A new miniaturized micro sequential injection coupled with the lab-on-valve (µSIA–LOV) technique was full-blown to perform inhibitory studies on dihydrofolate reductase (DHFR). The system was used to ...evaluate the DHFR inhibition activity of metal-based anticancer compounds. The metal complexes exhibited IC50 values in the range 1.3 ± 0.3–108 ± 7 μM, with half of the complexes lying in the low μM range, i.e., 1.3 ± 0.3–4.4 ± 0.2 μM. For comparison, methotrexate (MTX), a known inhibitor of DHFR, has an IC50 value of 0.38 ± 0.06 μM. The µSIA–LOV is a versatile, robust, rapid, and easy to operate, allowing automated determination of DHFR inhibition. Moreover, the automated system requires very little sample (approximately 40 µL per analysis), uses minimal reagents (5 times less than the batch procedure used), and generates very little waste (around 1.2 mL per analysis) compared with batch methods, considerably reducing costs.
•DHFR is an attractive promising target to differentiate cancer from normal cells.•µSIA-LOV is developed to test DHFR inhibition of synthesized organometallic compounds.•Some of the metal compounds evaluated exhibit noteworthy DHFR inhibitory activity.•The µSIA-LOV system is a useful tool for the evaluation of DHFR enzymatic reaction.•Automation to assess enzymatic assays proved to be advantageous.
ABSTRACT
The problems arising as a result of aging aircraft, rail and civil infrastructure have focused attention on tools for predicting the growth of cracks from small naturally occurring material ...discontinuities. To this end, the present paper discusses on the difference between the analysis tools needed for ab initio design and sustainment, modelling of cracks that grow from small naturally occurring material discontinuities and ways to determine the short crack da/dN versus ΔK data from long crack American Society for Testing and Materials (ASTM) tests. It also discusses how existing equations can be used to predict short crack growth and how to account for the variations seen in crack growth histories. Attention is also focused on the recent Federal Aviation Administration limit of validity ruling and the effect of the environment on widespread fatigue damage in civil transport aircraft.
Optical manipulations are widely used to analyze neuronal functions in vivo. Blue light is frequently used to activate channelrhodopsins or LOV domains, although the degrees of its absorption and ...scattering are higher than those of longer wavelength light. High spatial resolution of optical manipulation is easily achieved in vitro, while the light is unevenly scattered and absorbed in tissues due to many factors. It is difficult to spatially measure a blue light transmission area in vivo. Here, we propose a genetic method to visualize blue light transmission in the brain and other organs using light-induced nuclear translocation of fluorescent proteins with a LOV domain. A light-inducible nuclear localization signal (LINuS) consists of a LOV2 domain fused with a nuclear localization signal (NLS). We confirmed that blue light illumination induced reversible translocation of NES-tdTomato-LINuS from the cytosol to the nucleus within 30 min in HEK293 cells. By employing a PHP.eb capsid that can penetrate the blood-brain barrier, retro-orbital sinus injection of adeno-associated virus (AAV) vectors induced scattered expression of nuclear export signal (NES)-tdTomato-LINuS in the brain. We confirmed that 30-min transcranial blue light illumination induced nuclear translocation of NES-tdTomato-LINuS in the cortex, the hippocampus, and even the paraventricular nucleus of the thalamus. We also found that mice exposed to blue light in a shaved abdominal area exhibited a substantial increase in nuclear translocation in the ventral surface lobe of the liver. These results provide a simple way to obtain useful information on light transmission in tissues without any transgenic animals or skillful procedures.
•We achieved light-dependent nuclear translocation of tdTomato by employing a light-inducible nuclear localization signal.•We achieved scattered expression of NES-tdTomato-LINuS in the brain by simple intravenous injection of AAV vectors.•We achieved visualization of blue light transmission areas by quantifying nuclear translocation of NES-tdTomato-LINuS.
Light has emerged as a control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible ...transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we created a variant that returns to the inactive dark state within minutes once the blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators, which only require blue light from ordinary light-emitting diodes for induction, were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. Opto-T7RNAP, with minor alterations, should be extendable to other bacterial species as well as eukaryotes such as mammalian cells and yeast in which the T7 RNA polymerase and the light-inducible Vivid regulator have been shown to be functional. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation and allow for a more reliable dynamic control of synthetic and natural gene networks.
We report natural light–oxygen–voltage (LOV) photoreceptors with a blue light-switched, high-affinity (K
D ∼ 10−7 M), and direct electrostatic interaction with anionic phospholipids. Membrane ...localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure–function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of ...flavin‐binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time‐resolved and low‐temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein‐flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission, and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low‐temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine the x‐ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color‐tuning mechanisms of flavin‐binding proteins and could be helpful for the engineering of new variants with improved optical properties.
Community partition is of great importance in social networks because of the rapid increasing network scale, data and applications. We consider the community partition problem under Linear Threshold ...(LT) model in social networks, which is a combinatorial optimization problem that divides the social network to disjoint <inline-formula><tex-math notation="LaTeX">m</tex-math></inline-formula> communities. Our goal is to maximize the sum of influence propagation within each community. As the influence propagation function of community partition problem is supermodular under LT model, we use the method of Lov<inline-formula><tex-math notation="LaTeX">{\acute{a}}</tex-math></inline-formula>sz Extension to relax the target influence function and transfer our goal to maximize the relaxed function over a matroid polytope. Next, we propose a continuous greedy algorithm using the properties of the relaxed function to solve our problem, which needs to be discretized in concrete implementation. Then, random rounding technique is used to convert the fractional solution to the integer solution. We present a theoretical analysis with <inline-formula><tex-math notation="LaTeX">1-1/e</tex-math></inline-formula> approximation ratio for the proposed algorithms. Extensive experiments are conducted to evaluate the performance of the proposed continuous greedy algorithms on real-world online social networks datasets. The results demonstrate that continuous community partition method can improve influence spread and accuracy of the community partition effectively.
Phototropin (phot) is a blue light (BL) receptor and thermosensor that mediates chloroplast movements in plants. Liverworts, as early‐diverging plant species, have a single copy of PHOT gene, and the ...phot protein in each liverwort activates the signaling pathway adapted to its specific growing environment. In this study, we functionally compared phot from two different liverworts species: Apopellia endiviifolia (Aephot) and Marchantia polymorpha (Mpphot). The BL‐dependent photochemical activity of Aephot was similar to that of Mpphot, whereas the thermochemical activity of Aephot was lower than that of Mpphot. Therefore, the phot‐mediated signaling pathways of the two plant species may differ more in response to temperature than to BL. Furthermore, we analyzed the functional compatibility of Aephot and Mpphot in chloroplast movements by transiently expressing AePHOT or MpPHOT. The transient expression of AePHOT did not mediate chloroplast movement in M. polymorpha, showing the incompatibility of Aephot with the signaling pathway of M. polymorpha. By contrast, the transient expression of MpPHOT mediated chloroplast movement in A. endiviifolia, indicating the compatibility of Mpphot with the signaling pathway of A. endiviifolia. Our findings reveal both functional similarities and differences between Aephot and Mpphot proteins from the closely related liverworts.
The phototropin (phot) of two closed liverworts species: Apopellia endiviifolia (Aephot) and Marchantia polymorpha (Mpphot) have been functionally compared in this study. Both Aephot and Mpphot show similarities in their photocycle, but diversity in the functions of regulating chloroplast movement. The transient expression of AePHOT did not mediate chloroplast movement in M. polymorpha, showing the incompatibility of Aephot with the signaling pathway of M. polymorpha. By contrast, the transient expression of MpPHOT‐mediated chloroplast movement in A. endiviifolia indicates the compatibility of Mpphot with the signaling pathway of A. endiviifolia.
Drought tolerance is important for grain crops, including rice (Oryza sativa); for example, rice cultivated under intermittent irrigation produces less methane gas compared to rice grown in anaerobic ...paddy field conditions, but these plants require greater drought tolerance. Moreover, the roles of rice circadian‐clock genes in drought tolerance remain largely unknown. Here, we show that the mutation of LOV KELCH REPEAT PROTEIN 2 (OsLKP2) enhanced drought tolerance by increasing cuticular wax biosynthesis. Among ZEITLUPE family genes, OsLKP2 expression specifically increased under dehydration stress. OsLKP2 knockdown (oslkp2‐1) and knockout (oslkp2‐2) mutants exhibited enhanced drought tolerance. Cuticular waxes inhibit non‐stomatal water loss. Under drought conditions, total wax loads on the leaf surface increased by approximately 10% in oslkp2‐1 and oslkp2‐2 compared to the wild type, and the transcript levels of cuticular wax biosynthesis genes were upregulated in the oslkp2 mutants. Yeast two‐hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays revealed that OsLKP2 interacts with GIGANTEA (OsGI) in the nucleus. The osgi mutants also showed enhanced tolerance to drought stress, with a high density of wax crystals on their leaf surface. These results demonstrate that the OsLKP2‐OsGI interaction negatively regulates wax accumulation on leaf surfaces, thereby decreasing rice resilience to drought stress.
Summary statement
Loss of LOV KELCH REPEAT PROTEIN 2 (OsLKP2) function results in drought tolerance in rice due to an elevated total wax load on leaf surfaces through upregulated expression of wax‐related genes. In the nucleus, OsLKP2 interacts with GIGANTEA, whose null mutant also shows enhanced drought tolerance.
PDF
