Fluorescence Line Research Articles

Biocompatible Nanovector of siRNA Consisting of Arginine-Based Cationic Lipid for Gene Knockdown in Cancer Cells.

Despite the use of small interfering RNAs (siRNAs) as therapeutic agents through the knockdown expression of pathogenic proteins, transportation and delivery of such siRNAs into cells continue to be under investigation. Within nonviral vectors, cationic lipids that include amino acid residues in their structures, and that have already demonstrated their suitability as plasmid DNA nanocarriers, may be also considered as potential siRNA vehicles. A double-chain cationic lipid based on the amino acid arginine mixed with a helper lipid has been the object of this biophysical study. First, ζ-potential measurements and agarose gel electrophoresis experiments confirmed the siRNA compaction, while small-angle X-ray scattering analysis (SAXS) revealed the structural pattern of the lipoplexes. Two bicontinuous cubic phases were found to coexist: the double-gyroid phase (QIIG) and the double-diamond phase (QIID), with Pn3m and Ia3d as crystallographic space groups, respectively; the siRNA is known to be located inside their bicontinuous aqueous channels. Second, in vitro studies in HeLa-green fluorescent protein (GFP) and T731-GFP cell lines (modified for GFP overexpression) showed moderate to high gene knockdown levels (determined by flow cytometry and epifluorescence microscopy) with remarkable cell viabilities (CCK-8 assay). Finally, nano-liquid chromatography/mass spectrometry (nanoLC-MS/MS) was used to identify the nature of the proteins adhered to the surface of the lipoplexes after incubation with human serum, simulating their behavior in biological fluids. The abundant presence of lipoproteins and serum albumin in such protein corona, together with the coexistence of the bicontinuous cubic phases, may be behind the remarkable silencing activity of these lipoplexes. The results reported herein show that the use of amino-acid-based cationic lipids mixed with a suitable helper lipid, which have already provided good results as DNA plasmid nanocarriers in cellular transfection processes, may also be a biocompatible option, and so far little investigated, in gene silencing in vitro strategies.

Pro-resolving mediator protectin D1 promotes epimorphic regeneration by controlling immune cell function in vertebrates.

Specialized pro-resolving mediators (SPMs) are a family of lipids controlling the resolution of inflammation and playing a role in many processes including organ protection and tissue repair. While SPMs are potent bioactive molecules in vivo, their role in epimorphic regeneration of organs in vertebrates has not been tested. Using the zebrafish larva as a robust regenerative vertebrate system, we studied the role of the SPM neuroprotectin/protectin D1 (PD1) during the caudal fin fold regeneration. Regeneration of the fin fold was analysed when exposed to a synthetic PD1. The effect of PD1 on immune cell recruitment and activation was further investigated using live imaging combined with fluorescent reporter lines. Using genetic and pharmacological approaches, we dissected the role of neutrophils and macrophages on driving the pro-regenerative effect of PD1. We showed that PD1 improves fin fold regeneration. Acting in a narrow time window during regeneration, PD1 accelerates the resolution of inflammation without affecting the initial kinetic of neutrophil recruitment but instead, promotes their reverse migration potential. In addition, PD1 induces macrophage polarization switch towards non-inflammatory states in both zebrafish and mammalian system. Finally, macrophages but not neutrophils are essential for PD1-mediated regeneration. These results reveal the pro-regenerative action of PD1 and its role in regulating neutrophil and macrophage response in vertebrates. These findings strongly support the development of pro-resolving mediators as natural therapeutic candidates for degenerative disorders and the use of the zebrafish as a tool to investigate pro-regenerative drugs.

Accurate Treatment of Comptonization in X-Ray Illuminated Accretion Disks

Abstract A large fraction of accreting black hole and neutron star systems present clear evidence of the reprocessing of X-rays in the atmosphere of an optically thick accretion disk. The main hallmarks of X-ray reflection include fluorescent K-shell emission lines from iron (∼6.4–6.9 keV), the absorption iron K-edge (∼7–9 keV), and a broad featureless component known as the Compton hump (∼20–40 keV). This Compton hump is produced as the result of the scattering of high-energy photons (E ≳ 10 keV) of the relatively colder electrons (T e ∼ 105–107 K) in the accretion disk, in combination with photoelectric absorption from iron. The treatment of this process in most current models of ionized X-ray reflection has been done using an approximated Gaussian redistribution kernel. This approach works sufficiently well up to ∼100 keV, but it becomes largely inaccurate at higher energies and at relativistic temperatures (T e ∼ 109 K). We present new calculations of X-ray reflection using a modified version of our code xillver, including an accurate solution for Compton scattering of the reflected unpolarized photons in the disk atmosphere. This solution takes into account quantum electrodynamic and relativistic effects allowing the correct treatment of high photon energies and electron temperatures. We show new reflection spectra computed with this model, and discuss the improvements achieved in reproducing the correct shape of the Compton hump, the discrepancies with previous calculations, and the expected impact of these new models in the interpretation of observational data.

Pyroelectric X-ray Generator for XRF in Combination with Optical Microscopy

X-ray fluorescence spectroscopy (XRF) is a method for elemental analyses of materials. In comparison to other analysing methods such as energy dispersive X-Ray spectroscopy (EDS) within a scanning electron microscope (SEM) or optical emission spectroscopy (OES) there are several advantages. This paper contains experimental investigations to improve the emission behaviour of a pyroelectric X-ray source regarding X-ray energy and output intensity. Pyroelectric lithiumtantalate (LiTaO3) was used to emit and accelerate electrons and to generate primary X-rays subsequently. Geometrical conditions were altered and the design of the X-ray source was optimized. All steps of optimization have been performed with respect to an application in a spectroscopic device in combination with an optical microscope (OM). Based on this purpose, the miniaturization of all applied components is of major importance. Different pure metals, alloys, and a coated specimen were analysed to demonstrate potential applications and current limitations of the designed system. Materials with a wide range of necessary excitation energy for chemical analyses of 1.5 to 10 keV for the Kα and Lα X-ray fluorescent lines were used to demonstrate the technical relevance of this type of application. As a result, a prototype of a spectrometer that combines XRF and OM was designed and tested.

X-Ray Quasi-periodic Oscillations in the Lense–Thirring Precession Model. II. Variability of the Relativistic Iron Kα Line

Abstract The reprocessing of primary X-ray emission in the accretion disk of black hole X-ray binaries (BHXRBs) produces a reflection spectrum with the characteristic Fe Kα fluorescence line. Strong low-frequency quasi-periodic oscillations (QPOs) are observed from BHXRBs, and the dependence of QPO properties (e.g., phase lag) on the inclination angle suggests that the observed QPO may be associated with a geometrical effect, e.g., the precession of the X-ray source due to frame dragging near the spinning black hole. Here, in the scenario of the Lense–Thirring precession of the X-ray source, we use a Monte Carlo simulation of radiative transfer to study the irradiation/reflection and the resultant spectral properties including the Fe Kα line as a function of precession phase (time). We found that the reflection fraction, i.e., the ratio of incident flux toward the disk and the direct flux toward the observer at infinity, is modulated by the precession phase, which depends on the truncation radius (i.e., the spectral state in the truncated disk model) and the inclination angle. The Fe Kα line profile also changes as the primary X-ray source precesses, with the line luminosity and the flux-weighted centroid energy varying with the precession phase. The periodically modulated 2–10 keV continuum flux could apparently lag the line luminosity in phase, if the truncation radius is small enough for Doppler effects due to disk orbital motion to significantly affect the observed radiation.

An enhanced toolkit for the generation of knockout and marker-free fluorescent Plasmodium chabaudi.

The rodent parasite Plasmodium chabaudi is an important in vivo model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti- Plasmodium immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection. P. chabaudi also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable in vivo to in vitro model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids for P. chabaudi and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linear PlasmoGEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcoded P. chabaudi gene-deletion and -tagging vectors for transfection in our fluorescent P. chabaudi mother-lines. This produces a tool-kit of P. chabaudi lines, vectors and transfection approaches that will be of broad utility to the research community.

In-flight calibration of the Insight-Hard X-ray Modulation Telescope

We present the calibration of the Insight-Hard X-ray Modulation Telescope (Insight-HXMT) X-ray satellite, which can be used to perform timing and spectral studies of bright X-ray sources. Insight-HXMT carries three main payloads onboard: the High Energy X-ray telescope (HE), the Medium Energy X-ray telescope (ME) and the low Energy X-ray telescope (LE). In orbit, the radioactive sources, activated lines, the fluorescence lines and celestial sources are used to calibrate the energy scale and energy resolution of the payloads. The Crab nebular is adopted as the primary effective area calibrator and empirical functions are constructed to modify the simulated effective areas of the three payloads respectively. The systematic errors of HE, compared to the model of the Crab nebular, are less than 2% in 28–120 keV and 2%–10% above 120 keV. The systematic errors of ME are less than 1.5% in 10–35 keV. The systematic errors of LE are less than 1% in 1–7 keV except the Si K–edge (1.839 keV, up to 1.5%) and less than 2% in 7–10 keV.

Bright Fluorescent Vacuolar Marker Lines Allow Vacuolar Tracing Across Multiple Tissues and Stress Conditions in Rice

The vacuole is indispensable for cells to maintain their water potential and to respond to environmental changes. Nevertheless, investigations of vacuole morphology and its functions have been limited to Arabidopsis thaliana with few studies in the model crop rice (Oryza sativa). Here, we report the establishment of bright rice vacuole fluorescent reporter systems using OsTIP1;1, a tonoplast water channel protein, fused to either an enhanced green fluorescent protein or an mCherry red fluorescent protein. We used the corresponding transgenic rice lines to trace the vacuole morphology in roots, leaves, anthers, and pollen grains. Notably, we observed dynamic changes in vacuole morphologies in pollen and root epidermis that corresponded to their developmental states as well as vacuole shape alterations in response to abiotic stresses. Our results indicate that the application of our vacuole markers may aid in understanding rice vacuole function and structure across different tissues and environmental conditions in rice.

Real time visualization of cancer cell death, survival and proliferation using fluorochrome-transfected cells in an IncuCyte® imaging system.

Cancer immunotherapy is a rapidly advancing and viable approach to treating cancer along with more traditional forms of therapy. Real-time cell analysis technologies that examine the dynamic interactions between cancer cells and the cells of the immune system are becoming more important for assessment of novel therapeutics. In this report, we use the IncuCyte® imaging system to study the killing potential of various immune cells on cancer cell lines. The IncuCyte® system tracks living cells, labeled by a red fluorescent protein, and cell death, as indicated by the caspase-3/7 reagent, which generates a green fluorescent signal upon activation of apoptotic pathways. Despite the power of this approach, obtaining commercially fluorescent cancer cell lines is expensive and limited in the range of cell lines that are available. To overcome this barrier, we developed an inexpensive method using a lentiviral construct expressing nuclear localized mKate2 red fluorescent protein to stably label cancer cells. We demonstrate that this method is effective in labeling a wide variety of cell lines, allowing for analyses of different cancers as well as different cell lines of the same type of cancer.

Levels of Heterochiasmy During Arabidopsis Development as Reported by Fluorescent Tagged Lines.

Crossing over, the exchange of DNA between the chromosomes during meiosis, contributes significantly to genetic variation. The rate of crossovers (CO) varies depending upon the taxon, population, age, external conditions, and also, sometimes, between the sexes, a phenomenon called heterochiasmy. In the model plant Arabidopsis thaliana, the male rate of all crossover events (mCO) is typically nearly double the female rate (fCO). A previous, PCR-based genotyping study has reported that the disparity decreases with increasing parental age, because fCO rises while mCO remains stable. We revisited this topic using a fluorescent tagged lines approach to examine how heterochiasmy responded to parental age in eight genomic intervals distributed across the organism’s five chromosomes. We determined recombination frequency for, on average, more than 2000 seeds, for each interval, for each of four age groups, to estimate sex-specific CO rates. mCO did not change with age, as reported previously, but, here, fCO did not rise, and thus the levels of heterochiasmy were unchanged. We can see no methodological reason to doubt that our results reflect the underlying biology of the accessions we studied. The lack of response to age could perhaps be due to previously reported variation in CO rate among accessions of Arabidopsis.

X-Ray Emission from Jupiter’s Galilean Moons: A Tool for Determining Their Surface Composition and Particle Environment

Abstract We analyze archival Chandra X-ray Observatory observations of Jupiter to search for emission from the Galilean moons. X-ray emission has previously been reported from Io and Europa using a subset of these data. We confirm this detection, and marginally detect X-ray emission from both Ganymede and Callisto as well. The X-ray spectrum of Europa is strongly peaked around the neutral oxygen fluorescence line (525 eV), while Io’s has peaks at both oxygen and sulfur (2308 eV) plus a broad continuum between 350 and 5000 eV. Ganymede’s spectrum is similar to Io’s, but without the sulfur peak. A few events, mostly clustered around the oxygen line, are detected from Callisto. Using measurements by the Galileo mission of the specific intensity of ambient protons and electrons, we model the X-ray spectra and flux of the moons from two processes: particle-induced X-ray emission (PIXE) from the impact of energetic protons and X-ray emission from electron bremsstrahlung. With uncertainties of a factor of a few, the electron bremsstrahlung and PIXE models overestimate the X-ray flux from Europa, preventing us from making a definitive statement about the origin of the X-ray emission. The PIXE model of Io predicts emission lines at O and S similar to those observed, but underestimates their flux by nearly two orders of magnitude. Based on this discrepancy in the PIXE flux, combined with the detected broadband continuum in the spectrum, we conclude that the X-ray emission from Io is due to electron bremsstrahlung. Likewise, because of Ganymede’s broad continuum, we tentatively conclude that its X-ray emission is also due to electron bremsstrahlung. Callisto is too faint in the X-rays to draw any conclusion. Obtaining in situ X-ray observations of the moons would provide a direct measurement of their elemental composition.

In vivo dynamic cell tracking with long-wavelength excitable and near-infrared fluorescent polymer dots

Development of cell-based therapeutic systems has attracted great interest in biomedicine. In vivo cell tracking by fluorescence provides indispensable information for further advancing cell therapy in clinical applications. However, it is still challenging in many cases because of the limited light penetration depth as well as the variations in fluorescent probes, cell lines, and labeling brightness. Here, we designed highly fluorescent polymer dots (Pdots) with far-red-light absorption and near-infrared (NIR) emission for cell tracking. The Pdots consisted of a donor-acceptor polymer blending system where intra-particle energy transfer yielded a narrow-band emission at 800 nm with a high quantum yield of ~0.22. We investigated biocompatibility and cell labeling brightness of the Pdots coated with cell penetrating peptides. Flow cytometry indicated that the cell-labeling brightness of both stem cells and cancer cells increased as much as ~4 orders of magnitude comparing the intensity measurements of labeled cells and controls. Yet, in vivo cell tracking results revealed distinctive fluorescence distribution for the same number of cells that were administered into mice through the tail vein. The stem cells initially accumulated in the lung and remained for seven days, whereas the cancer cells tended to be cleared by the liver in four days. The difference is likely due to the fact that cancer cells are easily attacked by the immune system, whereas stem cells have low immunogenicity. Results obtained herein confirm that NIR-fluorescent Pdots are promising platforms for in vivo cell tracking in small animals.

Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200–1000 nm) and small EVs (50–200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

Chlorophyll-a Concentration Assessment Using Remotely Sensed Data over Multiple Years along the Coasts of the United Arab Emirates

The United Arab Emirates (UAE) recently witnessed algal/phytoplankton blooms attributed to the high concentrations of Chlorophyll-a associated with the spread and accumulation of a wide range of organisms with toxic effects that influence ecological and fishing economic activities and water desalination along coastal areas. This research explores the UAE coasts as a case study for the framework presented here. In this research, we argue that advances in satellite remote sensing and imaging of spatial and temporal data offer sufficient information to find the best-fit regression method and relationship between Chlorophyll-a concentration and a set of climatic and biological explanatory variables over time. Three functional forms of regression models were tested and analysed to reveal that the Log-Linear Model found to be the best fit providing the most statistically robust model compared to the Linear and the Generalised Least Square models. Besides, it is useful to identify the factors Sea Surface temperature, Calcite Concentration, Instantaneous Photosynthetically Available Radiation, Normalized Fluorescence Line Height, and Wind Speed that significantly influence Chlorophyll-a concentration. Research results can be beneficial to aid decision-makers in building a best-fit statistical system and models of algal blooms in the study area. The study found results to be sensitive to the study’s temporal time-period length and the explanatory variables selected for the analysis.

X-raying molecular clouds with a short flare: probing statistics of gas density and velocity fields

ABSTRACT We take advantage of a set of molecular cloud simulations to demonstrate a possibility to uncover statistical properties of the gas density and velocity fields using reflected emission of a short (with duration much less than the cloud’s light-crossing time) X-ray flare. Such a situation is relevant for the Central Molecular Zone (CMZ) of our Galaxy where several clouds get illuminated by an ∼110 yr-old flare from the supermassive black hole Sgr A* . Due to shortness of the flare (Δt ≲ 1.6 yr), only a thin slice (Δz ≲ 0.5 pc) of the molecular gas contributes to the X-ray reflection signal at any given moment, and its surface brightness effectively probes the local gas density. This allows reconstructing the density probability distribution function over a broad range of scales with virtually no influence of attenuation, chemo-dynamical biases, and projection effects. Such a measurement is key to understanding the structure and star formation potential of the clouds evolving under extreme conditions in the CMZ. For cloud parameters similar to the currently brightest in X-ray reflection molecular complex Sgr A, the sensitivity level of the best available data is sufficient only for marginal distinction between solenoidal and compressive forcing of turbulence. Future-generation X-ray observatories with large effective area and high spectral resolution will dramatically improve on that by minimizing systematic uncertainties due to contaminating signals. Furthermore, measurement of the iron fluorescent line centroid with sub-eV accuracy in combination with the data on molecular line emission will allow direct investigation of the gas velocity field.

Establishment and characterization of stable red, far-red (fR) and near infra-red (NIR) transfected canine prostate cancer cell lines

BackgroundCanine prostate cancer represents a unique model for human prostate cancer. In vitro systems offer various possibilities but Xenograft in vivo imaging allows studying complex tasks as tumor progression and drug intervention longitudinal. Herein, we established three canine prostate carcinoma cell lines stably expressing fluorescent proteins allowing deep tissue in vivo imaging.MethodsThree canine prostate carcinoma (cPC) cell lines were stably transfected with fluorescent proteins in red, far-red and near infra-red spectrum, followed by G418 selection. Fluorescent protein expression was demonstrated by microscopy, flow cytometry and a NightOWL LB 983 in vivo imaging system. Cellular and molecular characteristics of the generated cell lines were compared to the parental cell line CT1258. Cell proliferation, metabolic activity and sphere formation capacity were analyzed. Stem cell marker expression was examined by qPCR and genomic copy number variation by genomic DNA whole genome sequencing.ResultsThree stably fluorescent protein transfected cPC cell lines were established and characterized. Compared to the parental cell line, no significant difference in cell proliferation and metabolic activity were detected. Genomic copy number variation analyses and stem cell marker gene expression revealed in general no significant changes. However, the generated cell line CT1258-mKate2C showed uniquely no distal CFA16 deletion and an elevated metabolic activity. The introduced fluorescencent proteins allowed highly sensitive detection in an in vivo imaging system starting at cell numbers of 0.156 × 106. Furthermore, we demonstrated a similar sphere formation capacity in the fluorescent cell lines. Interestingly, the clone selected CT1258-mKate2C, showed increased sphere formation ability.DiscussionStarting from a well characterized cPC cell line three novel fluorescent cell lines were established showing high cellular and molecular similarity to the parental cell line. The introduction of the fluorescent proteins did not alter the established cell lines significantly. The red fluorescence allows deep tissue imaging, which conventional GFP labeling is not able to realize.ConclusionAs no significant differences were detected between the established cell lines and the very well characterized parental CT1258 the new fluorescent cell lines allow deep tissue in vivo imaging for perspective in vivo evaluation of novel therapeutic regimens.

An enhanced toolkit for the generation of knockout and marker-free fluorescent Plasmodium chabaudi.

The rodent parasite Plasmodium chabaudi is an important in vivo model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti- Plasmodium immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection. P. chabaudi also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable in vivo to in vitro model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids for P. chabaudi and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linear PlasmoGEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcoded P. chabaudi gene-deletion and -tagging vectors for transfection in our fluorescent P. chabaudi mother-lines. This produces a tool-kit of P. chabaudi lines, vectors and transfection approaches that will be of broad utility to the research community.

Iron line from neutron star accretion discs in scalar tensor theories

ABSTRACT The Fe Kα fluorescent line at 6.4 keV is a powerful probe of the space–time metric in the vicinity of accreting compact objects. We investigated here how some alternative theories of gravity, namely scalar tensor theories, that invoke the presence of a non-minimally coupled scalar field and predict the existence of strongly scalarized neutron stars (NSs), change the expected line shape with respect to General Relativity. By taking into account both deviations from the general relativistic orbital dynamics of the accreting disc, where the Fe line originates, and the changes in the light propagation around the NS, we computed line shapes for various inclinations of the disc with respect to the observer. We found that both the intensity of the low-energy tails and the position of the high-energy edge of the line change. Moreover, we verified that even if those changes are in general of the order of a few percent, they are potentially observable with the next generation of X-ray satellites.

Precisely Targeted Reporters Reveal ABCG2 Regulation in Live Lung Cancer Cells

Expression of the ABCG2 multidrug transporter is a marker of cancer stem cells and an indicator of poor disease outcome. Understanding the effect of pharmacotherapy on ABCG2 expression is critical in guiding therapy recommendations and may help rational drug development. Engineered reporter cell lines are useful in monitoring gene regulation and protein activity in live cells but rely on precise targeting to preserve native regulatory functions. Here, we generated a number of precision‐engineered, fluorescent human cell lines that interrogate ABCG2 regulation in live lung cancer cells. Using homology‐based CRISPR engineering, we targeted a promoterless eGFP to the translational start site of the ABCG2 gene, generating ABCG2 knockout and in situ tagged ABCG2 reporter cells. Using the engineered cell lines, we show that anti‐cancer medications, HDAC inhibitors and hypoxia‐mimicking agents induce ABCG2 expression. In addition, we provide evidence that the glucocorticoid receptor is a specific, positive regulator of the ABCG2 gene. To our knowledge, this is the first fluorescent reporter assay designed to follow the endogenous regulation of a human ABC transporter in live cells. The information gained may direct therapy recommendations and assist rational drug design.Support or Funding InformationThis research has been supported mainly by OTKA‐NKFIH grant NK 115375, but also by NKFIH K104903 and the NVKP_16‐1‐2016‐0005 program from the National Research, Development and Innovation Office. This work has also been supported by research grant OTKA‐NKFIH K128011, awarded to GV. A Borsy was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (HA: BO/00579/17/5), through the New National Excellence Program of the Ministry of Human Resources (HA: ÚNKP‐18‐4‐SE‐11).

Iron Line Tomography of General Relativistic Hydrodynamic Accretion around Kerr Black Holes

Abstract We consider a temporal response of relativistically broadened line spectrum of iron from black hole accretion irradiated by an X-ray echo under strong gravity. The physical condition of accreting gas is numerically calculated in the context of general relativistic hydrodynamics under steady-state axisymmetry in Kerr geometry. With the onset of a point-like X-ray flare of a short finite duration just above the accretion surface, the gas is assumed to be ionized to produce a neutral iron fluorescent line. Using a fully relativistic ray-tracing approach, the response of line photons due to the X-ray illumination is traced as a function of time and energy for different source configurations around Schwarzschild and Kerr black holes. Our calculations show that the X-ray echo on the accretion surface clearly imprints a characteristic time-variability in the line spectral features depending on those parameters. Simulated line profiles, aimed for the future microcalorimeter missions of large collecting area such as Athena/X-IFU for typical radio-quiet Seyfert galaxies, are presented to demonstrate that state-of-the-art new observations could differentiate various source parameters by such an X-ray tomographic line reverberation.