- A Novel Chemo-Enzymatic Cascade for Smart Detection of Nitro- and Halogenated Phenols. [Journal Article]
- ACAngew Chem Int Ed Engl 2019 Jun 24
- The flavin-dependent monooxygenase, HadA, catalyzes the dehalogenation and denitration of the toxicants, nitro- and halogenated phenols, to benzoquinone. The HadA reaction can be applied in one-pot r…
The flavin-dependent monooxygenase, HadA, catalyzes the dehalogenation and denitration of the toxicants, nitro- and halogenated phenols, to benzoquinone. The HadA reaction can be applied in one-pot reactions towards the de novo synthesis of D-luciferin by coupling with D-Cys condensation. D-luciferin, a valuable chemical widely used in biomedical applications, can be used as a substrate for the reaction of firefly luciferase to generate bioluminescence. As nitro- and halogenated phenols are key indicators of human overexposure to pesticides commonly used worldwide and indicators of pesticide contamination, the technology provides a sensitive and convenient tool for improved biomedical and environmental detection at ppb sensitivity in biological samples without the requirement for any pre-treatment. This newly developed methodology provides the dual-pronged advantage of waste biodetoxification to produce a valuable chemical as well as a smart detection tool for environmental and biomedical detection.
- In vitro and in vivo Efficacy of a Synergistic Combination of Itraconazole and Verapamil Against Aspergillus fumigatus. [Journal Article]
- FMFront Microbiol 2019; 10:1266
- The incidence of aspergillosis continues to rise sharply, while the progress made in expanding the antifungal drug arsenal remains extremely slow, indicating an urgent need for new strategies. Previo…
The incidence of aspergillosis continues to rise sharply, while the progress made in expanding the antifungal drug arsenal remains extremely slow, indicating an urgent need for new strategies. Previous studies have shown that the calcium signaling pathway, which is evolutionarily conserved in mammals and fungi, is involved in regulating the tolerance of azoles in fungi. In this study, we performed a preliminary screening among various combinations of different clinical calcium channel blockers and different antifungal drugs. We found that the combination of itraconazole and verapamil showed the best synergistic effect against Aspergillus fumigatus. Thereafter, using the checkboard assays we observed synergistic effects of the combination treatment against most of the A. fumigatus strains tested, including itraconazole-sensitive and itraconazole-resistant strains, with a fractional inhibitory concentration index (FICI) < 0.5. Furthermore, we showed that verapamil strongly decreased the cytosolic calcium transients following itraconazole stimulation by an aequorin-mediated method. Moreover, verapamil influenced the efflux of rhodamine 6G, an azole mimic substance. An ergosterol assay revealed that verapamil alone had no effect on ergosterol biosynthesis, but the combination of itraconazole and verapamil treatment decreased the ergosterol level. Further murine assays were performed using a luciferase-probed bioluminescence imaging method. Drug combination therapy reduced lung burden and improved survival rate. In conclusion, verapamil is a promising candidate to enhance the antifungal activity of itraconazole against A. fumigatus. In addition, our study suggests the effectiveness of an emerging approach based on bioluminescence imaging in monitoring the efficacy of drug combination therapy for invasive aspergillosis.
- Phrixotrix luciferase and 6'-aminoluciferins reveal a larger luciferin phenolate binding site and provide novel far-red combinations for bioimaging purposes. [Journal Article]
- SRSci Rep 2019 Jun 21; 9(1):8998
- How the unique luciferase of Phrixothrix hirtus (PxRE) railroad worm catalyzes the emission of red bioluminescence using the same luciferin of fireflies, remains a mystery. Although PxRE luciferase i…
How the unique luciferase of Phrixothrix hirtus (PxRE) railroad worm catalyzes the emission of red bioluminescence using the same luciferin of fireflies, remains a mystery. Although PxRE luciferase is a very attractive tool for bioanalysis and bioimaging in hemoglobin rich tissues, it displays lower quantum yield (15%) when compared to green emitting luciferases (>40%). To identify which parts of PxRE luciferin binding site (LBS) determine bioluminescence color, and to develop brighter and more red-shifted emitting luciferases, we compared the effects of site-directed mutagenesis and of larger 6'-substituted aminoluciferin analogues (6'-morpholino- and 6'-pyrrolidinyl-LH) on the bioluminescence properties of PxRE and green-yellow emitting beetle luciferases. The effects of mutations in the benzothiazolyl and thiazolyl parts of PxRE LBS on the KM and catalytic efficiencies, indicated their importance for luciferin binding and catalysis. However, the absence of effects on the bioluminescence spectrum indicated a less interactive LBS in PxRE during light emission. Mutations at the bottom of LBS of PxRE blue-shifted the spectra and increased catalytic efficiency, suggesting that lack of interactions of this part of LBS with excited oxyluciferin phenolate underlie red light emission. The much higher bioluminescence activity and red-shifted spectra of PxRE luciferase with 6'-morpholino- (634 nm) and 6'-pyrrolidinyl-luciferins (644 nm), when compared to other beetle luciferases, revealed a larger luciferin phenolate binding pocket. The size and orientation of the side-chains of L/I/H348 are critical for amino-analogues accommodation and modulate bioluminescence color, affecting the interactions and mobility of excited oxyluciferin phenolate. The PxRE luciferase and 6'-aminoluciferins provide potential far-red combinations for bioimaging applications.
- AGTR1 promotes lymph node metastasis in breast cancer by upregulating CXCR4/SDF-1α and inducing cell migration and invasion. [Journal Article]
- AAging (Albany NY) 2019 Jun 19; 11
- The angiotensin II type I receptor (AGTR1) has a strong influence on tumor growth, angiogenesis, inflammation and immunity. However, the role of AGTR1 on lymph node metastasis (LNM) in breast cancer,…
The angiotensin II type I receptor (AGTR1) has a strong influence on tumor growth, angiogenesis, inflammation and immunity. However, the role of AGTR1 on lymph node metastasis (LNM) in breast cancer, which correlates with tumor progression and patient survival, has not been examined. AGTR1 was highly expressed in lymph node-positive tumor tissues, which was confirmed by the Oncomine database. Next, inhibition of AGTR1 reduced tumor growth and LNM in orthotopic xenografts by bioluminescence imaging (BLI). Losartan, an AGTR1-specific inhibitor, decreased the chemokine pair CXCR4/SDF-1α levels in vivo and inhibited AGTR1-induced cell migration and invasion in vitro. Finally, the molecular mechanism of AGTR1-induced cell migration and LNM was assessed by knocking down AGTR1 in normal cells or CXCR4 in AGTR1high cells. AGTR1-silenced cells treated with losartan showed lower CXCR4 expression. AGTR1 overexpression caused the upregulation of FAK/RhoA signaling molecules, while knocking down CXCR4 in AGTR1high cells downregulated these molecules. Collectively, AGTR1 promotes LNM by increasing the chemokine pair CXCR4/SDF-1α and tumor cell migration and invasion. The potential mechanism of AGTR1-mediated cell movement relies on activating the FAK/RhoA pathway. Our study indicated that inhibiting AGTR1 may be a potential therapeutic target for LNM in early-stage breast cancer.
- Wide field of view quantitative imaging of cellular ATP release. [Journal Article]
- AJAm J Physiol Cell Physiol 2019 Jun 19
- While several mechanical stressors promote ATP secretion from eukaryotic cells, few mechanosensitive pathways for ATP release have been precisely characterized and none clearly identified. To facilit…
While several mechanical stressors promote ATP secretion from eukaryotic cells, few mechanosensitive pathways for ATP release have been precisely characterized and none clearly identified. To facilitate progress, we report here a wide field of view (~20x20 mm sample area) imaging technique paired with a quantitative image analysis to accurately map the dynamics of ATP release from a cell population. The approach has been tested on A549 cells stretched at high initial strain rate (2-5 s-1) or swelled by hypotonic shock. The amount of ATP secreted in response to a series of 5 graded stretch pulses (5-37% linear deformation, 1-s duration at 25°C) changed non-monotonically with respect to strain amplitude and was inhomogeneous across the cell monolayer. In a typical experiment, extracellular ATP density averaged 250 fmol/mm2 but the area of detectable signal covered only ~40% of the cells. In some areas, ATP accumulation peaked around 900 fmol/mm2 which corresponded to an estimated concentration of 4.5 µM. The total amount of ATP released from the combined stretch pulses reached 384±224 pmol/million cells (n=4). Compared to stretch, hypotonic shock (50%, 30°C) elicited a more homogeneous ATP secretion from the entire cell population but at lower yield totaling 28±12 pmol/million cells (n=4). The quantitative extracellular ATP mapping of several thousand cells at once, with this wide FOV imaging system, will help identify ATP release pathways by providing unique insights on the dynamics and inhomogeneities of the cellular ATP secretion otherwise difficult to assess within the smaller FOV of a microscope.
- Individual in vivo Profiles of Microglia Polarization After Stroke, Represented by the Genes iNOS and Ym1. [Journal Article]
- FIFront Immunol 2019; 10:1236
- Microglia are the brain-innate immune cells which actively surveil their environment and mediate multiple aspects of neuroinflammation, due to their ability to acquire diverse activation states and p…
Microglia are the brain-innate immune cells which actively surveil their environment and mediate multiple aspects of neuroinflammation, due to their ability to acquire diverse activation states and phenotypes. Simplified, M1-like microglia are defined as pro-inflammatory cells, while the alternative M2-like cells promote neuroprotection. The modulation of microglia polarization is an appealing neurotherapeutic strategy for stroke and other brain lesions, as well as neurodegenerative diseases. However, the activation profile and change of phenotype during experimental stroke is not well understood. With a combined magnetic resonance imaging (MRI) and optical imaging approach and genetic targeting of two key genes of the M1- and M2-like phenotypes, iNOS and Ym1, we were able to monitor in vivo the dynamic adaption of the microglia phenotype in response to experimental stroke.
- Evaluation of Functional Selectivity of Haloperidol, Clozapine, and LASSBio-579, an Experimental Compound With Antipsychotic-Like Actions in Rodents, at G Protein and Arrestin Signaling Downstream of the Dopamine D2 Receptor. [Journal Article]
- FPFront Pharmacol 2019; 10:628
- LASSBio-579, an N-phenylpiperazine antipsychotic lead compound, has been previously reported as a D2 receptor (D2R) ligand with antipsychotic-like activities in rodent models of schizophrenia. In ord…
LASSBio-579, an N-phenylpiperazine antipsychotic lead compound, has been previously reported as a D2 receptor (D2R) ligand with antipsychotic-like activities in rodent models of schizophrenia. In order to better understand the molecular mechanism of action of LASSBio-579 and of its main metabolite, LQFM 037, we decided to address the hypothesis of functional selectivity at the D2R. HEK-293T cells transiently coexpressing the human long isoform of D2 receptor (D2LR) and bioluminescence resonance energy transfer (BRET)-based biosensors were used. The antagonist activity was evaluated using different concentrations of the compounds in the presence of a submaximal concentration of dopamine (DA), after 5 and 20 min. For both signaling pathways, haloperidol, clozapine, and our compounds act as DA antagonists in a concentration-dependent manner, with haloperidol being by far the most potent, consistent with its nanomolar D2R affinity measured in binding assays. In our experimental conditions, only haloperidol presented a robust functional selectivity, being four- to fivefold more efficient for inhibiting translocation of β-arrestin-2 (β-arr2) than for antagonizing Gi activation. Present data are the first report on the effects of LASSBio-579 and LQFM 037 on the β-arr2 signaling pathway and further illustrate that the functional activity could vary depending on the assay conditions and approaches used.
- Near-Infrared Fluorescent Proteins and Their Applications. [Review]
- BBiochemistry (Mosc) 2019; 84(Suppl 1):S32-S50
- High transparency, low light-scattering, and low autofluorescence of mammalian tissues in the near-infrared (NIR) spectral range (~650-900 nm) open a possibility for in vivo imaging of biological pro…
High transparency, low light-scattering, and low autofluorescence of mammalian tissues in the near-infrared (NIR) spectral range (~650-900 nm) open a possibility for in vivo imaging of biological processes at the micro- and macroscales to address basic and applied problems in biology and biomedicine. Recently, probes that absorb and fluoresce in the NIR optical range have been engineered using bacterial phytochromes - natural NIR light-absorbing photoreceptors that regulate metabolism in bacteria. Since the chromophore in all these proteins is biliverdin, a natural product of heme catabolism in mammalian cells, they can be used as genetically encoded fluorescent probes, similarly to GFP-like fluorescent proteins. In this review, we discuss photophysical and biochemical properties of NIR fluorescent proteins, reporters, and biosensors and analyze their characteristics required for expression of these molecules in mammalian cells. Structural features and molecular engineering of NIR fluorescent probes are discussed. Applications of NIR fluorescent proteins and biosensors for studies of molecular processes in cells, as well as for tissue and organ visualization in whole-body imaging in vivo, are described. We specifically focus on the use of NIR fluorescent probes in advanced imaging technologies that combine fluorescence and bioluminescence methods with photoacoustic tomography.
- Grazer-induced bioluminescence gives dinoflagellates a competitive edge. [Letter]
- CBCurr Biol 2019 Jun 17; 29(12):R564-R565
- Bioluminescent dinoflagellates grow at one third the rate of their competitors of equivalent size, such as diatoms . Despite this disadvantage, dinoflagellates successfully persist within phytopla…
Bioluminescent dinoflagellates grow at one third the rate of their competitors of equivalent size, such as diatoms . Despite this disadvantage, dinoflagellates successfully persist within phytoplankton communities and even form large blooms during favourable conditions. One explanation for this paradox is that bioluminescence acts as a defence that reduces losses to zooplankton grazers, such as copepods [2,3]. Lindström et al. found that the dinoflagellate Lingulodinium polyedra (F.Stein) J.D.Dodge increase their bioluminescence in response to copepodamides , polar lipids exuded by copepod grazers, allowing for a brighter flash when bioluminescent capacity is stimulated. Here, we show that copepodamide-induced bioluminescence in L. polyedra causes a marked shift in the grazing preference of the copepod Acartia tonsa Dana. L. polyedra goes from being the preferred prey when non-bioluminescent to near complete rejection when pre-treated with copepodamides to induce a higher bioluminescent capacity. High-speed and low-light-sensitive videos show how L. polyedra cells flash upon contact with the copepod and are subsequently rejected, seemingly unharmed (Videos S1 and S2). Instead, A. tonsa shows compensatory feeding on the alternative prey.
New Search Next
- Evaluation of Luminogenic Substrates as Probe Substrates for Bacterial Cytochrome P450 Enzymes: Application to Mycobacterium tuberculosis. [Journal Article]
- SDSLAS Discov 2019 Jun 17; :2472555219853220
- Several cytochrome P450 enzymes (CYPs) encoded in the genome of Mycobacterium tuberculosis (Mtb) are considered potential new drug targets due to the essential roles they play in bacterial viability …
Several cytochrome P450 enzymes (CYPs) encoded in the genome of Mycobacterium tuberculosis (Mtb) are considered potential new drug targets due to the essential roles they play in bacterial viability and in the establishment of chronic intracellular infection. Identification of inhibitors of Mtb CYPs at present is conducted by ultraviolet-visible (UV-vis) optical titration experiments or by metabolism studies using endogenous substrates, such as cholesterol and lanosterol. The first technique requires high enzyme concentrations and volumes, while analysis of steroid hydroxylation is dependent on low-throughput analytical methods. Luciferin-based luminogenic substrates have proven to be very sensitive substrates for the high-throughput profiling of inhibitors of human CYPs. In the present study, 17 pro-luciferins were evaluated as substrates for Mtb CYP121A1, CYP124A1, CYP125A1, CYP130A1, and CYP142A1. Luciferin-BE was identified as an excellent probe substrate for CYP130A1, resulting in a high luminescence yield after addition of luciferase and adenosine triphosphate (ATP). Its applicability for high-throughput screening was supported by a high Z'-factor and high signal-to-background ratio. Using this substrate, the inhibitory properties of a selection of known inhibitors could be characterized using significantly less protein concentration when compared to UV-vis optical titration experiments. Although several luminogenic substrates were also identified for CYP121A1, CYP124A1, CYP125A1, and CYP142A1, their relatively low yield of luminescence and low signal-to-background ratios make them less suitable for high-throughput screening since high enzyme concentrations will be needed. Further structural optimization of luminogenic substrates will be necessary to obtain more sensitive probe substrates for these Mtb CYPs.