Label-free analysis of membrane protein binding kinetics and cell adhesions using evanescent scattering microscopy.

Measuring ligand interactions with membrane proteins in single live cells is critical for understanding many cellular processes and screening drugs. However, developing such a capability has been a difficult challenge. Here, we employ evanescent scattering microscopy (ESM) to show that ligand binding to membrane proteins can change the cell adhesion properties, which are intrinsic cell properties and independent of random cell micromotions and ligand mass, thus allowing the kinetics analyses of both proteins and small molecules binding to membrane proteins in both single fixed and live cells. In addition, utilizing the high spatiotemporal resolution of ESM, the positions of cell adhesion sites can be tracked in real-time to analyze the cell deformations and migrations, thus providing a potential approach for understanding the cell activity during the ligand binding process in detail. The presented method may pave the road for developing a versatile and easy-to-use label-free detection strategy for in situ analysis of molecular interaction dynamics in living biosystems with single-cell resolution.

Design, synthesis and bioevaluation of 2,7-diaryl-pyrazolo[1,5-a]pyrimidines as tubulin polymerization inhibitors.

Two series of 2,7-diaryl-pyrazolo[1,5-a]pyrimidines as tubulin polymerization inhibitors were designed to restrict bioactive configuration of (E,Z)-vinylogous CA-4. All of the target compounds were synthesized and then evaluated for their in vitro antiproliferative activities against three cancer cell lines (MCF-7, SGC-7901 and A549). Among them, 6d exhibited the most potent antiproliferative activity against the MCF-7 with IC50 value of 0.047 µM. Moreover, 6d significantly inhibited tubulin polymerization, disrupted microtubule networks, arrested cell cycle at G2/M phase, induced apoptosis and hindered cancer cell migration. Colchicine competition assay and molecular docking studies suggested that 6d could interact with tubulin by binding to the colchicine site.

One-pot sample preparation approach for profiling spatial distribution of gibberellins in a single shoot of germinating cereal seeds.

Sample preparation remains a bottleneck in the rapid and reliable quantification of gibberellins (GAs) for obtaining an insight into the physiological processes mediated by GAs. The challenges arise from not only the extremely low content of GAs in complex plant matrices, but the poor detectability of GAs by mass spectrometry (MS) in negative ion mode. In an effort to solve these urgent difficulties, we present a spatial-resolved analysis method to investigate the distribution of GAs in tiny plant tissues based on a simplified one-pot sample preparation approach coupled with ultrahigh-performance liquid chromatography-tandem MS. By integrating extraction and derivatization into one step, target GAs were effectively extracted from plant materials and simultaneously reacted with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, the sample preparation time was largely shortened, the probability of sample loss was minimized and the detection sensitivity of MS was also greatly improved compared with underivatized GAs. Under optimal conditions, the method was validated from the quantification linearity, limits of detection and limits of quantification in the presence of plant matrices, recoveries, and precision. With the proposed method, 15 endogenous GAs were detected and, among these, 11 GAs could be quantified in 0.50 mg fresh weight (FW) wheat shoot samples, and five GAs were quantified in only 0.15 mg FW developing seed samples of Arabidopsis thaliana. The distribution patterns of GAs along both the non-13-hydroxylation pathway and the early 13-hydroxylation pathway in a single shoot of germinating wheat, rice and maize seeds were finally profiled with a spatial resolution down to approximately 1 mm2 .

A bi-end injection capillary electrophoresis method for simultaneous determination of 37 cations and anions in beers.

Capillary electrophoresis (CE) is excellent at separating all the ions in a sample but is rarely used as a result of its detection issue and easy loss of very fast ions by common one-end injection methods. Herein we propose a newly developed method aimed at simultaneous determination of positive and negative ions with a home-made CE device, featuring bi-end injection and contactless conductivity detection at the middle. By simply using 2.5 M acetic acid as a running buffer, the method can separate 37 ions (3 inorganic anions, 8 inorganic cations, 10 biogenic amines, and 16 amino acids) per run, with linearity between 10 and 2000 µM (R2 > 0.99), limit of detection of 1.0-16.6 µM, and limit of quantification of 2.3-31.7 µM. The recovery measured by spiking standards into samples at high, middle, and low levels was between 73% and 110%. The intra- and interday repeatability of the 37 analytes ranged from 0.69% to 8.97% and from 0.68% to 11.04%, respectively. The proposed method was evaluated by analysis of 21 beers and, in addition to acquiring the concentration information, the brands of the tested beers were distinguished. This method is of high throughput, fast, and cost-effective. It could be a promising tool for ionomic analysis. Graphical abstract ᅟ.

Background subtraction via time continuity and texture consistency constraints.

Background subtraction has attracted enormous interest in the field of moving object detection. However, when there are complex scenarios such as illumination changes, dynamic background, and noise, the moving object area obtained by background subtraction often has holes, noise, and shadows. This paper proposes a novel background update model based on matrix factorization, which uses the temporal continuity of video content to solve the problems of holes, noise, and shadows. Moreover, in some cases, the texture consistency of the object is also a factor worth considering. The neighborhood weighed local binary pattern (NWLBP) is introduced to optimize the background update model, which is very effective for suppressing background or foreground shadow. The effectiveness of our method is confirmed by extensive experiments on public data sets and real shot video. Compared with the existing state-of-the-art moving object detection methods, the proposed methods can accurately establish the background model and locate the moving object region robustly.

Quantification of near-attomole gibberellins in floral organs dissected from a single Arabidopsis thaliana flower.

There remains a methodological bottleneck in the quantification of ultra-trace plant hormones in very tiny plant organs at fresh weights below a milligram. The challenge becomes even more serious in the determination of endogenous gibberellins (GAs), which are a class of compounds that are difficult to separate and detect. Herein, a quantification method using ultra-high-performance liquid chromatography-tandem mass spectrometry was developed, combined with a derivatization technique in which GAs react with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide in ethanol. The method was validated as capable of determining GAs in floral organs (about 80-250 µg) - pistil, stamens, petals, sepals and receptacle - which were dissected from only one flower of Arabidopsis thaliana. Substantially different abundance patterns of GAs were measured from the floral organs at floral stages 13, 14 and 15 along the non-13-hydroxylation pathway and the early 13-hydroxylation pathway in plants. This allows sub-flower-level insights into how GAs affect floral development. The method exhibited excellent limit of detection and limit of quantification down to 5.41 and 18.0 attomole, respectively, and offered a fairly wide linear range from 0.01 to 25 femtomole with linear coefficients above 0.9961. The precision of the method was evaluated with relative standard deviations below 10.6% for intra-day and 11.4% for inter-day assays, and recoveries ranged from 64.0% to 107%.

Facile derivatization of ultratrace carboxylic acids in saliva for quantification by HPLC-MS/MS.

It remains an issue to directly quantify trace biologically important carboxyl compounds in body fluids. Herein we propose an innovative method to determine α-lipoic acid, 2-(ß-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman, prostaglandin E2, cholic acid, and chenodeoxycholic acid in saliva. The method consists of two successive steps: fast and direct labeling of the target analytes with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide followed by ultrahigh-performance liquid chromatography-tandem mass spectrometry analysis. The method exhibited a wide linear range from 2.5 to 2500 pg/mL, with linear coefficients greater than 0.9963 and limits of detection and quantification as low as 0.10 and 0.33 pg/mL, respectively. The method precision was evaluated, with relative standard deviations ranging from 2.12% to 10.63% for intraday assays and from 2.98% to 12.88% for interday assays. The recoveries were measured by our spiking saliva samples with standards at three different levels, and ranged from 72.5% to 98.0%. Real applicability was validated by direct quantification of trace target analytes in human saliva, with simple pretreatment, use of a small sample volume, and a short analysis time. Graphical abstract Sequential steps to extract, label, and determine the ultratrace carboxylic acids in saliva. CDCA chenodeoxycholic acid, γ-CEHC 2-(ß-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman, α-LA α-lipoic acid, PGE2 prostaglandin E2, UHPLC-MS/MS ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Generic lures attract cerambycid beetles in a tropical montane rain forest in southern China.

The attraction of cerambycid beetles to 10 known cerambycid pheromones was tested in a tropical montane rain forest in southern China. From 28 May to 25 June 2010, 1,526 cerambycids representing 71 species were captured in pheromone-baited traps, with 14 species accounting for 92% of the specimens. Test compounds with a 3-hydroxyalkan-2-one or 2,3-alkanediol motif attracted significant numbers of both sexes for eight species in the subfamily Cerambycinae, including species of Demonax, Rhaphuma, and Xylotrechus. Rhaphuma horsfieldi (White) was the only species that was strongly attracted to more than one test compound, with significant attraction to both (2R*,3R*)-2,3-hexanediol and (2R*,3R*)-2,3-octanediol. Within the Lamiinae, males and females of five species were significantly attracted to 2-(undecyloxy) ethanol, including Acalolepta formosana (Breuning), Monochamus bimaculatus Gahan, Pharsalia subgemmata (Thomson), Pseudomacrochenus antennatus (Gahan), and Xenohammus bimaculatus Schwarzer. Only male Megopis costipennis White (Prioninae) were significantly attracted to (2R*,3S*)-2,3-octanediol, suggesting that this compound may be a sex pheromone component for this species. To date, 2,3-octanediols have only been reported as aggregation pheromone components for cerambycids in the subfamily Cerambycinae. Our results support the hypothesis that both closely related (congeners) and more distantly related cerambycid species (different tribes and even subfamilies) may share pheromone components. Our results also demonstrate that traps baited with even a limited number of different classes of pheromones may be useful tools for surveying cerambycid diversity, as well as for detecting and monitoring particular species, especially those that have the potential to be invasive pests in other parts of the world.

[Progress of highly reproducible capillary electrophoresis].

Following rapid developments in capillary electrophoresis (CE), this technology has become an established analytical technique owing to its microscale characteristics, high speed, high efficiency, and versatility. However, the challenges of poor peak stability and/or reproducibility have consistently hindered its wider applications. CE has long been used as a measurement tool for plotting signal intensities versus the migration time; however, the migration time is not an independent variable in CE, but is affected by many direct and indirect parameters, including capillary (length, diameter, and inner surface properties), electric field (or voltage, current, and/or power), temperature, and running buffer (electrolytes, additives, solvents, and their concentration, buffering pH, etc.). These intricacies render the acquisition of reproducible electropherograms difficult. Various studies ranging from those on the early stages of CE development to those on the exploration of three important strategies have been conducted to address this issue. In the first strategy, the CE conditions, especially those parameters that can maintain a stable electro-osmotic flow, are strictly controlled and stabilized to significantly improve peak repeatability. In the second strategy, either the peak position is corrected using internal standards or the peak time is converted into other variables, such as electrophoretic mobility, to offset or eliminate some unstable factors, thereby improving the repeatability and even reproducibility of the peaks; this strategy is useful when plotting signals versus the migration time ratio, correlated migration time, effective mobility, or temperature-correlated mobility. In the third strategy, a new methodology called highly reproducible CE (HRCE) is established using theoretical studies to explore better principles for real-time CE with the aim of the complete removal of the challenge from the root. This strategy includes the development of novel methods that plot electropherograms based on weighted mobility, migrated charge, charge density, or partial differential molar charge density. Similar to ordinary CE approaches, this strategy can also draw electropherograms based on the ratios of these properties. As theoretically predicted, these novel methods can offset or resist changes in critical CE conditions (mainly electric field strength, capillary length and diameter, and/or some buffer parameters such as concentration). Our experimental results demonstrate that given certain prerequisites, a new set of methods can produce highly reproducible electropherograms. This review focuses on the theoretical basis and advancements of HRCE, and elucidates the link between electrophoretic migration/peak expression theories and their impact on reproducibility. Studies on the transformation of time-scale electropherograms in the CE literature are summarized and analyzed in general. However, this review does not directly discuss research on and progress in improving CE repeatability or reproducibility through instrument upgrades, parameter optimization, or practical method refinements.

Stability assessment of surrounding rock in downward mining route supported by slab-wall backfill structure.

Characteristic of ground pressure in surrounding rock is generally considered as the theoretical basis of parameter optimization for stope structure and technology. To explore the feasibility of efficient method for the second-step downward route backfill stopes in Shanjin gold mine, various numerical simulation methods were used to investigate the effect of slab-wall backfill structure on stability of surrounding rock in downward route mining system. The maximum principal stress, artificial false roof stress, and displacement were analyzed to evaluate the level of ground pressure in different mining areas. These results indicate the optimized structural parameters for backfill stopes, which may also provide a low-cost way to achieve a high safety for downward route mining system.

Solar-assisted selective separation and recovery of precious group metals from deactivated air purification catalysts.

The mitigation of environmental and energy crises could be advanced by reclaiming platinum group precious metals (PGMs) from decommissioned air purification catalysts. However, the complexity of catalyst composition and the high chemical inertness of PGMs significantly impede this process. Consequently, recovering PGMs from used industrial catalysts is crucial and challenging. This study delves into an environmentally friendly approach to selectively recover PGMs from commercial air purifiers using photocatalytic redox technology. Our investigation focuses on devising a comprehensive strategy for treating three-way catalysts employed in automotive exhaust treatment. By meticulously pretreating and modifying reaction conditions, we achieved noteworthy results, completely dissolving and separating rhodium (Rh), palladium (Pd), and platinum (Pt) within a 12-h time frame. Importantly, the solubility selectivity persists despite the remarkably similar physicochemical properties of Rh, Pd, and Pt. To bolster the environmental sustainability of our method, we harness sunlight as the energy source to activate the photocatalysts, facilitating the complete dissolution of precious metals under natural light irradiation. This eco-friendly recovery approach demonstrated on commercial air purifiers, exhibits promise for broader application to a diverse range of deactivated air purification catalysts, potentially enabling implementation on a large scale.

Transferable multi-modal fusion in knee angles and gait phases for their continuous prediction.

Objective.The gait phase and joint angle are two essential and complementary components of kinematics during normal walking, whose accurate prediction is critical for lower-limb rehabilitation, such as controlling the exoskeleton robots. Multi-modal signals have been used to promote the prediction performance of the gait phase or joint angle separately, but it is still few reports to examine how these signals can be used to predict both simultaneously.Approach.To address this problem, we propose a new method named transferable multi-modal fusion (TMMF) to perform a continuous prediction of knee angles and corresponding gait phases by fusing multi-modal signals. Specifically, TMMF consists of a multi-modal signal fusion block, a time series feature extractor, a regressor, and a classifier. The multi-modal signal fusion block leverages the maximum mean discrepancy to reduce the distribution discrepancy across different modals in the latent space, achieving the goal of transferable multi-modal fusion. Subsequently, by using the long short-term memory-based network, we obtain the feature representation from time series data to predict the knee angles and gait phases simultaneously. To validate our proposal, we design an experimental paradigm with random walking and resting to collect data containing multi-modal biomedical signals from electromyography, gyroscopes, and virtual reality.Main results.Comprehensive experiments on our constructed dataset demonstrate the effectiveness of the proposed method. TMMF achieves a root mean square error of0.090±0.022s in knee angle prediction and a precision of83.7±7.7% in gait phase prediction.Significance.We demonstrate the feasibility and validity of using TMMF to predict lower-limb kinematics continuously from multi-modal biomedical signals. This proposed method represents application potential in predicting the motor intent of patients with different pathologies.

Separation and determination of chiral composition in penicillamine tablets by capillary electrophoresis in a broad pH range.

A chiral capillary electrophoretic method with nearly full pH window was explored for the separation and determination of dl-penicillamine. A facile one-pot labeling technique was coupled in the method for introduction of chromophore and charge groups onto the analytes to facilitate the electromigration and sensitive detection. By using simply a cost-effective neutral ß-cyclodextrin as chiral selector, baseline separation of the dl-penicillamine was achieved from pH 2.0 to over pH 10. Quantification of standard d- and l-penicillamines was demonstrated by taking pH 4.5, 7.4, and 9.7 as the representatives of acidic, neutral, and basic conditions. The working curves were constructed between peak area and concentration, having linear ranges of 8.56-8.56 × 10(2) µg/mL for pH 4.5 and 8.56-1.71 × 10(3) µg/mL for pH 7.4 and 9.7, with correlation coefficients all better than 0.999. The limit of detection (S/N = 3) was 2.58 µg/mL in acidic and neutral conditions or 1.41 µg/mL in basic condition. The method was further validated by assaying the commercial penicillamine tablets, applicable to quantification of the effective enantiomer and the trace impurity of l-penicillamine at a content of down to 0.2, 0.6, and 2.0% for pH 9.7, 4.5, and 7.4, respectively. The recovery determined by spiking technique was in a range from 93.1 to 105 %. The method is easily extendable to the analysis of other chiral amines or amino acids.

Capillary array electrophoresis imaging of biochemicals in tissue sections.

It is of great significance to reveal the molecular distribution images in biological tissues, which has led to the bloom of mass spectrometry imaging. Unfortunately, its application is encountering the resistance of high technical barriers and equipment cost, as well as the inability to image substances that cannot be desorbed or ionized, or cannot be separated by their mass-to-charge ratios. Herein presented is a complementary and cost-effective method called capillary array electrophoresis (CAE) imaging. To have the information of molecules and their spatial location, a gridding cutter was fabricated to orderly dissect a tissue section into a leakproof array of micro wells enclosed by the grid-blade arrays. After in situ extraction and fluorophore-labeling of analytes, the samples in the wells were directly subjected to CAE-LIF (laser-induced fluorescence), and the molecular distribution images were depicted with the separated peaks. The practicability was demonstrated by CAE imaging of rat brain tissue sections with amino acid neurotransmitters (e.g., glutamine, 4-aminobutyric acid, alanine, glutamic acid and aspartic acid) as targets. The resultant images showed the global differences of molecular distributions, with a spatial resolution of 1000 µm that was presently determined by the well width but ultimately by the bore size of capillary (down to 10-50 µm). CAE imaging can hence be promising for its low cost, low technical barriers and abundant mechanisms to separate the charged and non-charged, chiral and non-chiral substances.

A substantial increase of analytical throughput in capillary electrophoresis throughput by separation-interrupted sequential injections.

How to further improve the throughput of capillary electrophoresis (CE) is a fascinating question. Herein an idea to substantially increase the throughput of CE has been proposed together with theory and experimental demonstration. The key is to introduce samples for CE, one after another, by a short suspension of voltage application, which was hence termed separation-interrupted sequential injections (Sisi). The idea was demonstrated to be feasible on a laboratory-built CE instrument coupled with tandem C4D (contactless capacitively-coupled conductivity) detectors. At least 50 injections of a testing sample (mixture of NH4+, K+, Ca2+, Na+ and Mg2+) were successfully separated in only a single run. The separation took 145 min in total, equivalent to 2.9 min per analysis which is only 21% of that of normal CE. Quantification of the separated ions was performed, with a limit of detection of 1.1-2.6 µM, a limit of quantification of 3.2-8.9 µM, and a linear range up to 1000 µM (R2 > 0.99). The recovery was between 88% and 112% measured by spiking standards into samples at low, middle and high levels. The real applicability of Sisi-CE was evaluated by direct injection and analysis of 45 mineral water samples also in a single run. Its clinical application potential was demonstrated by high throughput assay of the calcium and zinc gluconate oral solution formula, and the blood potassium of hyperkalemia and hypokalemia from patients with renal failure disease. This method can be extended to other applications such as omics studies through the use of more suitable detectors. The theory proposed may also be applicable to other high throughput methods.

Electromigration diffusivity spectrometry: a way for simultaneous determination of diffusion coefficients from mixed samples.

A novel method was proposed for simultaneous measurement of diffusion coefficients, (D), from mixed samples by electrophoresis and termed electromigration-based diffusivity spectrometry. After theoretical treatment, D-equation for practical use has been deduced. With a modified CE system built in laboratory, electromigration-based diffusivity spectrometry has been realized and validated to suit for fast and accurate determination of diffusivities of mixed aromatic amino acids, phenols and aromatic organic acid, giving diffusivity spectra by peak area versus D, much similar to mass spectra. The precision of the measurement was found to critically depend on pH value of running buffer, which should be so selected that the analytes and internal standards could be charged at above 0.5e. The standards have to be selected at an electric flux far from each other and from analytes. In these cases, sample and running buffer concentrations, voltage and system temperature were found to have only negligible impact on the determination. In our test, the obtained measuring precision was generally kept within 1% for five runs, and the measured values of D agreed well with those from literature, with a deviation of less than 2.2% after the right use of calibration standards.

A stable version of capillary electrophoresis for determining human hemoglobin chains aiming at the screening and diagnosis of thalassemia.

As a fast, high-performance and cost-effective separation technique, capillary electrophoresis (CE) is applicable to the screening and diagnosis of diseases such as thalassemia. However, it is often not preferred due to its unrepeatable and/or irreproducible migration times. Herein, we propose a stable version of CE that uses migration charge density instead of the migration time to plot the electropherogram. The peak position is now independent of the applied voltage or current and the capillary geometry and is also insensitive to temperature. Its applicability was demonstrated in the quantitative analysis of human hemoglobin. On a laboratory-built device, with a running buffer simply consisting of 3.0 M acetic acid and 0.1% (w/v) hydroxyethyl cellulose, it allows a direct injection of whole blood samples and all the concerned globin chains, α, ß, Aγ and Gγ can be well separated in 15 minutes. The resolution of α/ß, ß/Aγ, and Aγ/Gγ reached 4.4, 3.1 and 5.3, respectively. The intra- and inter-day precisions for the peak position based on the migration charge densities were below 0.6%. Its diagnostic applicability was validated in the analysis of several real blood samples from newborns, children and adults, and its capacity was demonstrated to screen and define the type of thalassemia.

Sample preparation.

A panorama of sample preparation methods has been composed from 481 references, with a highlight of some promising methods fast developed during recent years and a somewhat brief introduction on most of the well-developed methods. All the samples were commonly referred to molecular composition, being extendable to particles including cells but not to organs, tissues and larger bodies. Some criteria to evaluate or validate a sample preparation method were proposed for reference. Strategy for integration of several methods to prepare complicated protein samples for proteomic studies was illustrated and discussed.

Degree of supersaturation-regulated chiral symmetry breaking in one crystal.

This paper aimed at studying chiral symmetry-breaking phenomena in one crystal. Preferential crystallization of racemic asparagines was carried out in nonseeded stagnant solutions through slow cooling. By varying the supersaturation, only one transparent crystal could be obtained at enough low supersaturation of dl-asparagine, and the crystal was not pure enantiomer with crystal enantiomeric excess increasing inversely with the degree of supersaturation. Crystal enantiomeric excess can amount up to 85% in one transparent crystal. Because no secondary nucleation occurred except for stochastic primary nucleation, we suggest that primary nucleation and competition between l- and d-nuclei were considered to be a mechanism for asymmetry amplification. High-performance capillary electrophoresis coupled with laser-induced fluorescence was used to separate and quantify l- and d-asparagine and the enantiomeric excess value can be calculated according to their concentration.

[Recent advancement of photonic-crystal-based analytical chemistry].

Photonic crystals are a type of novel materials with ordered structure, nanopores/channels and optical band gap. They have hence important applications in physics, chemistry, biological science and engineering fields. This review summarizes the recent advancement of photonic crystals in analytical chemistry applications, with focus on sensing and separating fields happening in the nearest 5 years.