scholarly journals Optogenetic regulation of endogenous gene transcription in mammals

2019 ◽  
Vol 23 (2) ◽  
pp. 219-225
Author(s):  
E. S. Omelina ◽  
A. V. Pindyurin
Keyword(s):  
Cell Biology ◽  
Near Infrared ◽  
Rapid Development ◽  
Infrared Light ◽  
Transcription Activator ◽  
Endogenous Gene ◽  
Advantages And Disadvantages ◽  
Ideal System ◽  
Endogenous Genes ◽  
Inducible Systems

Despite the rapid development of approaches aimed to precisely control transcription of exogenous genes in time and space, design of systems providing similar tight regulation of endogenous gene expression is much more challenging. However, finding ways to control the activity of endogenous genes is absolutely necessary for further progress in safe and effective gene therapies and regenerative medicine. In addition, such systems are of particular interest for genetics, molecular and cell biology. An ideal system should ensure tunable and reversible spatio-temporal control over transcriptional activity of a gene of interest. Although there are drug-inducible systems for transcriptional regulation of endogenous genes, optogenetic approaches seem to be the most promising for the gene therapy applications, as they are noninvasive and do not exhibit toxicity in comparison with druginducible systems. Moreover, they are not dependent on chemical inducer diffusion rate or pharmacokinetics and exhibit fast activation-deactivation switching. Among optogenetic tools, long-wavelength light-controlled systems are more preferable for use in mammalian tissues in comparison with tools utilizing shorter wavelengths, since far-red/near-infrared light has the maximum penetration depth due to lower light scattering caused by lipids and reduced tissue autofluorescence at wavelengths above 700 nm. Here, we review such light-inducible systems, which are based on synthetic factors that can be targeted to any desired DNA sequence and provide activation or repression of a gene of interest. The factors include zinc finger proteins, transcription activator-like effectors (TALEs), and the CRISPR/Cas9 technology. We also discuss the advantages and disadvantages of these DNA targeting tools in the context of the light-inducible gene regulation systems.

scholarly journals A simple cloning-free method to efficiently induce gene expression using CRISPR/Cas9

2018 ◽  
Author(s):  
Lyujie Fang ◽  
Sandy S.C. Hung ◽  
Jennifer Yek ◽  
Tu Nguyen ◽  
Shahnaz Khan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Neural Development ◽  
Cell Biology ◽  
Rapid Development ◽  
Gain Of Function ◽  
Simple Method ◽  
Endogenous Gene ◽  
Efficient Induction ◽  
Endogenous Genes ◽  
Endogenous Locus

AbstractGain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here we report a simple, cloning-free method to induce gene expression at endogenous locus using CRISPR/Cas9 activators. Our strategy utilises synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65 and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this cloning-free approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1 and NEUROD1) in human cells, whereas the rat cells exhibit a more variable and less efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applies as a rapid workflow to initiate gain-of-function studies for a range of molecular and cell biology disciplines.

scholarly journals Measurements of 3-D morphology of individual red blood cells upon localized photothermal effects using gold nanorods

2019 ◽  
Author(s):  
Chanwoo Song ◽  
Gu-Haeng Lee ◽  
Juntaek Oh ◽  
Moosung Lee ◽  
Seungwoo Shin ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Cell Biology ◽  
Near Infrared ◽  
Imaging Techniques ◽  
Hemoglobin Concentration ◽  
Local Heat ◽  
Dry Mass ◽  
Infrared Light ◽  
Phase Imaging

We investigated the in-situ photothermal response of human red blood cells (RBCs) by combining photothermal heat generation and 3-D quantitative phase imaging techniques. Gold-nanorod-coated substrates were excited using near-infrared light to generate local heat to RBCs, and response was measured by imaging 3-D refractive index tomograms of cells under various near infrared (NIR) excitation conditions. On photothermal treatment, cell morphology changed from discoid to crescent shapes, cell volume and dry mass decreased, and hemoglobin concentration increased. We also investigated the irreversible deformation of RBCs when multiple intense excitation shocks are applied. These results provide a new understanding of thermodynamic aspects of cell biology and hematology.

In Vivo Near Infrared Techniques for Protein Drug Development

2008 ◽  
pp. 1365-1373
Author(s):  
Yueqing Gu ◽  
Zhiyu Qian ◽  
Huimin Qian ◽  
Chunsheng Fang ◽  
Yulin Song
Keyword(s):  
Health Care ◽  
Near Infrared ◽  
Drug Carrier ◽  
Rapid Development ◽  
Infrared Light ◽  
Nir Light ◽  
Potential Applications ◽  
Living Tissues ◽  
Almost All

Near infrared (NIR) light (700 ~ 900 nm) possesses the capability of penetrating living tissues several centimeters due to the low absorbance of tissue intrinsic chromophores such as oxy- and deoxy-hemoglobin (the main absorber of visible light), melanin, water, and lipid (the principal absorber of infrared light). Featured with the deeper tissue penetration as well as nonionizing and nonradioactive, NIR light attracts extensive attentions on the development of noninvasive techniques for in vivo real time monitoring/tracing of biological signals in living tissues. Hitherto, NIR techniques have permeated to almost all aspects of health care, such as diagnosing disease (Nahum, Skippen, Gagnon, Macnab, & Skarsgard, 2006), designing the targeted molecular or drug carrier (Hsu et al., 2006), monitoring the response to therapeutic treatment (Tachtsidis et al., 2007), evaluating the rehabilitation, and so on. With the rapid development of various NIR techniques and more cooperation with clinic studies, more potential applications in health care will be exploited in the near future.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

An old friend is better than two new ones? Approaches to market research in the context of digital transformation for the antitrust laws enforcement

2020 ◽  
pp. 37-55 ◽  
Author(s):  
A. E. Shastitko ◽  
O. A. Markova
Keyword(s):  
Business Models ◽  
Rapid Development ◽  
Digital Transformation ◽  
Market Definition ◽  
Type I ◽  
Type Ii ◽  
Digital Platforms ◽  
Advantages And Disadvantages ◽  
Pass Through ◽  
Type Ii Errors

Digital transformation has led to changes in business models of traditional players in the existing markets. What is more, new entrants and new markets appeared, in particular platforms and multisided markets. The emergence and rapid development of platforms are caused primarily by the existence of so called indirect network externalities. Regarding to this, a question arises of whether the existing instruments of competition law enforcement and market analysis are still relevant when analyzing markets with digital platforms? This paper aims at discussing advantages and disadvantages of using various tools to define markets with platforms. In particular, we define the features of the SSNIP test when being applyed to markets with platforms. Furthermore, we analyze adjustment in tests for platform market definition in terms of possible type I and type II errors. All in all, it turns out that to reduce the likelihood of type I and type II errors while applying market definition technique to markets with platforms one should consider the type of platform analyzed: transaction platforms without pass-through and non-transaction matching platforms should be tackled as players in a multisided market, whereas non-transaction platforms should be analyzed as players in several interrelated markets. However, if the platform is allowed to adjust prices, there emerges additional challenge that the regulator and companies may manipulate the results of SSNIP test by applying different models of competition.

Catalyst Halogenation Enables Rapid and Efficient Polymerizations with Visible to Near-Infrared Light

2020 ◽  
Author(s):  
Alex Stafford ◽  
Dowon Ahn ◽  
Emily Raulerson ◽  
Kun-You Chung ◽  
Kaihong Sun ◽  
...  
Keyword(s):  
Near Infrared ◽  
Transient Absorption ◽  
Reaction Times ◽  
Infrared Light ◽  
Structure Property ◽  
Light Emitting ◽  
Structure Property Relationships ◽  
Nir Light ◽  
Light Intensities ◽  
Emission Quenching

Driving rapid polymerizations with visible to near-infrared (NIR) light will enable nascent technologies in the emerging fields of bio- and composite-printing. However, current photopolymerization strategies are limited by long reaction times, high light intensities, and/or large catalyst loadings. Improving efficiency remains elusive without a comprehensive, mechanistic evaluation of photocatalysis to better understand how composition relates to polymerization metrics. With this objective in mind, a series of methine- and aza-bridged boron dipyrromethene (BODIPY) derivatives were synthesized and systematically characterized to elucidate key structure-property relationships that facilitate efficient photopolymerization driven by visible to NIR light. For both BODIPY scaffolds, halogenation was shown as a general method to increase polymerization rate, quantitatively characterized using a custom real-time infrared spectroscopy setup. Furthermore, a combination of steady-state emission quenching experiments, electronic structure calculations, and ultrafast transient absorption revealed that efficient intersystem crossing to the lowest excited triplet state upon halogenation was a key mechanistic step to achieving rapid photopolymerization reactions. Unprecedented polymerization rates were achieved with extremely low light intensities (< 1 mW/cm<sup>2</sup>) and catalyst loadings (< 50 μM), exemplified by reaction completion within 60 seconds of irradiation using green, red, and NIR light-emitting diodes.

An ordered copper nanowell-array film with near-perfect broadband absorption from ultraviolet to near-infrared light

2020 ◽  
Vol 59 (11) ◽  
pp. 110906
Author(s):  
Juan Shen ◽  
Yong Ren ◽  
Xinxin Zhu ◽  
Min Mao ◽  
Quan Zhou ◽  
...  
Keyword(s):  
Near Infrared ◽  
Infrared Light ◽  
Near Infrared Light ◽  
Broadband Absorption

Recent Patents on the Angular Contact Ball Bearing of High-Speed Motorized Spindle

2020 ◽  
Vol 13 ◽  
Author(s):  
Yudong Bao ◽  
Linkai Wu ◽  
Yanling Zhao ◽  
Chengyi Pan
Keyword(s):  
High Speed ◽  
Ball Bearing ◽  
Rapid Development ◽  
Ceramic Materials ◽  
Development Trend ◽  
Numerical Control ◽  
Angular Contact Ball Bearing ◽  
Practical Applications ◽  
High Speed Cutting ◽  
Advantages And Disadvantages

Background:: Angular contact ball bearings are the most popular bearing type used in the high speed spindle for machining centers, The performance of the bearing directly affects the machining efficiency of the machine tool, Obtaining a higher value is the direction of its research and development. Objective:: By analyzing the research achievements and patents of electric spindle angular contact bearings, summarizing the development trend provides a reference for the development of electric spindle bearings. Methods:: Through the analysis of the relevant technology of the electric spindle angular contact ball bearing, the advantages and disadvantages of the angular contact ball bearing are introduced, and the research results are combined with the patent analysis. Results:: With the rapid development of high-speed cutting and numerical control technology and the needs of practical applications, the spindle requires higher and higher speeds for bearings. In order to meet the requirements of use, it is necessary to improve the bearing performance by optimizing the structure size and improving the lubrication conditions. Meanwhile, reasonable processing and assembly methods will also have a beneficial effect on bearing performance. Conclusion:: With the continuous deepening of bearing technology research and the use of new structures and ceramic materials has made the bearing's limit speed repeatedly reach new highs. The future development trend of high-speed bearings for electric spindles is environmental protection, intelligence, high speed, high precision and long life.

Current RNA-based Therapeutics in Clinical Trials

2019 ◽  
Vol 19 (3) ◽  
pp. 172-196 ◽  
Author(s):  
Ling-Yan Zhou ◽  
Zhou Qin ◽  
Yang-Hui Zhu ◽  
Zhi-Yao He ◽  
Ting Xu
Keyword(s):  
Clinical Trials ◽  
Rapid Development ◽  
Genetic Diseases ◽  
Three Dimensional ◽  
Therapeutic Effects ◽  
Messenger Rnas ◽  
Small Interfering Rnas ◽  
Rna Modifications ◽  
Guide Rna ◽  
Endogenous Genes

Long-term research on various types of RNAs has led to further understanding of diverse mechanisms, which eventually resulted in the rapid development of RNA-based therapeutics as powerful tools in clinical disease treatment. Some of the developing RNA drugs obey the antisense mechanisms including antisense oligonucleotides, small interfering RNAs, microRNAs, small activating RNAs, and ribozymes. These types of RNAs could be utilized to inhibit/activate gene expression or change splicing to provide functional proteins. In the meantime, some others based on different mechanisms like modified messenger RNAs could replace the dysfunctional endogenous genes to manage some genetic diseases, and aptamers with special three-dimensional structures could bind to specific targets in a high-affinity manner. In addition, the recent most popular CRISPR-Cas technology, consisting of a crucial single guide RNA, could edit DNA directly to generate therapeutic effects. The desired results from recent clinical trials indicated the great potential of RNA-based drugs in the treatment of various diseases, but further studies on improving delivery materials and RNA modifications are required for the novel RNA-based drugs to translate to the clinic. This review focused on the advances and clinical studies of current RNA-based therapeutics, analyzed their challenges and prospects.

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