Identification of Molecular Profile of Cell Membrane via Magnetic Plasmonic Nanoprobe.

Cell membranes are primarily composed of lipids, membrane proteins, and carbohydrates, and the related studies of membrane components and structures at different stages of disease development, especially membrane proteins, are of great significance. Here, we investigate the chemical signature profiles of cell membranes as biomarkers for cancer cells via label-free surface-enhanced Raman scattering (SERS). A magnetic plasmonic nanoprobe was proposed by decorating magnetic beads with silver nanoparticles, allowing self-driven cell membrane-targeted accumulation within 5 min. SERS profiles of three types of breast cells were achieved under the plasmon enhancement effect of these nanoprobes. Membrane fingerprint spectra from breast cell lines were further classified with the convolutional neural network model, which perfectly distinguished between two breast cancer subtypes. We further tested various clinical samples using this method and obtained fingerprint spectra from primary cells and frozen slices. This study presents a practical, user-friendly approach for label-free and in situ analysis of cell membranes, which can work for early tumor screening and treatment assessment for tumors reliant on the Molecular profiles of cell membranes. Additionally, this method can be applied universally to explore cell membrane components of other cells, thus assisting Human Cell Atlas.

Efficacy of the tetravalent protein COVID-19 vaccine, SCTV01E: a phase 3 double-blind, randomized, placebo-controlled trial.

Evolution of SARS-CoV-2 variants emphasizes the need for multivalent vaccines capable of simultaneously targeting multiple strains. SCTV01E is a tetravalent COVID-19 vaccine derived from the spike protein of SARS-CoV-2 variants Alpha, Beta, Delta, and Omicron BA.1. In this double-blinded placebo-controlled pivotal efficacy trial (NCT05308576), the primary endpoint was vaccine efficacy (VE) against COVID-19 seven days post-vaccination in individuals without recent infection. Other endpoints included evaluating safety, immunogenicity, and the VE against all SARS-CoV-2 infections in individuals meeting the study criteria. Between December 26, 2022, and January 15, 2023, 9,223 individuals were randomized at a 1:1 ratio to receive SCTV01E or a placebo. SCTV01E showed a VE of 69.4% (95% CI: 50.6, 81.0) 7 days post-vaccination, with 75 cases in the placebo group and 23 in the SCTV01E group for the primary endpoint. VEs were 79.7% (95% CI: 51.0, 91.6) and 82.4% (95% CI: 57.9, 92.6), respectively, for preventing symptomatic infection and all SARS-CoV-2 infections 14 days post-vaccination. SCTV01E elicited a 25.0-fold higher neutralizing antibody response against Omicron BA.5 28 days post-vaccination compared to placebo. Reactogenicity was generally mild and transient, with no reported vaccine-related SAE, adverse events of special interest (AESI), or deaths. The trial aligned with the shift from dominant variants BA.5 and BF.7 to XBB, suggesting SCTV01E as a potential vaccine alternative effective against present and future variants.

An atomic force microscopy and total internal reflection fluorescence microscopy correlated system (AFM-TIRF) for fluorescence imaging and spectroscopy of a single particle.

Combining atomic force microscopy (AFM) with other optical microscopic techniques is pivotal in nanoscale investigations, particularly leveraging the surface-sensitive properties of total internal reflection fluorescence microscopy (TIRF). A novel design that integrates AFM with a multi-wavelength TIRF is displayed, providing simultaneous fluorescence imaging and spectral acquisition capabilities. We elaborate on the considerations in the instrument design process and demonstrate the performance and potential applications of the instrument through fluorescence imaging and spectroscopy testing of individual nanoparticles. This AFM and TIRF correlated system (AFM-TIRF) emerges as a promising option for single-molecule fluorescence studies, enabling simultaneous manipulation and detection of fluorescence from individual molecules.

Comparison of a Supervised Home-Based Tele-Rehabilitation with Center-Based Pulmonary Rehabilitation: Protocol for a Randomized Non-Inferiority Multicenter Study in Ningxia.

Background: Pulmonary rehabilitation (PR) is an effective intervention for people with chronic obstructive pulmonary disease (COPD). However, fewer than 5% of eligible individuals receive pulmonary rehabilitation, largely due to limited by the accessibility of rehabilitation and difficulties associated with travel and transport. Supervised home-based tele-rehabilitation (SHTR) is an alternative model to center-based pulmonary rehabilitation. We will determine whether supervised home-based tele-rehabilitation is non-inferior to center-based pulmonary rehabilitation. Methods: The participants will undergo an 8-week rehabilitation program. Pulmonary rehabilitation comprises four main modules: exercise training, education, nutritional support, and psychological and behavioral interventions. We mainly focus on the module of exercise training and education. The education module includes information on exercise training, nutrition, and psychology, which are presented in an educational booklet provided to each participant. Blinded assessors will evaluate the outcomes at baseline, post-intervention, and 6 months after the intervention. The primary outcome is the change in the 6-minute walking distance. Secondary outcomes will assess changes in the patients' 1-minute sit-to-stand test, maximal inspiratory pressure (MIP), scales (CAT, mMRC, HAD), diaphragm ultrasound (TD, DE, DIF), changes in extrathoracic muscle volume and mass, completion rate of patient exercise prescriptions, occurrence of adverse events, as well as disease exacerbation and rehospitalization rates after rehabilitation and during the 6-month follow-up. Discussion: In order to improve the accessibility of pulmonary rehabilitation and patient-related outcomes, it is necessary to propose an alternative model of pulmonary rehabilitation. This trial will establish whether a supervised home-based tele-rehabilitation is not inferior to traditional center-based pulmonary rehabilitation. Trial Registration: Chinese Clinical Trial Registry ChiCTR2300076969. Registered on October 25, 2023.

In-droplet multiplex immunoassays for hypoxia-induced single-cell cytokines.

Cytokine expression is an important biomarker in understanding hypoxia microenvironments in tumor growth and metastasis. In-droplet-based immunoassays performed above the target cell membrane were employed to track the cytokines of single cells with the aid of three types of immuno-nanoprobes (one capture nanoprobe and two reporter nanoprobes). Single cells and nanoprobes were co-packaged in water-in-oil microdroplets (about 100 µm in diameter) using a cross-shaped microfluidic chip. In each droplet, capture nanoprobes would be first fixed to the cell surface by linking to membrane proteins that have been streptavidinized. Then, the capture nanoprobes can collect cell-secreted cytokines (VEGF and IL-8) by the antibodies, followed by two reporter nanoprobes that emit distinguishable fluorescence. Fluorescence imaging was utilized to record the signal outputs of two reporter probes, which reflect cytokine expressions secreted by a single tumor cell. The cytokine levels at different degrees of hypoxia induction were assessed. Multiple chemometric methods were adopted to distinguish differences in the secretion of two cytokines and the results demonstrated a positive correlation. This study developed an in-droplet, dual-target, simultaneous biosensing strategy for a single cell, which is helpful for understanding the impacts of hypoxia microenvironments on cell cytokines that are vital for assessing early cancer diagnosis and prognosis.

Fabrication of High-Negatively Charged Bicelle-Mediated Supported Lipid Bilayer.

Supported lipid bilayers (SLBs), two-dimensional lipid films formed on a solid-supporting substrate, serve as models for biomembranes and exhibit remarkable potential in chemistry, biology, and medicine. However, preparing SLBs with highly negatively charged contents on the negatively charged surface by overcoming electrostatic repulsion remains a challenge. Here, a creative bicelle-mediated and divalent cation-free SLB preparation method with the assistance of phosphate-buffered saline (PBS) solution was proposed, which can form the SLBs containing 50% DOPS or 30% CL on the silica surface monitored by a quartz crystal microbalance with dissipation (QCM-D). Results of molecular dynamics (MD) simulation indicate that electrostatic repulsion can be overcome by the increased number of hydrogen bonds caused by the adsorption of dihydrogen phosphate ions onto the headgroups of lipids. In addition, the negatively charged SLB formation was identified to be a three-step kinetic process, which differs from a two-step mechanism in the case of amphoteric SLB. The extra kinetic step can be attributed to the reduction in the number of intermolecular hydrogen bonds and the ordering of water molecules in the hydration layer. This investigation resolves the challenge of fabricating SLB over negatively charged surfaces and offers a fresh perspective on the SLB assembly methodology.

A review of clinical use of surface-enhanced Raman scattering-based biosensing for glioma.

Glioma is the most common malignant tumor of the nervous system in recent centuries, and the incidence rate of glioma is increasing year by year. Its invasive growth and malignant biological behaviors make it one of the most challenging malignant tumors. Maximizing the resection range (EOR) while minimizing the impact on normal brain tissue is crucial for patient prognosis. Changes in metabolites produced by tumor cells and their microenvironments might be important indicators. As a powerful spectroscopic technique, surface-enhanced Raman scattering (SERS) has many advantages, including ultra-high sensitivity, high specificity, and non-invasive features, which allow SERS technology to be widely applied in biomedicine, especially in the differential diagnosis of malignant tumor tissues. This review first introduced the clinical use of responsive SERS probes. Next, the sensing mechanisms of microenvironment-responsive SERS probes were summarized. Finally, the biomedical applications of these responsive SERS probes were listed in four sections, detecting tumor boundaries due to the changes of pH-responsive SERS probes, SERS probes to guide tumor resection, SERS for liquid biopsy to achieve early diagnosis of tumors, and the application of free-label SERS technology to detect fresh glioma specimens. Finally, the challenges and prospects of responsive SERS detections were summarized for clinical use.

Engineering of 4-hydroxyphenylacetate 3-hydroxylase derived from Pseudomonas aeruginosa for the ortho-hydroxylation of ferulic acid.

Polyphenolic compounds have natural antioxidant properties, and their antioxidant activity is usually related to the number and position of hydroxyls. Here, we successfully applied the engineered 4-hydroxyphenylacetate 3-hydroxylases (4HPA3Hs) derived from Pseudomonas aeruginosa to catalyze ferulic acid (FA) synthesis of ortho-hydroxyferulic acid (5-hydroxyferulic acid, 5-OHFA). Through optimization of co-expression, the oxygenase component (PaHpaB) and the reductase component (PaHpaC) in E. coli, and optimization of whole-cell catalytic conditions, the engineered strain BC catalyzed ortho-hydroxylation of 2 g/L of FA with a yield of 75 % from 39 %. Through tunnel engineering of PaHpaB, the obtained mutants F301A and Q376A almost completely transformed 2 g/L of FA. Further, a multiple mutant L214A/F301A/Q376A converted 4 g/L FA into 5-OHFA within 12 h, and the yield reached 99.9 %, which was approximately 2.39-fold of the wild type. The kcat/Km value of L214A/F301A/Q376A was about 307 times greater than that of the wide type. Analysis of three-dimensional structural models showed that L214, F301, and Q376 mutated into Ala, which greatly shortened the side chain and broadened the tunnel size, thereby significantly improving the catalytic efficiency of L214A/F301A/Q376A. This biosynthesis of 5-OHFA is simple, efficient, and green, suggesting that it is useful for efficient biosynthesis of polyphenolic compounds.

Policies for recovery from drug use: Differences between public stigma and perceived stigma and associated factors.

INTRODUCTION: Public stigma towards people who use drugs is widespread and places obstacles in way of their recovery. Previous studies have used different approaches to measure public stigma, resulting in a notable gap in the understanding of the relationship between it and its associated factors. Some studies measure public stigma by assessing stigma perceived by those who use drugs, while others investigate attitudes towards them among the general public. This study aimed to compare perceived and public stigma, and factors related to these two variables. METHODS: The study comprised a cross-sectional survey in China of two samples: males who used drugs (N = 257) and the general public (N = 376). The survey assessed demographic variables, social distance, public stigma and perceived stigma of those who use drugs. The data were analysed using t-tests and linear regression. RESULTS: Public stigma was significantly higher than perceived stigma. The findings indicated that gender, knowledge of drugs, family relationships with people who use drugs, attributions of drug use and social distance were significantly related to levels of public stigma. Among those who use drugs, perceived stigma was significantly correlated with age, marital status, duration of drug abstinence and social distance. DISCUSSION AND CONCLUSIONS: Findings indicate that public stigma undermines the recovery of people who use drugs and highlight the importance of interaction between them and the social environment. The study also underscores the necessity of developing policies to enhance their integration into mainstream culture and provide access to social support and life activities.

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o-position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o-hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o-hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented.

Sensitive detection of genetically modified maize based on a CRISPR/Cas12a system.

With the vigorous development of biotechnology, genetically modified organisms (GMOs) have become more and more common. In order to effectively supervise and administrate them, the rapid and accurate detection of GMOs is urgently demanded. Here, GMO gene-specific sensing methods based on colorimetry and surface-enhanced Raman scattering (SERS) were proposed based on the lateral branch cleavage function of the CRISPR/Cas12a system. Two transgenes, pCaMV35S and M810 Cry1Ab, were chosen as targets for transgenic crops. By using these methods, we performed transgenic detection on five types of maize leaves and successfully distinguished transgenic from non-transgenic samples. The colorimetric method is rapid, economical and available for field detection. The SERS approach, giving a higher sensitivity to 100 fM, is more suitable for laboratory application scenarios. This study explores practical transgenic detection approaches and will be valuable for the supervision of GMOs.

Surface Plasmon Field-Enhanced Raman Scattering Co-Excited by P-Polarized and S-Polarized Light Based on Waveguide-Coupled Surface Plasmon Resonance Configuration.

We constructed a waveguide-coupled surface plasmon resonance (WCSPR) structure to enhance Raman scattering. In this structure, P-polarized and S-polarized incident lasers can simultaneously coexcite the evanescent field, thereby further enhancing Raman scattering. This configuration is a five-phase Kretschmann resonance setup that consists of a SF10 prism/inner Ag film/SiO2 film/outer Ag film/water structure. The WCSPR configuration effectively concentrates and confines the evanescent field excited by the incident light. Ag nanoparticles assembled on the outer Ag film surface enhance the evanescent field further by means of surface plasmon resonance. By finely tuning the thickness of the Ag and SiO2 films, it is possible to achieve a coincidence between the SPR angle of P-polarized light and that of S-polarized light. At this angle, both P- and S-polarized light can jointly elevate the evanescent field intensity, leading to the simultaneous enhancement of the electric fields at the upper, lower, left, and right parts of the silver nanoparticles and generating maximum evanescent field enhancement. We achieved an electric field enhancement of up to 103 around the nanoparticles, leading to more SERS hotspots and comparable SERS enhancement capability to gap-type hotspots. Our WCSPR structure combined with the nanoparticles offers a feasible strategy for the SERS detection of large molecules that cannot be placed in traditional gap-type hotspots. It is highly convenient for SERS detection of large molecules.

Biosynthesis of 3-Hydroxyphloretin Using Rational Design of 4-Hydroxyphenylacetate 3-Monooxygenase.

The compound 3-hydroxyphloretin is a typical dihydrochalcone that can be obtained in plants by the 3-hydroxylation of phloretin. Here, the flavin-dependent two-component monooxygenase (HpaBC) derived from Pseudomonas aeruginosa was used to convert phloretin into 3-hydroxyphloretin. Following molecular docking and sequence alignment, modifications to the substrate pocket and loop of PaHpaBC were rationally designed, and mutant residues were selected. The results showed that the mutant Q212G/F292A/Q376N gave the best yield of 3-hydroxyphloretin and showed improved catalytic efficiency. Under optimal reaction condition, 2.03 g/L of 3-hydroxyphloretin was produced in the whole-cell catalysis experiment. Molecular docking and molecular dynamics simulations were used to analyze mutants and elucidate the potential mechanism. It was found that the increase in 3-hydroxyphloretin yield was due to the improvement in the flexibility of the loop and the expansion of its substrate pocket. This strategy based on loop and substrate pocket modification has significance in the engineering of PaHpaB.

Single-Cell Analysis and Classification according to Multiplexed Proteins via Microdroplet-Based Self-Driven Magnetic Surface-Enhanced Raman Spectroscopy Platforms Assisted with Machine Learning Algorithms.

A microdroplet-based surface-enhanced Raman spectroscopy (microdroplet SERS) platform was constructed to envelop individual cells in microdroplets, followed by the SERS detection of their extracellular vesicle-proteins (EV-proteins) via the in-drop immunoassays by use of immunomagnetic beads (iMBs) and immuno-SERS tags (iSERS tags). A unique phenomenon is found that iMBs can start a spontaneous reorientation on the probed cell surface based on the electrostatic force-driven interfacial aggregation effect, which leads EV-proteins and iSERS tags to be gathered from a liquid phase to a cell membrane interface and significantly improves SERS sensitivity to the single-cell analysis level due to the formation of numbers of SERS hotspots. Three EV-proteins from two breast cancer cell lines were collected and further analyzed by machine learning algorithmic tools, which will be helpful for a deeper understanding of breast cancer subtypes from the view of EV-proteins.

Safety and immunogenicity of a tetravalent and bivalent SARS-CoV-2 protein booster vaccine in men.

The safety and immunogenicity of a protein-based tetravalent vaccine SCTV01E that contains spike protein ectodomain (S-ECD) of Alpha, Beta, Delta and Omicron BA.1 are assessed and compared with bivalent protein vaccine SCTV01C (Alpha and Beta variants) and monovalent mRNA vaccine (NCT05323461). The primary endpoints are the geometric mean titers (GMT) of live virus neutralizing antibodies (nAb) to Delta (B.1.617.2) and Omicron BA.1 at day 28 post-injection. The secondary endpoints include the safety, day 180 GMTs against Delta and Omicron BA.1, day 28 GMTs to BA.5, and seroresponse rates of neutralizing antibodies and T cell responses at day 28 post-injection. 450 participants, comprising of 449 males and 1 female, with a median age (range) of 27 (18-62) years, are assigned to receive one booster dose of BNT162b2, 20 µg SCTV01C or 30 µg SCTV01E and completed 4-week follow-up. All SCTV01E related adverse events (AEs) are mild or moderate and no Grade ≥3 AE, serious AE or new safety concerns are identified. Day 28 GMT of live virus neutralizing antibodies and seroresponse against Omicron BA.1 and BA.5 with SCTV01E are significantly higher than those with SCTV01C and BNT162b2. These data indicate an overall neutralization superiority with tetravalent booster immunization in men.

Asymmetric Heterodimer-Regulated Surface Plasmon Coupling ECL Polarization Strategy for MiRNA-182 Detection.

In this work, a novel plasmonic heterodimer with controllable hot spot was designed and applied to regulate surface plasmon coupling electrochemiluminescence (SPC-ECL) polarization sensing system. The heterodimer nanostructure consisted of individual Au-Ag core-shell nanocubes (Au@Ag NC) and Au nanospheres (Au NS), which were precisely assembled by thiol-DNA and biotin-streptavidin. The asymmetric nanostructure can significantly modulate the ECL intensity and emission polarization angle based on the synergy of the surface plasmon coupling (SPC) effect and the lightning rod effect with extraordinary field enhancement in the hot spot region. As a result, the isotropic ECL signal of zinc-doped nitrogen dots (Zn-N dots) was regulated in the directional emission. Furthermore, the SPC-ECL biosensor was successfully applied to detect miRNA-182 in triple-negative breast cancer (TNBC) tissues. The research on the established relationship between ECL polarization analysis and plasmonic heterodimers can provide a new pathway for the development of ECL sensing platforms.

Carbonized Polymer Dot Probe for Two-Photon Fluorescence Imaging of Lipid Droplets in Living Cells and Tissues.

As a dynamic and multifunctional organelle, lipid droplets (LDs) are essential in maintaining lipid balance and transducing biological signals. LD accumulation and catabolism are closely associated with energy metabolism and cell signaling. In order to easily trace LDs in living cells, a novel carbonized polymer dot (CPD)-based fluorescent nanoprobe is reported to serve the needs of LD-targeting imaging. This probe exhibits the advantages of excellent biocompatibility, simple preparation, good lipophilicity, and high compatibility with commercial dyes. Transient absorption spectroscopy was employed to discuss the luminescence mechanism of CPDs, and the results indicate that the excellent fluorescence property and the environment-responsive feature of our CPDs are derived from the intramolecular charge transfer (ICT) characteristics and the D-π-A structure that possibly formed in CPD. This nanoprobe is available for one-photon fluorescence (OPF) and two-photon fluorescence (TPF) imaging and is also practicable for staining LDs in living/fixed cells and lipids in tissue sections. The staining process is completed within several seconds, with no washing step. The intracellular LDs involving the intranuclear LDs (nLDs) can be selectively lit up. This probe is feasible for visualizing dynamic interactions among LDs, which suggests its great potential in revealing the secret of LD metabolism. The in situ TPF spectra were analyzed to determine surrounding microenvironment according to the polarity-responsive feature of our CPDs. This work expands the applications of CPDs in biological imaging, helps design new LD-selective fluorescent probes, and has implications for studying LD-related metabolism and diseases.

Pretreatment-free, on-site separation and sensitive identification of methamphetamine in biological specimens by SERS-active hydrogel microbeads.

The worldwide abuse of illicit drugs led to severe consequences for human health, and society environment. Therefore, urgently required are effective and efficient on-site detection methods for illicit drugs of interest in various matrices, e.g., police samples, biofluids, and hairs. Although surface-enhanced Raman spectroscopy (SERS) shows power in many analytical fields, the cumbersome pretreatment of various matrices restricts its use in the easy-to-operate and on-site detection of illicit drugs. To address this problem, we adopted pore-size selectivity SERS-active hydrogel microbeads, whose meshes are adjustable to allow small molecules to access and to exclude large molecules. Meanwhile, Ag nanoparticles were uniformly dispersed and wrapped in the hydrogel matrix, providing excellent SERS performances with high sensitivity, reproducibility, and stability. By using these SERS hydrogel microbeads, one of the illicit drugs, methamphetamine (MAMP), can be rapidly and reliably detected in various biological specimens (blood, saliva, and hair) without sample pretreatment. The minimum detectable concentration is 0.1 ppm for MAMP in three biological specimens with a linear range of 0.1-100 ppm, which is lower than the maximum allowable level of 0.5 ppm set by the department of the health and human service. The SERS detection results were consistent with the gas chromatographic (GC) data. Thanks to its operational simplicity, fast response, high throughput and low cost, our established SERS hydrogel microbeads can be used as a sensing platform for facile analysis of illicit drugs through simultaneous separation, preconcentration, and optical detection, which shall be provided practically for front-line narcotics squad and resistance to the overwhelmed drug abuses.

3D Plasmonic Nanostructure-Based Polarized ECL Sensor for Exosome Detection in Tumor Microenvironment.

Exosomes of cancer cells play an important role in the proliferation, adhesion, and migration of tumors. Especially, exosomes in the tumor microenvironment can reflect the proliferation of tumors directly, thus serving as ideal referenced markers of the possibility and grade of malignancy in neoplasms. However, the sensitive and accurate detection of exosomes remains challenging. In this work, a novel three-dimensional (3D) plasmonic nanostructure was constructed for exosomal miRNA detection. It combined the advantages of Au nanostar monolayer and Ag nanowire monolayer to provide multiple hot spots. Moreover, Au nanostar monolayer changed the isotropic electrochemiluminescence (ECL) into polarized emission. The Ag nanowire monolayer worked as waveguides for the light direction. As a result, the polarized resolution and intensity of ECL signal were improved. The polarized ECL emission was significantly increased by 47.1 times. This high-resolution polarized ECL sensor was used for detecting exosomal miRNA-146b-5p in the thyroid tumor microenvironment. This sensor showed the linear range from 1 fM to 1 nM with a detection limit of 0.3 fM. The satisfactory results indicated the developed 3D plasmonic nanostructure-based ECL sensor had great potential in biosensing and clinical diagnosis.