keyword
https://read.qxmd.com/read/38526790/crispr-cas9-mediated-bioluminescent-tagging-of-endogenous-proteins-by-fluorescent-protein-assisted-cell-sorting
#1
JOURNAL ARTICLE
Robert G Hawley, Teresa S Hawley
Oncogenic fusion genes are attractive therapeutic targets because of their tumor-specific expression and central "driver" roles in various human cancers. However, oncogenic fusions involving transcription factors such as PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) have been difficult to inhibit due to the apparent lack of tractable drug-like binding sites comparable to that recognized by Gleevec (imatinib mesylate) on the BCR-ABL1 tyrosine kinase fusion protein. Toward the identification of novel small molecules that selectively target PAX3-FOXO1, we used CRISPR-Cas9-mediated knock-in to append the pro-luminescent HiBiT tag onto the carboxy terminus of the endogenous PAX3-FOXO1 fusion protein in two human FP-RMS cell lines (RH4 and SCMC)...
2024: Methods in Molecular Biology
https://read.qxmd.com/read/38525648/an-efficient-peptide-ligase-engineered-from-a-bamboo-asparaginyl-endopeptidase
#2
JOURNAL ARTICLE
Xin-Bo Wang, Cong-Hui Zhang, Teng Zhang, Hao-Zheng Li, Ya-Li Liu, Zeng-Guang Xu, Gang Lei, Chun-Ju Cai, Zhan-Yun Guo
In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves...
March 25, 2024: FEBS Journal
https://read.qxmd.com/read/38521872/molecular-mechanism-of-muscarinic-acetylcholine-receptor-m3-interaction-with-gq
#3
JOURNAL ARTICLE
Donghee Ham, Asuka Inoue, Jun Xu, Yang Du, Ka Young Chung
Muscarinic acetylcholine receptor M3 (M3) and its downstream effector Gq/11 are critical drug development targets due to their involvement in physiopathological processes. Although the structure of the M3-miniGq complex was recently published, the lack of information on the intracellular loop 3 (ICL3) of M3 and extensive modification of Gαq impedes the elucidation of the molecular mechanism of M3-Gq coupling under more physiological condition. Here, we describe the molecular mechanism underlying the dynamic interactions between full-length wild-type M3 and Gq using hydrogen-deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cell systems...
March 23, 2024: Communications Biology
https://read.qxmd.com/read/38502493/crispr-based-split-luciferase-as-a-biosensor-for-unique-dna-sequences-in-situ
#4
JOURNAL ARTICLE
Nicholas G Heath, David J Segal
To date, CRISPR-based DNA targeting approaches have typically used fusion proteins between full fluorescent reporters and catalytically inactive Cas9 (dCas9) for imaging rather than detection of endogenous genomic DNA sequences. A promising alternative strategy for DNA targeting is the direct biosensing of user-defined sequences at single copy with single-cell resolution. Our recently described DNA biosensing approach using a dual fusion protein biosensor comprised of two independently optimized fragments of NanoLuc luciferase (NLuc) directionally fused to dCas9 paired with user-defined single-guide RNAs (sgRNAs) could allow users to sensitively detect unique copies of a target sequence in individual living cells using common laboratory equipment such as a microscope or a luminescence-equipped microplate reader...
2024: Methods in Molecular Biology
https://read.qxmd.com/read/38500100/nanoluc-binary-technology-as-a-methodological-approach-an-important-new-tool-for-studying-the-localization-of-androgen-receptor-and-androgen-receptor-splice-variant-v7-homo-and-heterodimers
#5
JOURNAL ARTICLE
Juan Guzman, Katrin Weigelt, Angela Neumann, Philipp Tripal, Benjamin Schmid, Zoltán Winter, Ralph Palmisano, Zoran Culig, Marcus V Cronauer, Paul Muschler, Bernd Wullich, Helge Taubert, Sven Wach
BACKGROUND: The androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT). The clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens...
March 19, 2024: BMC Cancer
https://read.qxmd.com/read/38499155/integration-of-hibit-into-enteroviruses-a-universal-tool-for-advancing-enterovirus-virology-research
#6
JOURNAL ARTICLE
Rui Yu, Xiaohong Li, Peng Zhang, Minghao Xu, Jitong Zhao, Jingjing Yan, Chenli Qiu, Jiayi Shu, Shuo Zhang, Miaomiao Kang, Xiaoyan Zhang, Jianqing Xu, Shuye Zhang
The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications, enabling the development of effective therapeutic and preventive strategies. In this study, our initial attempts to introduce a NanoLuc luciferase (NLuc) reporter gene into recombinant enteroviruses were unsuccessful in rescuing viable progenies. We hypothesized that the size of the inserted tag might be a determining factor in the rescue of the virus...
March 16, 2024: Virologica Sinica
https://read.qxmd.com/read/38484568/is-myeloid-derived-growth-factor-a-ligand-of-the-sphingosine-1-phosphate-receptor-2
#7
JOURNAL ARTICLE
Yong-Shan Zheng, Ya-Li Liu, Zeng-Guang Xu, Cheng He, Zhan-Yun Guo
Secretory myeloid-derived growth factor (MYDGF) exerts beneficial effects on organ repair, probably via a plasma membrane receptor; however, the identity of the expected receptor has remained elusive. In a recent study, MYDGF was reported as an agonist of the sphingosine-1-phosphate receptor 2 (S1PR2), an A-class G protein-coupled receptor that mediates the functions of the signaling lipid, sphingosine-1-phosphate (S1P). In the present study, we conducted living cell-based functional assays to test whether S1PR2 is a receptor for MYDGF...
March 10, 2024: Biochemical and Biophysical Research Communications
https://read.qxmd.com/read/38478279/mitoluc-a-luminescence-based-assay-to-study-real-time-protein-import-into-mitochondria
#8
JOURNAL ARTICLE
Holly C Ford, Ian Collinson
All but a few mitochondrial proteins are translated into the cytosol and imported in via complicated and varied pathways. These processes occur over short time frames and, as such, are difficult to monitor with classical approaches such as Western blotting or autoradiography that require sample collection at discrete time points. The development of an assay based on a split version of the small luciferase-Mitoluc-has allowed us to monitor the import of proteins into mitochondria in high resolution and real time (Pereira et al...
2024: Methods in Molecular Biology
https://read.qxmd.com/read/38419322/understanding-the-basis-of-thermostability-for-enzyme-nanoluc-towards-designing-industry-competent-engineered-variants
#9
JOURNAL ARTICLE
Adwaita S R Nair, Arup Samanta, Saugata Hazra
As a leading contender in the study of luminescence, nanoluciferase has recently attracted attention and proven effective in a wide variety of research areas. Although numerous attempts have been made to improve activity, there has yet to be a thorough exploration of further possibilities to improve thermostability. In this study, protein engineering in tandem with molecular dynamics simulation at various temperatures (300 K, 400 K, 450 K and 500 K) was used to improve our understanding of nanoluciferase dynamics and identification of factors that could significantly enhance the thermostability...
February 28, 2024: Journal of Biomolecular Structure & Dynamics
https://read.qxmd.com/read/38414102/rapid-quantitative-detection-system-for-measles-virus-neutralizing-antibodies-using-hibit-tagged-virus-like-particles
#10
JOURNAL ARTICLE
Takashi Okura, Kei Miyakawa, Maino Tahara, Kenji Someya, Fumio Seki, Mayuko Nishi, Noriyuki Otsuki, Akihide Ryo
Immunological testing to detect neutralizing antibodies (NAbs) is important in measles (MV) infection control. Currently, the plaque reduction neutralization test is the only credible method for measuring actual virus NAbs; however, its feasibility is hampered by drawbacks, such as long turnaround times, low throughput, and the need for laboratory biosafety equipment. To solve these problems, we developed a simple and rapid MV-NAb detection system using lentivirus-based virus-like particles incorporated with the NanoLuc fragment peptide HiBiT comprising the MV fusion protein and hemagglutinin on their exterior surface...
February 27, 2024: Microbiology and Immunology
https://read.qxmd.com/read/38390557/gesicles-packaging-dcas9-vpr-ribonucleoprotein-complexes-can-combine-with-vorinostat-and-promote-hiv-proviral-transcription
#11
JOURNAL ARTICLE
Michaela A Fisher, Waj Chaudhry, Lee A Campbell
Despite the success of combination antiretroviral therapy (cART) in HIV treatment, a cure for HIV remains elusive. Scientists postulate that HIV latent reservoirs may be a vital target in curative strategies. Vorinostat is a latency-reversing agent that has demonstrated some effectiveness in reactivating latent HIV, but complementary therapies may be essential to enhance its efficacy. One such approach may utilize the CRISPR-Cas9 system, which has evolved to include transcriptional activators such as dCas9-VPR...
March 14, 2024: Molecular Therapy. Methods & Clinical Development
https://read.qxmd.com/read/38363137/nanobody-engineering-for-sars-cov-2-neutralization-and-detection
#12
JOURNAL ARTICLE
Liina Hannula, Suvi Kuivanen, Jonathan Lasham, Ravi Kant, Lauri Kareinen, Mariia Bogacheva, Tomas Strandin, Tarja Sironen, Jussi Hepojoki, Vivek Sharma, Petri Saviranta, Anja Kipar, Olli Vapalahti, Juha T Huiskonen, Ilona Rissanen
In response to the ongoing COVID-19 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, i.e., "nanobodies." Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants at high potency, with IC50 values as low as 50 pM...
February 16, 2024: Microbiology Spectrum
https://read.qxmd.com/read/38355118/establishment-of-a-superinfection-exclusion-method-for-pestivirus-titration-using-a-recombinant-reporter-pestiviruses
#13
JOURNAL ARTICLE
Yume Mimura, Takahiro Hiono, Loc Tan Huynh, Saho Ogino, Maya Kobayashi, Norikazu Isoda, Yoshihiro Sakoda
Pestiviruses are classified into two biotypes based on their cytopathogenicity. As the majority of pestivirus field isolates are noncytopathogenic, their titration requires alternative methods rather than direct observation of cytopathogenic effects, such as immunostaining using specific antibodies or interference with cytopathogenic strains. However, these methods require microscopic observation to assess virus growth, which is time- and labor-intensive, especially when handling several samples. In this study, we developed a novel luciferase-based pestivirus titration method using the superinfection exclusion phenomenon with recombinant reporter pestiviruses that possessed an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT)...
February 14, 2024: Journal of Veterinary Medical Science
https://read.qxmd.com/read/38353566/engineering-recombinant-replication-competent-bluetongue-viruses-expressing-reporter-genes-for-in-vitro-and-non-invasive-in-vivo-studies
#14
JOURNAL ARTICLE
Sergio Utrilla-Trigo, Luis Jiménez-Cabello, Alejandro Marín-López, Miguel Illescas-Amo, Germán Andrés, Eva Calvo-Pinilla, Gema Lorenzo, Piet A van Rijn, Javier Ortego, Aitor Nogales
Bluetongue virus (BTV) is the causative agent of the important livestock disease bluetongue (BT), which is transmitted via Culicoides bites. BT causes severe economic losses associated with its considerable impact on health and trade of animals. By reverse genetics, we have designed and rescued reporter-expressing recombinant (r)BTV expressing NanoLuc luciferase (NLuc) or Venus fluorescent protein. To generate these viruses, we custom synthesized a modified viral segment 5 encoding NS1 protein with the reporter genes located downstream and linked by the Porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site...
February 14, 2024: Microbiology Spectrum
https://read.qxmd.com/read/38338806/development-of-novel-bioluminescent-biosensors-monitoring-the-conformation-and-activity-of-the-merlin-tumour-suppressor
#15
JOURNAL ARTICLE
Alexander Pipchuk, Tynan Kelly, Madeleine Carew, Christopher Nicol, Xiaolong Yang
Solid tumours can universally evade contact inhibition of proliferation (CIP), a mechanism halting cell proliferation when cell-cell contact occurs. Merlin, an ERM-like protein, crucially regulates CIP and is frequently deactivated in various cancers, indicating its significance as a tumour suppressor in cancer biology. Despite extensive investigations into Merlin's role in cancer, its lack of intrinsic catalytic activity and frequent conformation changes have made it notoriously challenging to study. To address this challenge, we harnessed innovative luciferase technologies to create and validate a NanoBiT split-luciferase biosensor system in which Merlin is cloned between two split components (LgBiT and SmBiT) of NanoLuc luciferase...
January 26, 2024: International Journal of Molecular Sciences
https://read.qxmd.com/read/38315567/bioluminescent-intercalating-dyes-for-ratiometric-nucleic-acid-detection
#16
JOURNAL ARTICLE
Yosta de Stigter, Harmen J van der Veer, Bas J H M Rosier, Maarten Merkx
Rapid and sensitive DNA detection methods that can be conducted at the point of need may aid in disease diagnosis and monitoring. However, translation of current assays has proven challenging, as they typically require specialized equipment or probe-specific modifications for every new target DNA. Here, we present Lu minescent M ultivalent I ntercalating D ye (LUMID), off-the-shelf bioluminescent sensors consisting of intercalating dyes conjugated to a NanoLuc luciferase, which allow for nonspecific detection of double-stranded DNA through a blue-to-green color change...
February 5, 2024: ACS Chemical Biology
https://read.qxmd.com/read/38284490/tandem-repeats-of-highly-bioluminescent-nanoluc-are-refolded-noncanonically-by-the-hsp70-machinery
#17
JOURNAL ARTICLE
Dimitra Apostolidou, Pan Zhang, Devanshi Pandya, Kaden Bock, Qinglian Liu, Weitao Yang, Piotr E Marszalek
Chaperones are a large family of proteins crucial for maintaining cellular protein homeostasis. One such chaperone is the 70 kDa heat shock protein (Hsp70), which plays a crucial role in protein (re)folding, stability, functionality, and translocation. While the key events in the Hsp70 chaperone cycle are well established, a relatively small number of distinct substrates were repetitively investigated. This is despite Hsp70 engaging with a plethora of cellular proteins of various structural properties and folding pathways...
February 2024: Protein Science
https://read.qxmd.com/read/38275943/development-of-a-replication-deficient-bacteriophage-reporter-lacking-an-essential-baseplate-wedge-subunit
#18
JOURNAL ARTICLE
Jose Gil, John Paulson, Henriett Zahn, Matthew Brown, Minh M Nguyen, Stephen Erickson
Engineered bacteriophages (phages) can be effective diagnostic reporters for detecting a variety of bacterial pathogens. Although a promising biotechnology, the large-scale use of these reporters may result in the unintentional release of genetically modified viruses. In order to limit the potential environmental impact, the ability of these phages to propagate outside the laboratory was targeted. The phage SEA1 has been previously engineered to facilitate food safety as an accurate and sensitive reporter for Salmonella contamination...
December 20, 2023: Viruses
https://read.qxmd.com/read/38272933/monitoring-phosphorylation-and-acetylation-of-crispr-mediated-hibit-tagged-endogenous-proteins
#19
JOURNAL ARTICLE
Juliano Alves, Marie Schwinn, Thomas Machleidt, Said A Goueli, James J Cali, Hicham Zegzouti
Intracellular pathways transduce signals through changes in post-translational modifications (PTMs) of effector proteins. Among the approaches used to monitor PTM changes are immunoassays and overexpression of recombinant reporter genes. Genome editing by CRISPR/Cas9 provides a new means to monitor PTM changes by inserting reporters onto target endogenous genes while preserving native biology. Ideally, the reporter should be small in order not to interfere with the processes mediated by the target while sensitive enough to detect tightly expressed proteins...
January 25, 2024: Scientific Reports
https://read.qxmd.com/read/38223674/comparative-study-on-estrogen-receptor-alpha-dimerization-and-transcriptional-activity-of-parabens
#20
JOURNAL ARTICLE
Jong-Yeon Kim, Yooheon Park, Seok-Hee Lee, Eun-Jung Park, Hae-Jeung Lee
Parabens are used as preservatives in various household products, including oral products, cosmetics, and hair/body washes. In recent years, the widespread use of parabens has raised concerns due to the potential health risks associated with their estrogenic effects. In the present study, we evaluated and compared the estrogenic activity of parabens using two cell-based in vitro tests: (1) bioluminescence resonance energy transfer (BRET)-based estrogen receptor alpha (ERα) dimerization using HEK293 cells that were stably transfected with ERα-fused NanoLuc luciferase (Nluc) and HaloTag (HT) expression vector, and (2) stably transfected transcriptional activation (STTA) assays using ERα-HeLa9903 cells...
January 2024: Toxicological Research
keyword
keyword
102517
1
2
Fetch more papers »
Fetching more papers... Fetching...
Remove bar
Read by QxMD icon Read
×

Save your favorite articles in one place with a free QxMD account.

×

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"

We want to hear from doctors like you!

Take a second to answer a survey question.