcl-xL was evaluated as the model target. The sensor was proved to be sensitive and applied for the detection of the target proteins in buffer, 2% serum and living cells.
Antirrhinum (snapdragon) species are models for genetic and evolutionary research but recalcitrant to genetic transformation, limiting use of transgenic methods for functional genomics. Transient gene expression from viral vectors and virus-induced gene silencing (VIGS) offer transformation-free alternatives. Here we investigate the utility of Tobacco rattle virus (TRV) for homologous gene expression in Antirrhinum and VIGS in Antirrhinum and its relative Misopates.A. majus proved highly susceptible to systemic TRV infection. TRV carrying part of the Phytoene Desaturase (PDS) gene triggered efficient PDS silencing, visible as tissue bleaching, providing a reporter for the extent and location of VIGS. VIGS was initiated most frequently in young seedlings, persisted into inflorescences and flowers and was not significantly affected by the orientation of the homologous sequence within the TRV genome. Its utility was further demonstrated by reducing expression of two developmental regulators that act either in the protoderm of young leaf primordia or in developing flowers. The effects of co-silencing PDS and the trichome-suppressing Hairy (H) gene from the same TRV genome showed that tissue bleaching provides a useful marker for VIGS of a second target gene acting in a different cell layer. The ability of TRV-encoded H protein to complement the h mutant phenotype was also tested. TRV carrying the native H coding sequence with PDS to report infection failed to complement h mutations and triggered VIGS of H in wild-type plants. However, a sequence with 43% synonymous substitutions encoding H protein, was able to complement the h mutant phenotype when expressed without a PDS VIGS reporter.We demonstrate an effective method for VIGS in the model genus Antirrhinum and its relative Misopates that works in vegetative and reproductive tissues. We also show that TRV can be used for complementation of a loss-of-function mutation in Antirrhinum. These methods make rapid tests of gene function possible in these species, which are difficult to transform genetically, and opens up the possibility of using additional cell biological and biochemical techniques that depend on transgene expression.
Abstract The clinical advantage of co‐targeting cancer drug escape has been indicated by the percentage of these co‐targeting drugs among all multi‐target drugs in clinics and clinical trials. This clinical advantage needs to be further interrogated from such perspectives as the clinical impact of multi‐target inhibition of drug‐escape mediators. This impact may be reflected by drug sales data, that is, multi‐target inhibition of higher number of drug‐escape mediators favors the expanded coverage of drug‐resistant patients leading to higher sales. We investigated whether this expectation is followed by the 25 FDA‐approved anticancer kinase inhibitors, which were divided into 11 groups of comparable therapeutic mechanisms and approval years. We found 19 (76%) drugs to follow and 3 (12%) drugs not to follow this expectation. The remaining two (8%) and one (4%) drugs cannot be assessed due to insufficient data and incomparability. Therefore, drug sales strongly indicate the clinical advantage of multi‐target inhibition of cancer drug escapes.
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