High-throughput screening of chemicals with
reporter gene assays in Tox21 has produced a large database on cytotoxicity and specific modes of action. However, the validity of some of the reported ...activities is questionable due to the "cytotoxicity burst," which refers to the supposition that many stress responses are activated in a nonspecific way at concentrations close to cell death.
We propose a pragmatic method to identify whether reporter gene activation is specific or cytotoxicity-triggered by comparing the measured effects with baseline toxicity.
Baseline toxicity, also termed narcosis, is the minimal toxicity any chemical causes. Quantitative structure-activity relationships (QSARs) developed for baseline toxicity in mammalian reporter gene cell lines served as anchors to define the chemical-specific threshold for the cytotoxicity burst and to evaluate the degree of specificity of the reporter gene activation. Measured 10% effect concentrations were related to measured or QSAR-predicted 10% cytotoxicity concentrations yielding specificity ratios (SR). We applied this approach to our own experimental data and to
chemicals that were tested in six of the high-throughput Tox21 reporter gene assays.
Confirmed baseline toxicants activated reporter gene activity around cytotoxic concentrations triggered by the cytotoxicity burst. In six Tox21 assays, 37%-87% of the active hits were presumably caused by the cytotoxicity burst (
) and only 2%-14% were specific with
against experimental cytotoxicity but 75%-97% were specific against baseline toxicity. This difference was caused by a large fraction of chemicals showing excess cytotoxicity.
The specificity analysis for measured
effects identified whether a cytotoxicity burst had likely occurred. The SR-analysis not only prevented false positives, but it may also serve as measure for relative effect potency and can be used for quantitative
extrapolation and risk assessment of chemicals. https://doi.org/10.1289/EHP6664.
Celotno besedilo
Dostopno za:
CEKLJ, DOBA, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
Positron emission tomography (PET) reporter systems are a valuable means of estimating the level of expression of a transgene in vivo. For example, the safety and efficacy of gene therapy approaches ...for the treatment of neurological and neuropsychiatric disorders could be enhanced via the monitoring of exogenous gene expression levels in the brain. The present study evaluated the ability of a newly developed PET reporter system 18Ffluoroestradiol (18FFES) and the estrogen receptor-based PET reporter ChRERα, to monitor expression levels of a small hairpin RNA (shRNA) designed to suppress choline acetyltransferase (ChAT) expression in rhesus monkey brain. The ChRERα gene and shRNA were expressed from the same transcript via lentivirus injected into monkey striatum. In two monkeys that received injections of viral vector, 18FFES binding increased by 70% and 86% at the target sites compared with pre-injection, demonstrating that ChRERα expression could be visualized in vivo with PET imaging. Post-mortem immunohistochemistry confirmed that ChAT expression was significantly suppressed in regions in which 18FFES uptake was increased. The consistency between PET imaging and immunohistochemical results suggests that 18FFES and ChRERα can serve as a PET reporter system in rhesus monkey brain for in vivo evaluation of the expression of potential therapeutic agents, such as shRNAs.
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Eldridge, Michaelides, and colleagues successfully applied 18FFES-ChRERα in rhesus monkeys, which increases the likelihood that this new PET reporter system will translate effectively to the human brain. PET reporter systems could be valuable for gene therapy-based treatments of neuropsychiatric and neurological disorders.
MRI-based gene reporters allow imaging of gene expression at depth (tens of centimetres) and at relatively high resolution (~10–100 μm) and have the potential to be translated to the clinic. The ...reporters exploit either endogenous contrast mechanisms or they modulate the response to an introduced exogenous contrast agent.
Kinase translocation reporters (KTRs) are genetically encoded fluorescent activity sensors that convert kinase activity into a nucleocytoplasmic shuttling equilibrium for visualizing single-cell ...signaling dynamics. Here, we adapt the first-generation KTR for extracellular signal-regulated kinase (ERK) to allow easy implementation in vivo. This sensor, “ERK-nKTR,” allows quantitative and qualitative assessment of ERK activity by analysis of individual nuclei and faithfully reports ERK activity during development and neural function in diverse cell contexts in Caenorhabditis elegans. Analysis of ERK activity over time in the vulval precursor cells, a well-characterized paradigm of epidermal growth factor receptor (EGFR)-Ras-ERK signaling, has identified dynamic features not evident from analysis of developmental endpoints alone, including pulsatile frequency-modulated signaling associated with proximity to the EGF source. The toolkit described here will facilitate studies of ERK signaling in other C. elegans contexts, and the design features will enable implementation of this technology in other multicellular organisms.
•ERK-nKTR is a genetically encoded biosensor for extracellular signal-regulated kinase•Qualitative or quantitative assessment is possible by analyzing individual nuclei•ERK-nKTR is a faithful reporter for ERK activity in several different cell types•In the vulval precursor cells, there is pulsatile, frequency-modulated signaling
A genetically encoded biosensor for ERK activity allows qualitative or quantitative assessment by analysis of individual nuclei during development and neural function in diverse cell contexts in C. elegans. In-depth analysis of ERK activity over time in the vulval precursor cells has identified dynamic features including pulsatile frequency-modulated signaling.
Aflatoxin contamination causes tremendous economic losses and seriously threatens human health due to its prevalence in agricultural products and foods coupled with the severe toxicity of aflatoxins. ...Thus, sensitive, reliable, and rapid methods for aflatoxin analysis are urgently required. In recent years, electrochemical biosensors have emerged as a suitable alternative to gold standard methods that rely on sophisticated instruments (e.g. HPLC-MS). This review aims to describe recent advances (2016–2019) in electrochemical biosensors for aflatoxins detection in novel sensing strategies, recognition reporters, and integrated platforms. Particular emphasis is placed on the utilization of novel sensing strategies including dual-mode detection and ratiometric sensing in the fabrication of biosensors based on electrochemistry, photoelectrochemistry, and electrochemiluminescence. Meanwhile, a comprehensive discussion is provided on the challenges and perspectives related to electrochemical biosensing of aflatoxins.
•This review aims to provide the recent advances of electrochemical biosensors for aflatoxins.•Particular emphasis is given to the ratiometric electrochemical biosensing and dual-mode detection.•Challenges and perspectives related to electrochemical biosensing of aflatoxin are discussed.
Transcriptional reporters are reliable and time-tested tools to study gene regulation. In Staphylococcus aureus, β-galactosidase (
)-based genetic screens are not widely used because of the necessity ...of selectable markers for strain construction and the production of staphyloxanthin pigment, which obfuscates results. We describe a series of vectors that allow for markerless insertion of codon-optimized
-based transcriptional reporters. The vectors code for different ribosomal binding sites, allowing for tailored
expression. A Δ
::
deletion insertion mutant was constructed that prevents the synthesis of staphyloxanthin, thereby permitting blue-white screening without the interference of carotenoid production. We demonstrate the utility of these vectors to monitor aerobic and anaerobic transcriptional activities. For the latter, we describe the use of a ferrocyanide-ferricyanide redox system Fe(CN)
permitting blue-white screening in the absence of oxygen. We also describe additional reporter systems and methods for monitoring transcriptional activity during anaerobic culture, including an FAD-binding fluorescent protein (
), alpha-hemolysin (
), or lipase (
). The systems and methods described are compatible with vectors utilized to create and screen high-density transposon mutant libraries.
Staphylococcus aureus is a human pathogen and a leading cause of infectious disease-related illness and death worldwide. For S. aureus to successfully colonize and invade host tissues, it must tightly control the expression of genes encoding virulence factors. Oxygen tension varies greatly at infection sites, and many abscesses are devoid of oxygen. In this study, we have developed novel tools and methods to study how and when S. aureus alters transcription of genes. A key advantage of these methods and tools is that they can be utilized in the presence and absence of oxygen. A better understanding of anaerobic gene expression in S. aureus will provide important insights into the regulation of genes in low-oxygen environments.
The diversity of cardiac lineages contributes to the heterogeneity of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs). Here, we report the generation of a hiPSC TBX5Clover2 ...and NKX2-5TagRFP double reporter to delineate cardiac lineages and isolate lineage-specific subpopulations. Molecular analyses reveal that four different subpopulations can be isolated based on the differential expression of TBX5 and NKX2-5, TBX5+NKX2-5+, TBX5+NKX2-5−, TBX5−NKX2-5+, and TBX5−NKX2-5−, mimicking the first heart field, epicardial, second heart field, and endothelial lineages, respectively. Genetic and functional characterization indicates that each subpopulation differentiates into specific cardiac cells. We further identify CORIN as a cell-surface marker for isolating the TBX5+NKX2-5+ subpopulation and demonstrate the use of lineage-specific CMs for precise drug testing. We anticipate that this tool will facilitate the investigation of cardiac lineage specification and isolation of specific cardiac subpopulations for drug screening, tissue engineering, and disease modeling.
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•TBX5Clover2 and NKX2-5TagRFP reporter enables purification of 4 cardiac subpopulations•Different cardiac lineages differentiate into specific cardiac cell types•CORIN is a cell-surface marker for the TBX5+NKX2-5+ subpopulation•Lineage-specific cardiomyocyte subtypes can be used for precise drug testing
Wu and colleagues create a double-knockin human TBX5Clover2 and NKX2-5TagRFP reporter to delineate cardiac lineages during differentiation. Molecular and functional analyses uncover that differential expression of TBX5 and NKX2-5 specifies different cardiac fate. This reporter is applicable for studying cardiac lineage determination in vitro and isolating cardiac subpopulations.
Hypoxia has been reported to promote tumor progression and metastasis in murine models, and patients with hypoxic tumors have a worse prognosis. Besides its effect on cancer, normal processes like ...embryogenesis, or other pathologies such as ischemia, depend on hypoxia-regulated mechanisms. Given the degradable nature of HIF-1/2α in the presence of oxygen, defining the role of hypoxia in modeling biological processes becomes challenging when a cell enters oxygen-rich regions within a tissue. Here, we describe a unique approach to permanently mark cells that experience hypoxia with a fluorescent protein switch that is maintained even after a cell is reoxygenated. This method consists of a dual-viral delivery system that can be transduced into any mammalian cell line.
Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. ...However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.
Senecavirus A (SVA) is an emerging picornavirus that has been associated with vesicular disease and neonatal mortality in swine. The construction of SVA virus carrying foreign reporter gene provides ...a powerful tool in virus research. However, it is often fraught with rescuing a recombinant picornavirus harboring a foreign gene or maintaining the stability of foreign gene in the virus genome. Here, we successfully generated recombinant SVA GD05/2017 viruses (V-GD05-clone) expressing the green fluorescent protein (iLOV), red fluorescent protein (RFP), or NanoLuc luciferase (Nluc). These recombinant viruses have comparable growth kinetics to the parental virus. Genetic stability analysis indicated that V-GD05-iLOV was highly stable in retaining iLOV gene for more than 10 passages, while V-GD05-RFP and V-GD05-Nluc lost the foreign genes in five passages. In addition, high-intensity fluorescent signals were found in the V-GD05-RFP- and V-GD05-iLOV-infected cells by fluorescence observation and flow cytometry analysis, and the luciferase activity assay could quantitatively monitor the replication of V-GD05-Nluc. In order to identify the porcine cell receptor for SVA, anthrax toxin receptor 1 (ANTXR1) was knocked out or overexpressed in the ST-R cells. The ANTXR1 knock-out cells lost the ability for SVA infection, while overexpression of ANTXR1 significantly increased the cell permissivity. These results confirmed that ANTXR1 was the receptor for SVA to invade porcine cells as reported in the human cells. Overall, this study suggests that these SVA reporter viruses will be useful tools in elucidating virus pathogenesis and developing control measures.
Key points
• We successfully generated SVA viruses expressing the iLOV, RFP, or Nluc.
• The iLOV was genetically stable in the V-GD05-iLOV genome over ten passages.
• ANTXR1 was the receptor for SVA to invade porcine cells.
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