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Fraction unbound in plasma (fu,p) of drugs is an significant factor for drug delivery and other biological incidences related to the pharmacokinetic behaviours of drugs. Exploration of different molecular fragments for fu,p of different small molecules/agents can facilitate in identification of suitable candidates in the preliminary stage of drug discovery. Different researchers have implemented strategies to build several prediction models for fu,p of different drugs. However, these studies did not focus on the identification of responsible molecular fragments to determine the fraction unbound in plasma. In the current work, we tried to focus on the development of robust classification-based QSAR models and evaluated these models with multiple statistical metrics to identify essential molecular fragments/structural attributes for fractions unbound in plasma. The study unequivocally suggests various N-containing aromatic rings and aliphatic groups have positive influences and sulphur-containing thiadiazole rings have negative influences for the fu,p values. The molecular fragments may help for the assessment of the fu,p values of different small molecules/drugs in a speedy way in comparison to experiment-based in vivo and in vitro studies. [ABSTRACT FROM AUTHOR]

BACKGROUND: Protein-ligand binding affinity is of significant importance in structure-based drug design. Recently, the development of machine learning techniques has provided an efficient and accurate way to predict binding affinity. However, the prediction performance largely depends on how molecules are represented. OBJECTIVE: Different molecular descriptors are designed to capture different features. The study aims to identify the optimal circular fingerprints for predicting protein-ligand binding affinity with matched neural network architectures. METHODS: Extended-connectivity fingerprints (ECFP) and protein-ligand extended connectivity fingerprints (PLEC) encode circular atomic and bonding connectivity environments with the preference for intra- and inter-molecular features, respectively. Densely-connected neural networks are employed to map the circular fingerprints of protein-ligand complexes to binding affinities RESULTS: The performance of neural networks is sensitive to the parameters used for ECFP and PLEC fingerprints. The R2_score of the evaluated ECFP and PLEC fingerprints reaches 0.52 and 0.49, higher than that of the improperly set ECFP and PLEC fingerprints with R2_score of 0.45 and 0.38, respectively. Additionally, compared to the predictions from the standalone fingerprints, the ECFP+PLEC conjoint ones slightly improve the prediction accuracy with R2_score of approximately 0.55. CONCLUSION: Both intra- and inter-molecular structural features encoded in the circular fingerprints contribute to the protein-ligand binding affinity. Optimizing the parameters of ECFP and PLEC can enhance performance. The conjoint fingerprint scheme can be generally extended to other molecular descriptors for enhanced feature engineering and improved predictive performance. [ABSTRACT FROM AUTHOR]

Abstract This study aims at improving upon existing activity predictions methods by augmenting chemical structure fingerprints with bio-activity based fingerprints derived from high-throughput screening (HTS) data (HTSFPs) and thereby showcasing the benefits of combining different descriptor types. This type of descriptor would be applied in an iterative screening scenario for more targeted compound set selection. The HTSFPs were generated from HTS data obtained from PubChem and combined with an ECFP4 structural fingerprint. The bioactivity-structure hybrid (BaSH) fingerprint was benchmarked against the individual ECFP4 and HTSFP fingerprints. Their performance was evaluated via retrospective analysis of a subset of the PubChem HTS data. Results showed that the BaSH fingerprint has improved predictive performance as well as scaffold hopping capability. The BaSH fingerprint identified unique compounds compared to both the ECFP4 and the HTSFP fingerprint indicating synergistic effects between the two fingerprints. A feature importance analysis showed that a small subset of the HTSFP features contribute most to the overall performance of the BaSH fingerprint. This hybrid approach allows for activity prediction of compounds with only sparse HTSFPs due to the supporting effect from the structural fingerprint.

Cysteine plays a critical role in maintaining normal human metabolism, redox homeostasis, and immune regulation. Despite its functional versatility, cysteine metabolism in the human body is not well understood because of the lack of a robust tool for real-time measurement of cysteine at the cellular and sub-cellular level. In the present study, a genetically encoded nanosensor was developed using Cj0982 protein of Campylobacter jejuni , Enhanced Cyan Fluorescent Protein (ECFP) and Venus. The Cj0982 was sandwiched between ECFP and Venus for the construction of the nanosensor, named as Cys-FS (Cysteine-Fluorescent-Sensor). The Cys-FS is pH stable, specific to cysteine and has an affinity of 1.2 × 10−5 M. A range of affinity mutants were also developed with a cumulative cysteine detection range from 800 nM to 3.5 mM. The Cys-FS nanosensor was expressed in bacterial, yeast and mammalian cells, and the dynamics of cysteine level was measured in living cells using the confocal microscopy. The results showed that the Cys-FS nanosensor successfully monitored the dynamics of cysteine in both prokaryotic and eukaryotic systems without disrupting the cell. Thus, this study presents a novel nanosensor that can measure cysteine in living cells. This nanosensor is minimally invasive and non-toxic. • A genetically encoded FRET based nanosensor was developed for real-time monitoring of cysteine in living cells. • The sensor consists of Cj0982 protein from Campylobacter jejuni flanked by Enhanced Cyan Fluorescent Protein and Venus. • Conformational Change in Cys-FS sensor in presence of cysteine results in modulation of FRET ratio. • The sensor has a highly specific response to cysteine with an in vitro dissociation constant of 1.2 × 10−5 M. • The sensor is appropriate for real-time imaging of cysteine in bacterial, yeast and mammalian cells [ABSTRACT FROM AUTHOR]

The comprehensive evaluation of the quality of traditional Chinese medicines (TCM) is an important issue for the continuous progress and exploration of TCM. In this study, a "Yiqing" tablet (YQT) was taken as an example, and the sample quality was comprehensively investigated by multi-component quantification, multi-dimensional fingerprint construction, and antioxidant activity analysis. Based on high performance liquid chromatography (HPLC) and fourier transform infrared spectroscopy (FTIR) fingerprint, accurate and fast multi-component quantification is achieved by reliable Multi-markers assay by mono-linear method (MAML) method and verified partial least squares regression (PLSR) model. The basic HPLC fingerprint and the special FTIR quantitative fingerprint were evaluated by SQFM, and the rich fingerprint qualitative and quantitative information of the sample was obtained. The characteristic parameter (blocking rate (BR)) characterizing antioxidant activity in the electrochemical (EC) fingerprint was excavated for the first time, and the fingerprint-efficacy analysis results with HPLC and FTIR were obtained through bivariate correlation analysis (BCA). The results showed that 25 components in the HPLC fingerprint and had antioxidant activity, and most bands of FTIR showed antioxidant activity. Finally, by combining the evaluation results of HPLC and FTIR fingerprint using the mean method, all samples were classified as first level, except for S1, demonstrating the consistency of sample quality. Based on the comprehensive quality evaluation system combining vertical and horizontal combination, this study provides a new idea for achieving comprehensive quality evaluation of TCM. [Display omitted] • MAML for multi-component quantification was established. • PLSR model for FTIR quantitative has been established. • The electrochemical fingerprint of "Yiqing" tablets was established for the first time. • A new electrochemical parameters was found to reveal antioxidant activity of samples. • The fingerprint-efficacy relationship of EC (BR) and FTIR was explored for the first time. [ABSTRACT FROM AUTHOR]

• A novel molecular characterization method inspired by coral software. • Designed a novel neural network based QSAR modeling scheme. • Seven commonly used machine learning methods for modeling are used and comparison. In supervised model training, QSAR modeling of drug analogous texture is commonly used, mostly using RNN for learning structural information of molecules, but a problem is a large time cost in fitting temporal data by recurrent training units. In this paper, we propose a novel molecular canonicalization method, GAFMol, leveraging the Gramian Angular Field (GAF) algorithm and Extended-Connectivity Fingerprint (ECFP) algorithm. GAFMol transforms molecular sequences into graphs, providing an innovative approach to capturing structural information. We apply the GAFMol-CNN scheme to construct a robust QSAR model for SARS-CoV-2 Mpro inhibitors, and thoroughly investigate the model's interpretability. Our method demonstrates superior performance compared to existing QSAR models, as discussed in detail in the Results and Discussion section. Compared with previous QSAR models (Monte Carlo, Mol2vec, FCNN, LSTM, bi-directional LSTM and CNN methods), the GAFMol-CNN scheme has significant improvement, and achieved relatively good results in the test set. The source code of the molecular canonicalization method GAFMol is freely available on https://github.com/Xuhaiyin/. [ABSTRACT FROM AUTHOR]

This article supplies raw data related to a research article entitled “Joint refinement of FRET measurements using spectroscopic and computational tools” (Kyrychenko et al., 2017) [1], in which we demonstrate the use of molecular dynamics simulations to estimate FRET orientational factors in a benchmark donor-linker-acceptor system of enhanced cyan (ECFP) and enhanced yellow (EYFP) fluorescent proteins. This can improve the recalculation of donor-acceptor distance information from single-molecule FRET measurements.

In multitarget drug design, it is critical to identify active and inactive compounds against a variety of targets and antitargets. Multitarget strategies thus test the limits of available technology, be that in screening large databases of compounds vs. a large number of targets, or in using in silico methods for understanding and reliably predicting these pharmacological outcomes. In this paper, we have evaluated the potential of several in silico approaches to predict the target, antitarget and physicochemical profile of (S)-blebbistatin, the best-known myosin II ATPase inhibitor, and a series of analogs thereof. Standard and augmented structure-based design techniques could not recover the observed activity profiles. A ligand-based method using molecular fingerprints was, however, able to select actives for myosin II inhibition. Using further ligand- and structure-based methods, we also evaluated toxicity through androgen receptor binding, affinity for an array of antitargets and the ADME profile (including assay-interfering compounds) of the series. In conclusion, in the search for (S)-blebbistatin analogs, the dissimilarity distance of molecular fingerprints to known actives and the computed antitarget and physicochemical profile of the molecules can be used for compound design for molecules with potential as tools for modulating myosin II and motility-related diseases.

The extensive demand of large scale therapeutic protein production in Chinese Hamster Ovarian cell lines increased the need of developing efficient and cost effective transfection reagent. Polyethylenimine has recently emerged as an attractive alternative to currently employed conventional transfection methods. In the present report, we attempted to develop inexpensive and easy to prepare polyethylenimine-based transfection reagent and compared its transfection efficiency with calcium phosphate, electroporation and lipoplex mediated transfection methods. Our results indicated highest transfection efficiency with in-house prepared polyplexes as marked by maximum percentage of enhanced cyan fluorescence protein positive cells. These findings contribute towards the development of economical and effective solution for high protein expression in Chinese hamster ovarian system. [ABSTRACT FROM AUTHOR]

The variability of the orientation factor is a long-standing challenge in converting FRET efficiency measurements into donor-acceptor distances. We propose the use of molecular dynamics (MD) simulations to characterize orientation distributions and thus improve the accuracy of distance measurements. Here, we test this approach by comparing experimental and simulated FRET efficiencies for a model donor-acceptor pair of enhanced cyan and enhanced yellow FPs connected by a flexible linker. Several spectroscopic techniques were used to characterize FRET in solution. In addition, a series of atomistic MD simulations of a total length of 1.5 μs were carried out to calculate the distances and the orientation factor in the FRET-pair. The resulting MD-based and experimentally measured FRET efficiency histograms coincided with each other, allowing for direct comparison of distance distributions. Despite the fact that the calculated average orientation factor was close to 2/3, the application of the average κ 2 to the entire histogram of FRET efficiencies resulted in a substantial artificial broadening of the calculated distribution of apparent donor-acceptor distances. By combining single pair-FRET measurements with computational tools, we demonstrate that accounting for the donor and acceptor orientation heterogeneity is critical for accurate representation of the donor-acceptor distance distribution from FRET measurements. [ABSTRACT FROM AUTHOR]

We constructed the fibroin H-chain expression system to produce enhanced cyan fluorescent proteins (ECFP) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing ECFP cDNA to the heavy chain gene and injecting it into a silkworm. The ECFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the ECFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworms. The EGFP fluorescence became visible in the ocelli and in the central and peripheral nervous system on the seventh day of embryonic development. A mixture of the donor and helper vector was micro-injected into 1,020 Kumokjam, bivoltin silkworm eggs. We obtained 6 broods. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the ECFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk. [ABSTRACT FROM AUTHOR]

A broad range of genetically-encoded fluorescence biosensors has been developed, allowing the detection of signaling intermediates and metabolites in real time. Many of these biosensors are based on Foerster Resonance Energy Transfer (FRET). The two biosensors of the well-known “Venus-flytrap” type exemplarily studied in this work are composed of a central sugar binding protein flanked by two green fluorescent protein derivatives, namely ECFP as well as Citrine and EYFP, respectively. In order to evaluate FRET-based biosensors as an in vivo tool for quantitative metabolite analyses, we have thoroughly studied the effects of pH, buffer salts, ionic strength, temperature and several intracellular metabolites on the signal intensity of both biosensors and both fluorescence proteins. Almost all micro-environmental variations led to considerably different FRET signals, because either the fluorescent proteins or the metabolite binding domains were affected by the tested parameters. This resulted not only in altered FRET ratios between the apo state and the saturated state but also in significant shifts of the apparent binding constant. This underlines the necessity of careful controls in order to allow reliable quantitative measurements in vivo . [ABSTRACT FROM AUTHOR]

Genetically encoded cyan fluorescent proteins (CFPs) bearing a tryptophan-derived chromophore are commonly used as energy-donor probes in Förster resonance energy transfer (FRET) experiments useful in live cell-imaging applications. In recent years, significant effort has been expended on eliminating the structural and excited-state heterogeneity of these proteins, which has been linked to undesirable photophysical properties. Recently, mCerulean3, a descendant of enhanced CFP, was introduced as an optimized FRET donor protein with a superior quantum yield of 0.87. Here, the 1.6 Å resolution X-ray structure of mCerulean3 is reported. The chromophore is shown to adopt a planar trans configuration at low pH values, indicating that the acid-induced isomerization of Cerulean has been eliminated. β-Strand 7 appears to be well ordered in a single conformation, indicating a loss of conformational heterogeneity in the vicinity of the chromophore. Although the side chains of Ile146 and Leu167 appear to exist in two rotamer states, they are found to be well packed against the indole group of the chromophore. The Ser65 reversion mutation allows improved side-chain packing of Leu220. A structural comparison with mTurquoise2 is presented and additional engineering strategies are discussed. [ABSTRACT FROM AUTHOR]

Summary Heavy metal accumulation in the environment poses great risks to flora and fauna. However, monitoring sites prone to accumulation poses scale and economic challenges. In this study, we present and test a method for monitoring these sites using fluorescent resonance energy transfer (FRET) change in response to zinc (Zn) accumulation in plants as a proxy for environmental health. We modified a plant Zn transport protein by adding flanking fluorescent proteins (FPs) and deploying the construct into two different species. In Arabidopsis thaliana, FRET was monitored by a confocal microscope and had a 1.4-fold increase in intensity as the metal concentration increased. This led to a 16.7% overall error-rate when discriminating between a control (1 μ m Zn) and high (10 m m Zn) treatment after 96 h. The second host plant ( Populus tremula × Populu salba) also had greater FRET values (1.3-fold increase) when exposed to the higher concentration of Zn, while overall error-rates were greater at 22.4%. These results indicate that as plants accumulate Zn, protein conformational changes occur in response to Zn causing differing interaction between FPs. This results in greater FRET values when exposed to greater amounts of Zn and monitored with appropriate light sources and filters. We also demonstrate how this construct can be moved into different host plants effectively including one tree species. This chimeric protein potentially offers a method for monitoring large areas of land for Zn accumulation, is transferable among species, and could be modified to monitor other specific heavy metals that pose environmental risks. [ABSTRACT FROM AUTHOR]

Is oxytocin the hormone of happiness? Probably not. However, this small nine amino acid peptide is involved in a wide variety of physiological and pathological functions such as sexual activity, penile erection, ejaculation, pregnancy, uterus contraction, milk ejection, maternal behavior, osteoporosis, diabetes, cancer, social bonding, and stress, which makes oxytocin and its receptor potential candidates as targets for drug therapy. In this review, we address the issues of drug design and specificity and focus our discussion on recent findings on oxytocin and its heterotrimeric G protein-coupled receptor OTR. In this regard, we will highlight the following topics: (i) the role of oxytocin in behavior and affectivity, (ii) the relationship between oxytocin and stress with emphasis on the hypothalamo–pituitary–adrenal axis, (iii) the involvement of oxytocin in pain regulation and nociception, (iv) the specific action mechanisms of oxytocin on intracellular Ca2+ in the hypothalamo neurohypophysial system (HNS) cell bodies, (v) newly generated transgenic rats tagged by a visible fluorescent protein to study the physiology of vasopressin and oxytocin, and (vi) the action of the neurohypophysial hormone outside the central nervous system, including the myometrium, heart and peripheral nervous system. As a short nine amino acid peptide, closely related to its partner peptide vasopressin, oxytocin appears to be ideal for the design of agonists and antagonists of its receptor. In addition, not only the hormone itself and its binding to OTR, but also its synthesis, storage and release can be endogenously and exogenously regulated to counteract pathophysiological states. Understanding the fundamental physiopharmacology of the effects of oxytocin is an important and necessary approach for developing a potential pharmacotherapy. [ABSTRACT FROM AUTHOR]

Abstract: The lifetime variations of enhanced cyan fluorescence protein (ECFP) in relatively short integration time bins were studied via time-correlated single photon counting (TCSPC) measurement. We observed that minimum photon counts are necessary for the lifetime estimation to achieve a certain range of variance. The conditions to decrease the variance of lifetime were investigated and the channel width of the measurement of TCSPC data was found to be another important factor for the variance of lifetime. Though the lifetime of ECFP is best fit by a double exponential, a mono exponential fit for the same integration time is more stable. The results may be useful in the analysis of photophysical dynamics for ensemble molecules in short measurement time windows. [Copyright &y& Elsevier]

Extracellular pH, especially in relatively inaccessible microdomains between cells, affects transport membrane protein activity and might have an intercellular signaling role. We have developed a genetically encoded extracellular pH sensor capable of detecting pH changes in basolateral spaces of epithelial cells. It consists of a chimerical membrane protein displaying concatenated enhanced variants of cyan fluorescence protein (ECFP) and yellow fluorescence protein (EYFP) at the external aspect of the cell surface. The construct, termed pHCECSensor01, was targeted to basolateral membranes of Madin–Darby canine kidney (MDCK) cells by means of a sequence derived from the aquaporin AQP4. The fusion of pH-sensitive EYFP with pH-insensitive ECFP allows ratiometric pH measurements. The titration curve of pHCECSensor01 in vivo had a p K a value of 6.5 ± 0.04. Only minor effects of extracellular chloride on pHCECSensor01 were observed around the physiological concentrations of this anion. In MDCK cells, the sensor was able to detect changes in pH secondary to H+ efflux into the basolateral spaces elicited by an ammonium prepulse or lactate load. This genetically encoded sensor has the potential to serve as a noninvasive tool for monitoring changes in extracellular pH microdomains in epithelial and other tissues in vivo. [ABSTRACT FROM AUTHOR]

Protein-protein binding and signaling pathways are important fields of biomedical science. Here we report simple optical methods for the determination of the equilibrium binding constant Kd of protein-protein interactions as well as quantitative studies of biochemical cascades. The techniques are based on steady-state and time-resolved fluorescence resonance energy transfer (FRET) between ECFP and Venus-YFP fused to proteins of the SUMO family. Using FRET has several advantages over conventional free-solution techniques such as isothermal titration calorimetry (ITC): Concentrations are determined accurately by absorbance, highly sensitive binding signals enable the analysis of small quantities, and assays are compatible with multi-well plate format. Most importantly, our FRET-based techniques enable us to measure the effect of other molecules on the binding of two proteins of interest, which is not straightforward with other approaches. These assays provide powerful tools for the study of competitive biochemical cascades and the extent to which drug candidates modify protein interactions. [ABSTRACT FROM AUTHOR]

Abstract: Gene expression in eukaryotes is largely controlled at the level of transcriptional initiation by gene-specific activators. Transcriptional activators stimulate the assembly of general transcription factors on the promoter to form a preinitiation complex (PIC). Such a stimulated assembly of PIC is believed to result from a direct interaction between the activator and one or more components of the transcription machinery, termed the “target”. Based primarily upon in vitro protein–protein interaction experiments, a variety of factors have been proposed to be the direct targets of activators. However, whether any of these are bona fide in vivo targets required for stimulation of PIC assembly and hence transcriptional activation remains mostly unknown, primarily because of lack of appropriate experimental methods. Using a confocal microscopy-based FRET (fluorescence resonance energy transfer) assay, we have recently identified the target of a prototypic activator, Gal4p, in living yeast cells. Here, we describe the FRET assay in general for analysis of the targets of transcriptional activators in living yeast cells. Such an assay can also be used as a general method to monitor protein–protein interactions in vivo. [Copyright &y& Elsevier]

P2X receptor function in the CNS is poorly understood, and currently available data are partly inconsistent. In the presented study, we investigated P2X 3 receptors stably expressed in HEK293 cells. Non-stationary noise analysis of whole cell currents and rapid ATP application through flash photolysis allowed for assessing the single channel conductance (6.6  pS) and the fast activation kinetics of the receptor (20  ms). The characteristics of channel desensitization and pharmacological properties matched previous findings. The properties of wild type receptors were compared with P2X 3 constructs carrying a fluorescent tag (ECFP or DsRed 2 ) at the C-terminus. These fluorescently labeled subunits formed functional receptors, with neither the affinity of the ligand binding site nor channel properties (ion selectivity, gating kinetics, single channel conductance) differing from wild type. We conclude that both fusion proteins tested here are suitable for generating transgenic mice, which can be expected to promote understanding of the physiological role of P2X 3 receptors in CNS signaling. [ABSTRACT FROM AUTHOR]

Abstract: Here, we report the properties of a FRET-based calcium indicator protein. We constructed a tandem fusion protein, named F2C, of ECFP and EYFP combined with calpain-sensitive sequences of α-spectrin, with N-terminal palmitoylation signal of GAP-43. It was previously reported that calpain cleaved a similar ECFP–EYFP fusion protein linked by a calpain-sensitive sequence of α-spectrin (fodrin). Unexpectedly, F2C was not cleaved by calpain, but demonstrated properties of a Ca2+ indicator when transiently infected in Purkinje cells of rat primary cerebellar culture or in the brainstem neurons infected in vivo using Sindbis virus encoding F2C. The emission ratio of 480nm/535nm was repeatedly increased when the intracellular Ca2+ concentration ([Ca2+]i) was raised. F2C had a Ca2+ sensitivity with an apparent dissociation constant (K d for Ca2+) of 150nM, and demonstrated kinetics that paralleled Fura-2 when [Ca2+]i was measured simultaneously. These properties of F2C are useful to be a Ca2+ indicator. [Copyright &y& Elsevier]

Abstract: The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes–macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals. [Copyright &y& Elsevier]

Organisms use synonymous codons in a highly non-random fashion. These codon usage biases sometimes frustrate attempts to express high levels of exogenous genes in hosts of widely divergent species. The Caenorhabditis elegans GluClα1 and GluClβ genes form a functional glutamate and ivermectin-gated chloride channel when expressed in Xenopus oocytes, but expression is weak in mammalian cells. We have constructed synthetic genes that retain the amino acid sequence of the wild-type GluCl channel proteins, but use codons that are optimal for mammalian cell expression. We have tagged the native and codon-optimized GluCl cDNAs with enhanced yellow fluorescent protein (EYFP, GluClα1 subunit) and enhanced cyan fluorescent protein (EFCP, GluClβ subunit), expressed the channels in E18 rat hippocampal neurons and measured the relative expression levels of the two genes with fluorescence microscopy as well as with electrophysiology. Codon optimization provides a 6- to 9-fold increase in expression, allowing the conclusions that the ivermectin-gated channel has an EC50 of 1.2 nM and a Hill coefficient of 1.9. We also confirm that the Y182F mutation in the codon-optimized β subunit results in a heteromeric channel that retains the response to ivermectin while reducing the response to 100 μM glutamate by 7-fold. The engineered GluCl channel is the first codon-optimized membrane protein expressed in mammalian cells and may be useful for selectively silencing specific neuronal populations in vivo. [Copyright &y& Elsevier]

The first effectively achieved germ-line transformations of non-drosophilid insects were based on mutant rescue of eye color phenotypes. However, for most insect species neither visible mutants nor corresponding cloned genes are available. Therefore, the development of broadly applicable and reliable transformation markers will be of great importance to fully exploit the enormous potential transgenic insect technology has to offer. Here we review transposon-mediated germ-line transformation approaches that employ green fluorescent protein (GFP) variants to identify successful gene transfer. Furthermore, we provide novel data on the use of DsRed as an additional red fluorescent transformation marker for insect transgenesis. In conclusion, fluorescent proteins controlled by suitable strong promoters possess ideal characteristics to serve as transformation markers for a wide range of insect species. [Copyright &y& Elsevier]

Many chemicals that enter the environment, food chain, and the human body can disrupt androgen-dependent pathways and mimic hormones and therefore, may be responsible for multiple diseases from reproductive to tumor. Thus, modeling and predicting androgen receptor activity is an important area of research. The aim of the current study was to find a method or combination of methods to predict compounds that can bind to and/or disrupt the androgen receptor, and thereby guide decision making and further analysis. A stepwise procedure proceeded from analysis of protein structures from human, chimp, and rat, followed by docking and subsequent ligand, and statistics based techniques that improved classification gradually. The best methods used multivariate logistic regression of combinations of chimpanzee protein structural docking scores, extended connectivity fingerprints, and naïve Bayesians of known binders and non-binders. Combination or consensus methods included data from a variety of procedures to improve the final model accuracy. [ABSTRACT FROM AUTHOR]

Recently, in order to elucidate the role of rab3B in porosome, we have observed the incorporation of rab3B in the secretion of GH through porosome under confocal laser scanning microscopy (CLSM). Transfected cells with GH-EYFP fusion protein and rab3B-ECFP fusion protein were observed under CLSM, which showed the colocalization of EYFP-GH and ECFP-rab3B in the budding configuration of secretory process. These structural and functional images of rab3B imply the incorporation of rab3B in the secretion of GH through porosome. [ABSTRACT FROM AUTHOR]

Abstract: We successfully fabricated a protein pattern using a microstructured organosilane layer fabricated by vacuum ultraviolet light (VUV) lithography and subsequently by chemical modification of organosilane layer for immobilization of proteins. An amino-terminated organosilane layer was formed using aminopropyltriethoxysilane (APTES) by the liquid method on quartz glass. This organosilane layer was patterned by VUV etching through a metal mask. The residual amino groups were chemically modified for immobilization of proteins. Fluorescent protein (ECFP) was immobilized to the chemically modified pattern, and a clear ECFP pattern was confirmed with a fluorescent microscope after the removal of non-specifically bound ECFP. This technique can be applied to almost all the proteins and will be useful for biotechnology, such as neuronal cell arrangement. [Copyright &y& Elsevier]

Genes are expressed to proteins for a wide variety of fundamental biological processes at the cellular and organismal levels. However, a protein rarely functions alone, but rather acts through interactions with other proteins to maintain normal cellular and organismal functions. Therefore, it is important to analyze the protein-protein interactions to determine functional mechanisms of proteins, which can also guide to develop therapeutic targets for treatment of diseases caused by altered protein-protein interactions leading to cellular/organismal dysfunctions. There is a large number of methodologies to study protein interactions in vitro, in vivo and in silico, which led to the development of many protein interaction databases, and thus, have enriched our knowledge about protein-protein interactions and functions. However, many of these interactions were identified in vitro, but need to be verified/validated in living cells. Furthermore, it is unclear whether these interactions are direct or mediated via other proteins. Moreover, these interactions are representative of cell- and time-average, but not a single cell in real time. Therefore, it is crucial to detect direct protein-protein interactions in a single cell during biological processes in vivo, towards understanding the functional mechanisms of proteins in living cells. Importantly, a fluorescence resonance energy transfer (FRET)-based methodology has emerged as a powerful technique to decipher direct protein-protein interactions at a single cell resolution in living cells, which is briefly described in a limited available space in this mini-review., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

In multitarget drug design, it is critical to identify active and inactive compounds against a variety of targets and antitargets. Multitarget strategies thus test the limits of available technology, be that in screening large databases of compounds vs. a large number of targets, or in using in silico methods for understanding and reliably predicting these pharmacological outcomes. In this paper, we have evaluated the potential of several in silico approaches to predict the target, antitarget and physicochemical profile of ( S )-blebbistatin, the best-known myosin II ATPase inhibitor, and a series of analogs thereof. Standard and augmented structure-based design techniques could not recover the observed activity profiles. A ligand-based method using molecular fingerprints was, however, able to select actives for myosin II inhibition. Using further ligand- and structure-based methods, we also evaluated toxicity through androgen receptor binding, affinity for an array of antitargets and the ADME profile (including assay-interfering compounds) of the series. In conclusion, in the search for ( S )-blebbistatin analogs, the dissimilarity distance of molecular fingerprints to known actives and the computed antitarget and physicochemical profile of the molecules can be used for compound design for molecules with potential as tools for modulating myosin II and motility-related diseases.

Eukaryotic cells consist of a complex network of thousands of proteins present in different organelles where organelle-specific cellular processes occur. Identification of the subcellular localization of a protein is important for understanding its potential biochemical functions. In the post-genomic era, localization of unknown proteins is achieved using multiple tools including a fluorescent-tagged protein approach. Several fluorescent-tagged protein organelle markers have been introduced into dicot plants, but its use is still limited in monocot plants. Here, we generated a set of multicolored organelle markers (fluorescent-tagged proteins) based on well-established targeting sequences. We used a series of pGWBs binary vectors to ameliorate localization and co-localization experiments using monocot plants. We constructed different fluorescent-tagged markers to visualize rice cell organelles, i.e., nucleus, plastids, mitochondria, peroxisomes, golgi body, endoplasmic reticulum, plasma membrane, and tonoplast, with four different fluorescent proteins (FPs) (G3GFP, mRFP, YFP, and CFP). Visualization of FP-tagged markers in their respective compartments has been reported for dicot and monocot plants. The comparative localization of the nucleus marker with a nucleus localizing sequence, and the similar, characteristic morphology of mCherry-tagged Arabidopsis organelle markers and our generated organelle markers in onion cells, provide further evidence for the correct subcellular localization of the Oryza sativa (rice) organelle marker. The set of eight different rice organelle markers with four different FPs provides a valuable resource for determining the sub-cellular localization of newly identified proteins, conducting co-localization assays, and generating stable transgenic localization in monocot plants.

This article supplies raw data related to a research article entitled "Joint refinement of FRET measurements using spectroscopic and computational tools" (Kyrychenko et al., 2017) [1], in which we demonstrate the use of molecular dynamics simulations to estimate FRET orientational factors in a benchmark donor-linker-acceptor system of enhanced cyan (ECFP) and enhanced yellow (EYFP) fluorescent proteins. This can improve the recalculation of donor-acceptor distance information from single-molecule FRET measurements.

A-Kinase Anchoring Proteins (AKAPs) direct the flow of cellular information by positioning multiprotein signaling complexes into proximity with effector proteins. However, certain AKAPs are not stationary but can undergo spatiotemporal redistribution in response to stimuli. Gravin, a 300kD AKAP that intersects with a diverse signaling array, is localized to the plasma membrane but has been shown to translocate to the cytosol following the elevation of intracellular calcium ([Ca(2+)]i). Despite the potential for gravin redistribution to impact multiple signaling pathways, the dynamics of this event remain poorly understood. In this study, quantitative microscopy of cells expressing gravin-EGFP revealed that Ca(2+) elevation caused the complete translocation of gravin from the cell cortex to the cytosol in as little as 60s of treatment with ionomycin or thapsigargin. In addition, receptor mediated signaling was also shown to cause gravin redistribution following ATP treatment, and this event required both [Ca(2+)]i elevation and PKC activation. To understand the mechanism for Ca(2+) mediated gravin dynamics, deletion of calmodulin-binding domains revealed that a fourth putative calmodulin binding domain called CB4 (a.a. 670-694) is critical for targeting gravin to the cell cortex despite its location downstream of gravin's membrane-targeting domains, which include an N-terminal myristoylation site and three polybasic domains. Finally, confocal microscopy of cells co-transfected with gravin-EYFP and PKA RII-ECFP revealed that gravin redistribution mediated by ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localized at the cell cortex. Our results support the hypothesis that gravin redistribution regulates cross-talk between PKA-dependent signaling and receptor-mediated events involving Ca(2+) and PKC., (© 2013.)

Ectopic coexpression of the two chains of the Type I and Type III interferon (IFN) receptor complexes (IFN-αR1 and IFN-αR2c, or IFN-λR1 and IL-10R2) yielded sensitivity to IFN-alpha or IFN-lambda in only some cells. We found that IFN-αR1 and IFN-αR2c exhibit FRET only when expressed at equivalent and low levels. Expanded clonal cell lines expressing both IFN-αR1 and IFN-αR2c were sensitive to IFN-alpha only when IFN-αR1 and IFN-αR2c exhibited FRET in the absence of human IFN-alpha. Coexpression of RACK-1 or Jak1 enhanced the affinity of the interaction between IFN-αR1 and IFN-αR2c. Both IFN-αR1 and IFN-αR2c exhibited FRET with Jak1 and Tyk2. Together with data showing that disruption of the preassociation between the IFN-gamma receptor chains inhibited its biological activity, we propose that biologically active IFN receptors require ligand-independent juxtaposition of IFN receptor chains assisted by their associated cytosolic proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Inositol 1,4,5-trisphosphate (IP₃) receptors consist of three subtypes: IP₃R1, IP₃R2, and IP₃R3. Although numerous IP₃ receptor ligands have been synthesized, none of the subtype-selective ligands are known. We have developed a simple fluorescence method to examine the subtype selectivity of IP₃ receptor ligands using FRET-based IP₃ biosensors LIBRAvI, LIBRAvII, and LIBRAvIII. The addition of IP₃ or adenophostin A (ADA) to permeabilized biosensor-expressing cells increased the fluorescence ratios of these biosensors in a concentration-dependent manner, and the potency of ADA relative to that of IP₃ in terms of the changes in the fluorescence ratios of LIBRAvI, LIBRAvII, and LIBRAvIII was 43-, 22-, and 28-fold, respectively. This fluorescence-based method further showed that several ADA analogs had significant differences with respect to subtype selectivity and potency. These results highlight the important role played by the O-glycosidic structure of ADA in the selectivity of the ligands for IP₃R1, as evidenced by the modified selectivity following replacement of the 5'-hydroxyl with a phenyl or phenethyl group. We also found that one ADA analog 5'-deoxy-5'-phenyladenophostin A possessed a partial agonistic effect on IP₃R1. Together, the novel fluorescent methods described herein are useful for the evaluation of properties of IP₃R ligands, including potency, efficacy, and subtype selectivity., (Copyright © 2013 Elsevier B.V. All rights reserved.)