Actin-nucleation promoting factor N-WASP influences alpha-synuclein condensates and pathology.

Abnormal intraneuronal accumulation of soluble and insoluble α-synuclein (α-Syn) is one of the main pathological hallmarks of synucleinopathies, such as Parkinson's disease (PD). It has been well documented that the reversible liquid-liquid phase separation of α-Syn can modulate synaptic vesicle condensates at the presynaptic terminals. However, α-Syn can also form liquid-like droplets that may convert into amyloid-enriched hydrogels or fibrillar polymorphs under stressful conditions. To advance our understanding on the mechanisms underlying α-Syn phase transition, we employed a series of unbiased proteomic analyses and found that actin and actin regulators are part of the α-Syn interactome. We focused on Neural Wiskott-Aldrich syndrome protein (N-WASP) because of its association with a rare early-onset familial form of PD. In cultured cells, we demonstrate that N-WASP undergoes phase separation and can be recruited to synapsin 1 liquid-like droplets, whereas it is excluded from α-Syn/synapsin 1 condensates. Consistently, we provide evidence that wsp-1/WASL loss of function alters the number and dynamics of α-Syn inclusions in the nematode Caenorhabditis elegans. Together, our findings indicate that N-WASP expression may create permissive conditions that promote α-Syn condensates and their potentially deleterious conversion into toxic species.

Structuring electronic dental records through deep learning for a clinical decision support system.

Extracting information from unstructured clinical text is a fundamental and challenging task in medical informatics. Our study aims to construct a natural language processing (NLP) workflow to extract information from Chinese electronic dental records (EDRs) for clinical decision support systems (CDSSs). We extracted attributes, attribute values, and tooth positions based on an existing ontology from EDRs. A workflow integrating deep learning with keywords was constructed, in which vectors representing texts were unsupervised learned. Specifically, we implemented Sentence2vec to learn sentence vectors and Word2vec to learn word vectors. For attribute recognition, we calculated similarity values among sentence vectors and extracted attributes based on our selection strategy. For attribute value recognition, we expanded the keyword database by calculating similarity values among word vectors to select keywords. Performance of our workflow with the hybrid method was evaluated and compared with keyword-based method and deep learning method. In both attribute and value recognition, the hybrid method outperforms the other two methods in achieving high precision (0.94, 0.94), recall (0.74, 0.82), and F score (0.83, 0.88). Our NLP workflow can efficiently structure narrative text from EDRs, providing accurate input information and a solid foundation for further data-based CDSSs.

Hydrogel-based microbeads for Raman-encoded suspension array using the reversed-phase suspension polymerization method and ultraviolet light curing.

A one-step synthesis using the reversed-phase suspension polymerization method and ultraviolet light curing is proposed for preparing the Raman-encoded suspension array (SA). The encoded microcarriers are prepared by doping the Raman reporter molecules into an aqueous phase, and then dispersing the aqueous phase in an oil phase and curing by ultraviolet light irradiation. The multiplexed biomolecule detection and various concentration experiments confirm the qualitative and quantitative analysis capabilities of the Raman-encoded SA with a limit of detection of 52.68 pM. The narrow bandwidth of the Raman spectrum can achieve a large number of codes in the available spectral range and the independence between the encoding channel and the fluorescent label channel provides the encoding method with high accuracy. This preparation method is simple and easy to operate, low in cost, and high in efficiency. A large number of hydrogel-based encoding microbeads could be quickly obtained with good biocompatibility. Most importantly, concentrating plenty of Raman reporter molecules inside the microbeads increases the signal intensity and means the molecular assembly is not limited by the functional groups; thus, the types of materials available for Raman encoding method are expanded. Furthermore, the signal intensity-related encoding method is verified by doping different proportions of Raman reporter molecules with our proposed synthesis method, which further increases the detection throughput of Raman-encoded SA. Graphical Abstract.

Weak measurement-based sensor for the rapid identification of L(+)-ascorbic acid and D(-)-isoascorbic acid.

The ability to identify L(+)-ascorbic acid from D(-)-isoascorbic acid in medicinal products is of practical interest. Based on the method of frequency domain weak measurement, a set of common optical path sensors for identification of L(+)-ascorbic acid and D(-)-isoascorbic acid is established. By quantificationally analyzing the magnitude and offset direction of the spectral central wavelength, a good identification of the concentration and the optically active forms of ascorbic acid has been achieved. The sensitivity and resolution of the sensor for optical rotation can reach 34.35 nm/° and ${5.53} \times {{10}^{ - 5}}^\circ $5.53×10-5 ∘, respectively. The detection resolution for L(+)-ascorbic acid is ${2.00} \times {{10}^{ - 4}}\;{\rm mol}/{\rm mL}$2.00×10-4mol/mL, and that for D(-)-isoascorbic acid is ${2.73} \times {{10}^{ - 4}}\;{\rm mol}/{\rm mL}$2.73×10-4mol/mL. The potential of the sensor in the detection of transparent but optically inactive impurities has been verified by comparative experiments of sodium chloride solution. The sensor also has been applied to identify medicinal vitamin C tablets, which verified the feasibility of the method in optically active pharmaceutical solutions with water-insoluble, optically inactive impurities. Since the sensor has the advantages of high precision, real-time, high robustness, and being non-destructive, it has a great prospect in the field of drug detection containing chiral molecules.

Detection of Macromolecular Content in a Mixed Solution of Protein Macromolecules and Small Molecules Using a Weak Measurement Linear Differential System.

In this paper, we propose a linear differential detection system based on a frequency domain weak measurement. The system can be used for detecting optical substances. Moreover, we completed an experiment to detect himan serum albumin (HSA) content in a mixture of human serum albumin and l-proline via dialysis. This work also proves the differential function of the system. This experiment can be further extended to detecting protein content in a mixed solution that contains protein macromolecules and various small molecules. It is very important for detecting molecules without photomarking in solutions of complex biological samples. In this paper, the system has an optical resolution of 1.39 × 10-5, and resolution of 4.06 × 10-8 mol/L for himan serum protein solution.

Gold-nanorod-enhanced Raman spectroscopy encoded micro-quartz pieces for the multiplex detection of biomolecules.

The rapid analysis and detection of biomolecules has become increasingly important in biological research. Hence, here we propose a novel suspension array method that is based on gold nanorod (AuNR)-enhanced Raman spectroscopy and uses micro-quartz pieces (MQPs) as microcarriers. AuNRs and Raman reporter molecules are coupled together by Au-S bonds to obtain surface-enhanced Raman scattering labels (SERS labels). The SERS labels are then assembled on the surfaces of the MQPs via electrostatic interactions, yielding encoded MQPs. Experimental results showed that the encoded MQPs could be decoded using a Raman spectrometer. A multiplex immunoassay experiment demonstrated the validity and specificity of these encoded MQPs when they were used for bioanalysis. In concentration gradient experiments, the proposed method was found to give a linear concentration response to the target biomolecule at target concentrations of 0.46875-30 nM, and the detection limit was calculated to be 1.78 nM. The proposed method utilizes MQPs as carriers rather than conventional microbeads, which allows the interference caused by the background fluorescence of microbeads to be eliminated. The fluorescence of the encoded MQPs can be simply, rapidly, and inexpensively quantified using fluorescence microscopy. By dividing the quantitative and qualitative detection of biomolecules into two independent channels, crosstalk between the encoded signal and the labeled signal is averted and high decoding accuracy and detection sensitivity are guaranteed. Graphical abstract.

Ion-chelation based digital barcodes for multiplexing of a suspension array.

Ion-chelated microbeads (ICMs) for suspension arrays can be prepared by chelating metal ions (MIs), which are used as encoding materials. Stimulating the ICMs, laser induced breakdown spectra (LIBs) can be obtained and the atomic spectra of the chelated ions are chosen as the decoding signals. Our ICMs show digital characteristics with high stability due to the properties of LIBs. And, since there are many available coding materials and different kinds of coding materials can be easily combined, the coding capacity can be considerably enlarged. Further, the background interference in fluoroimmunoassay detection could be avoided, because the ICMs contain no fluorescence emission. In our studies, we achieved a total of 15 types of barcodes by taking full advantage of 4 kinds of ions, then a fluoroimmunoassay was performed to demonstrate the specificity and detection performance of our ICMs in multiplexing and the detection limit could reach 1.49 × 10-10 M, showing promising potential in applications.

A Differential Detection Method Based on a Linear Weak Measurement System.

Self-reference detection is necessary and important to a biosensor. The linear weak measurement system based on total internal reflection has attracted widespread attention due to its high stability, label-free detection, and easy integration. In this paper, we propose a differential detection method based on the linear total internal reflection weak measurement system. We introduce the half-wave plate (HWP) to convert the H light and the V light to each other, thereby obtaining the difference in phase change of the optical path before and after the HWP. Experiments show that the system can not only achieve differential detection, but also has high stability. The linear differential weak measurement system proposed in this paper not only provides a new differential measurement method for real-time biosensors, but also enriches the types of weak measurement sensors.

Fast and accurate decoding of Raman spectra-encoded suspension arrays using deep learning.

A deep learning network called "residual neural network" (ResNet) was used to decode Raman spectra-encoded suspension arrays (SAs). With narrow bandwidths and stable signals, Raman spectra have ideal encoding properties. The different Raman reporter molecules assembled micro-quartz pieces (MQPs) were grafted with various biomolecule probes, which enabled simultaneous detection of numerous target analytes in a single sample. Multiple types of mixed MQPs were measured by Raman spectroscopy and then decoded by ResNet to acquire the type information of analytes. The good classification performance of ResNet was verified by a t-distributed stochastic neighbor embedding (t-SNE) diagram. Compared with other machine learning models, these experiments showed that ResNet was obviously superior in terms of classification stability and training convergence to different datasets. This method simplified the decoding process and the classification accuracy reached 100%.

An approach for medical event detection in Chinese clinical notes of electronic health records.

BACKGROUND: Medical event detection in narrative clinical notes of electronic health records (EHRs) is a task designed for reading text and extracting information. Most of the previous work of medical event detection treats the task as extracting concepts at word granularity, which omits the overall structural information of the clinical notes. In this work, we treat each clinical note as a sequence of short sentences and propose an end-to-end deep neural network framework. METHODS: We redefined the task as a sequence labelling task at short sentence granularity, and proposed a novel tag system correspondingly. The dataset were derived from a third-level grade-A hospital, consisting of 2000 annotated clinical notes according to our proposed tag system. The proposed end-to-end deep neural network framework consists of a feature extractor and a sequence labeller, and we explored different implementations respectively. We additionally proposed a smoothed Viterbi decoder as sequence labeller without additional parameter training, which can be a good alternative to conditional random field (CRF) when computing resources are limited. RESULTS: Our sequence labelling models were compared to four baselines which treat the task as text classification of short sentences. Experimental results showed that our approach significantly outperforms the baselines. The best result was obtained by using the convolutional neural networks (CNNs) feature extractor and the sequential CRF sequence labeller, achieving an accuracy of 92.6%. Our proposed smoothed Viterbi decoder achieved a comparable accuracy of 90.07% with reduced training parameters, and brought more balanced performance across all categories, which means better generalization ability. CONCLUSIONS: Evaluated on our annotated dataset, the comparison results demonstrated the effectiveness of our approach for medical event detection in Chinese clinical notes of EHRs. The best feature extractor is the CNNs feature extractor, and the best sequence labeller is the sequential CRF decoder. And it was empirically verified that our proposed smoothed Viterbi decoder could bring better generalization ability while achieving comparable performance to the sequential CRF decoder.

Dual-wavelength digital holographic phase and fluorescence microscopy combining with Raman spectroscopy for micro-quartz pieces-based dual-channel encoded suspension array.

Dual-wavelength digital holographic phase and fluorescence microscopy (DW-DHPFM), combining with Raman spectroscopy, is designed to achieve the detection and analysis of biomolecules with a new dual-channel encoding method. This employs the Raman reporter molecules assembled micro-quartz pieces (MQPs) as microcarriers of suspension array (SA). The dual-wavelength digital holographic phase microscopy (DW-DHPM) and Raman spectroscopy are served as the decoding platforms, and the fluorescence microscopy is used to quantify target analytes. Considering the independence between encoding and label signal, the above two encoding channels could effectively avoid the crosstalk in immunoassay process, and the combination of two encoding methods expand the encoding capacity with a considerable magnitude. Accurate and stable decoding abilities are verified by multiplexed immunoassay experiment and the quantitative analysis of targets with high-sensitivity is confirmed by concentration gradient experiments.

Spectral-optical-tweezer-assisted fluorescence multiplexing system for QDs-encoded bead-array bioassay.

As an efficient tool in the multiplexed detection of biomolecules, bead-array could achieve separation-free detection to multiple targets, making it suitable to analyze valuable and scarce samples like antigen and antibody from living organism. Herein, we propose a spectral-optical-tweezer-assisted fluorescence multiplexing system to analyze biomolecule-conjugated bead-array. Using optical tweezer, we trapped and locked beads at the focus to accept stimulation, offering a stable and optimized analysis condition. Moving the system focus and scanning the sample slide, we achieved emissions collection to QDs-encoded bead-array after the multiplexed detection. The emission spectra of fluorescence were collected and recorded by the spectrometer. By recognizing locations of decoding peaks and counting the intensities of label signals of emission spectra, we achieved qualitative and quantitative detection to targets. As proof-of-concept studies, we use this system to carry out multiplexed detection to various types of anti-IgG in the single sample and the detection limit reaches 1.52 pM with a linear range from 0.31 to 10 nM. Through further optimization of experimental conditions, we achieved specific detection to target IgG with sandwich method in human serum and the detection limit reaches as low as 0.23 pM with a linear range from 0.88 to 28 pM, validating the practical application of this method in real samples.

Rapid Separation of Enantiomeric Impurities in Chiral Molecules by a Self-Referential Weak Measurement System.

We propose a self-referential fast detection scheme for a frequency domain weak measurement system for the detection of enantiomeric impurities in chiral molecules. In a transmissive weak measurement system, the optical rotation (OR) is used to modify the pre-selected polarization state and the post-selection polarization state. We obtained the sum and difference of the optical rotations produced by the sample and the standard by rotating the quarter wave plate in the system. Then, we estimate the ratio of chiral molecules to enantiomeric impurities using the ratio of the central wavelength shifts caused by the addition and subtraction states described above. In this paper, our system has an optical resolution of 1.88 × 10-5°. At the same time, we completed the detection of the ratio of the two substances in the mixture of L-proline and D-proline in different proportions, which proved that our system can quickly detect the content of enantiomeric impurities in chiral molecules.

Optimization of a quantum weak measurement system with digital filtering technology.

In this paper, we propose a post-Gaussian filtering theory for weak measurement in the frequency domain, and propose a highly deformed digital filtering technique that can be used to optimize sensors based on weak-frequency measurement techniques. We completed the experimental verification based on the weak measurement total internal reflection sensor. The experimental results show that digital filtering technology can optimize the system in the working range, sensitivity, and resolution of the frequency domain weak measurement system, so that it can reach 0.210 rad, 3210.9 nm/RIU, and 7.12×10-7 RIU, respectively.

Ubiquitin Carboxyl Terminal Hydrolase L1 Attenuates TNF-α-Mediated Vascular Smooth Muscle Cell Migration Through Suppression of NF-κB Activation.

Ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) is one of the deubiquitinating enzymes in the ubiquitin-proteasome system. It has been shown that UCH-L1 could markedly decrease neointima formation through suppressing vascular smooth muscle cell (VSMC) proliferation in the balloon-injured rat carotid. However, whether UCH-L1 plays roles in VSMC migration remains to be determined. In this study, the primary VSMCs were isolated from aortic media of rats and TNF-α to was used to induce VSMC migration. Using a modified Boyden chamber and wound healing assay, it was found that TNF-α can dose and time-dependently induce VSMC migration with a maximal effect at 10 ng/mL. Moreover, UCH-L1 expression increased gradually with the prolonged induction time at 10 ng/mL of TNF-α. UCH-L1 content in VSMC was then modulated by recombinant adenoviruses expressing UCH-L1 or RNA interference to evaluate its roles in cell migration. The results showed that over-expression of UCH-L1 attenuated VSMC migration, while knockdown of it enhanced cell migration significantly no matter whether TNF-α treatment or not. Finally, the effect of UCH-L1 on NF-κB activation was demonstrated by NF-κB nuclear translocation and DNA binding activity, and the levels of IL-6 and IL-8 in cell culture media were examined by ELISA. It was showed that UCH-L1 over-expression inhibited NF-κB activation and decrease IL-6 and IL-8 levels, while knockdown of it enhanced NF-κB activation and increase IL-6 and IL-8 levels during TNF-α treatment. These data suggest that UCH-L1 can inhibit TNF-α-induced VSMCs migration, and this kind of effect may partially due to its suppression role in NF-κB activation.

Protective effect and mechanism of rat recombinant S100 calcium-binding protein A4 on oxidative stress injury of rat vascular endothelial cells.

The aim of the present study was to examine the protective effects and mechanisms of S100 calcium-binding protein A4 (S100A4) on endothelial cell apoptosis induced by oxidative stress injury. Endothelial cells were cultured and divided into control and oxidative stress injury groups, with the latter state induced by H2O2. Endothelial cells in every group were incubated with or without 50 or 100 µM S100A4. The cell viability and amounts of malondialdehyde, nitric oxide and lactate dehydrogenase in the culture medium were measured. The apoptotic index was detected by TUNEL staining. Western blot and immunoprecipitation analyses were used to detect the expression levels and the association between S100A4 and P53. H2O2 treatment led to oxidative stress injury in the cultured vascular endothelial cells, a decrease in the cell viability and an increase in the rate of apoptosis of vascular endothelial cells compared with the negative control group. Exogenous S100A4 serves a significant function against oxidative stress injury (P<0.05), increasing the viability and attenuating the apoptotic rate of endothelial cells. Western blotting results suggested that the protein levels of S100A4 and P53 increased subsequent to oxidative stress injury and that exogenous S100A4 increased the expression of P53 in the cytoplasm and decreased the expression of P53 in nucleus. The immunoprecipitation assay results revealed a protein-protein interaction between S100A4 and P53. These results suggested that rat recombinant S100A4 serves an anti-apoptotic function in oxidative stress injury. This effect of S100A4 is mediated, at least in part, via the inhibition of the translocation of P53 to the nucleus.

Functional expression, characterization, and application of human S100B.

The EF-hand calcium-binding protein S100B presents a wide range of biological activities and functions. This binding protein is involved in various human diseases, including cancer, brain trauma and ischemia, neuro-degenerative disease (Alzheimer's disease), and psychiatric disorders. In this study, we prepared human S100B protein and its monoclonal antibodies. Human S100B protein was expressed in Escherichia coli, successfully purified by diethylaminoethyl cellulose anion-exchange chromatography, and then identified by western blot analysis. Monoclonal antibodies (mAbs) were produced by the standard hybridoma method and validated by enzyme-linked immunosorbent assay and western blot analysis. The prepared human S100B protein and its mAbs demonstrated potential biological activities. The KD of one mAb is approximately 4.72x10-8 mol/l, and its cross reactivity is low with human S100A4, mouse S100A4, and human S100A1. Recombinant Soluble S100B can promote the migration and invasion of HeLa cells. The expression of S100B protein in tumor tissues can be detected effectively by using the prepared monoclonal antibodies. Increasing concentration of the anti-human S100B mAbs showed a reduced expression of the S100B protein. Subsequently, the expression of p53 increased significantly (P<0.05) in A375 cells. A significant increase in apoptosis in A375 cells was observed with increasing S100B mAb concentration. Results showed that our prepared S100B mAbs were suitable for detecting S100B expression in human tissues, furnishing promising tools for further functional investigation and clinical applications.

[Establishment and evaluation of oxidative stress injury model of in vitro rat aortic endothelial cells].

OBJECTIVE: To establish a model of oxidative stress injury in cultured rat aortic endothelial cells, and to provide a basis for the research of cell injury and apoptosis. METHODS: The rats were decapitated to get the aorta in thoracic operation under aseptic conditions. By subculture after tissue block culture method to get sufficient aortic endothelial cells, cultured in 96-well plates or grow on cover glass for the following test. Without H2O2 group as a control group, with different doses of H2O2 (100,200,300,400,500 µmol/L) treated endothelialcells in 12 h to screen the optimal dose. Based on the results, with the same dose of H2O2 (100 or 200 µmol/L) acted on endothelial cells respectively in different time (3, 6, 9, 12 and 24 h) to screen the optimal duration. Each group was made in sextuplicate. The establishment of the model was evaluated by immunofluorescence,cell viability testing, biochemical indicators detection (lactate dehydrogenase(LDH), nitric oxide(NO), malondialdehyde(MDA), superoxidedismutase(SOD))and apoptosis index testing. RESULTS: Endothelial cells were cultured successfully and verified by immunofluorescence staining of intracellular antigen Ⅷ collagen. With the increase of H2O2 doses at the same action time 12 h, the cell viability was significantly decreased (77.63%±5.20% to 40.90%±2.10%). The same dose(100 µmol/L group and 200 µmol/L group)with the action time increasing, the cellviability was significantly decreased (100 µmol/L group was 86.83%±12.11% to 44.26%±5.70%, 200 µmol/L group was 78.28%±11.98% to 34.45%±5.87%). At dose of H2O2 was 100 µmol/L and treated in 3,6,9,12 and 24 h, LDH-L and MDA were significantly increased after 9 h while NO and SOD were significantly decreased. In H2O2 dose of 100 µmol/L and action time 12 h, flow cytometry showed endothelial cellapoptosis rate was 16.92%±2.37%, significantly higher than the control group of 2.68%±0.47%(P<0.01); TUNEL detected endothelial cell apoptosis index was17.65%±2.36%, which was significantly higher than that in the control group of 3.23%±0.57%(P<0.01). CONCLUSIONS: The method was successfullyestablished a model of oxidative stress injury in cultured rat aortic endothelial cells, explore the moderate conditions that induced cells injury and apoptosis which could be a basis for the research.

[Protective effects of histone deacetylase inhibitor on stress-induced myocardial injury in rats].

OBJECTIVE: To observe the protective effects of histone deacetylase inhibitor on stress-induced myocardial injury. METHODS: Healthy male Wistar rats were randomly divided into 3 groups( n = 6), and the stress-induced myocardial injury model was established with chronic restraint stress method. The protective effects of histone deacetylase inhibitor on stress-induced myocardial injury were observed with Trichostatin A (TSA) intervention. Histone acetylation levels in myocardium of rats were detected by Western blot method, spectrophotometry method was used to dynamically determine the activity of rat serum lactate dehydrogenase (LDH), serum creatine kinase isoenzyme-MB (CK-MB) and Caspase 3, and nagar Olsen staining were used to observe the early myocardial damage. RESULTS: Restraint stress could significantly reduce the level of histone acetylation of myocardium in rats, and TSA intervention could inhibit the stress-induced reduction of myocardial levels of histone acetylation. Restraint stress could cause the significant increase of serum LDH activity ( P < 0.05), serum CK-MB activity ( P < 0.05), and the Caspase 3 activity of myocardial tissue (P < 0.05), and early myocardial damage also occurred during restraint stress. ISA intervention could significantly reduce the serum LDH activity (P < 0.05), the serum CK-MB activity (P < 0.05), the activity of myocardial tissue caspase 3 induced by restraint stress (P < 0.05), and the stress-induced myocardial injury was also attenuated by TSA intervention. CONCLUSION: The histone deacetylase inhibitor TSA can protect stress-induced myocardial injury.

Functional expression, characterization and application of the human S100A4 protein.

Preparations utilizing monoclonal antibodies against S100A4 provide useful tools for functional studies to investigate the clinical applications of the human S100A4 protein. In the present study, human S100A4 protein was expressed in Escherichia coli (E. coli) BL21 (DE3), successfully purified by diethylaminoethyl cellulose anion-exchange chromatography and identified by western blot analysis. Soluble S100A4 bioactivity was confirmed by Transwell migration and invasion assays in the human HeLa cell line. Monoclonal antibodies (mAbs) were generated utilizing the standard hybridoma method and were validated by enzyme-linked immunosorbent assay and western blot analysis. The antibody was then used to examine human gastric carcinoma specimens by immunohistochemistry. Recombinant S100A4 was functionally expressed in E. coli and promoted the migration and invasion of HeLa cells. Four hybridoma cell lines, which secreted mAbs specifically against human S100A4 protein, were obtained. One of the four mAbs, namely 2A12D10B2, recognized human S100A4 as indicated by immunohistochemical staining of human gastric carcinoma specimens and recombinant S100A4 was functionally expressed in E. coli. The mAbs of recombinant S100A4 were suitable for detecting S100A4 expression in human tissues and for investigating the subsequent clinical applications of the protein.