Extracellular Vesicle Preparation and Analysis: A State-of-the-Art Review.

In recent decades, research on Extracellular Vesicles (EVs) has gained prominence in the life sciences due to their critical roles in both health and disease states, offering promising applications in disease diagnosis, drug delivery, and therapy. However, their inherent heterogeneity and complex origins pose significant challenges to their preparation, analysis, and subsequent clinical application. This review is structured to provide an overview of the biogenesis, composition, and various sources of EVs, thereby laying the groundwork for a detailed discussion of contemporary techniques for their preparation and analysis. Particular focus is given to state-of-the-art technologies that employ both microfluidic and non-microfluidic platforms for EV processing. Furthermore, this discourse extends into innovative approaches that incorporate artificial intelligence and cutting-edge electrochemical sensors, with a particular emphasis on single EV analysis. This review proposes current challenges and outlines prospective avenues for future research. The objective is to motivate researchers to innovate and expand methods for the preparation and analysis of EVs, fully unlocking their biomedical potential.

[Research progress on bioactive constituents and pharmacological effects of Tibetan medicine Sinopodophylli Fructus].

Sinopodophylli Fructus is a traditional medicine used by the Tibetan people. It is known for its ability to regulate menstruation and promote blood circulation. Presently, bioactive constituents that have been isolated and identified from Sinopodophylli Fructus mainly include 15 lignans(e.g., podophyllotoxin, deoxypodophyllotoxin, and 4'-demethylpodophyllotoxin) and 20 flavonoids(e.g., quercetin, kaempferol, and rutin). These components exhibit pharmacological effects such as anticancer, antibacterial, and lipid-lowering activities. Additionally, Sinopodophylli Fructus contains other components such as proteins, fatty acids, polysaccharides, vitamins, amino acids, and trace elements. According to the relevant literature reports in China and abroad, this article reviewed the chemical constituents and pharmacological effects of Sinopodophylli Fructus, aiming to provide references for the development and rational clinical application of this medicinal resource.

Optical pH Monitoring in Microdroplet Platforms for Live Cell Experiments Using Colloidal Surfactants.

Droplet microfluidic technology facilitates the development of high-throughput screening applications in nanoliter volumes. Surfactants provide stability for emulsified monodisperse droplets to carry out compartmentalization. Fluorinated silica-based nanoparticles are used; they can minimize crosstalk in microdroplets and provide further functionalities by surface labeling. Here we describe a protocol for monitoring pH changes in live single cells by fluorinated silica nanoparticles, for their synthesis, chip fabrication, and optical monitoring on the microscale. The nanoparticles are doped with ruthenium-tris-1,10-phenanthroline dichloride on the inside and conjugated with fluorescein isothiocyanate on the surface. This protocol may be used more generally to detect pH changes in microdroplets. The fluorinated silica nanoparticles can also be used as droplet stabilizers with an integrated luminescent sensor for other applications.

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations.

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric field of metallic nanostructures. Salt-induced silver nanoparticle aggregation is the most popular method for generating SERS-active substrates; however, it is limited by poor reproducibility, stability, and biocompatibility. The present protocol integrates optical manipulation and SERS detection to develop an efficient analytical platform to address this. A 1064 nm trapping laser and a 532 nm Raman probe laser are combined in a microscope to assemble silver nanoparticles, which generate plasmonic hotspots for in situ SERS measurements in aqueous environments. Without aggregating agents, this dynamic plasmonic silver nanoparticle assembly enables an approximately 50-fold enhancement of the analyte molecule signal. Moreover, it provides spatial and temporal control to form the SERS-active assembly in as low as 0.05 nM analyte-coated silver nanoparticle solution, which minimizes the potential perturbation for in vivo analysis. Hence, this optical trapping-integrated SERS platform holds great potential for efficient, reproducible, and stable molecular analyses in liquids, especially in aqueous physiological environments.

Automated Miniaturized Digital Microfluidic Antimicrobial Susceptibility Test Using a Chip-Integrated Optical Oxygen Sensor.

We present the first digital microfluidic (DMF) antimicrobial susceptibility test (AST) using an optical oxygen sensor film for in-situ and real-time continuous measurement of extracellular dissolved oxygen (DO). The device allows one to monitor bacterial growth across the entire cell culture area, and the fabricated device was utilized for a miniaturized and automated AST. The oxygen-sensitive probe platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin was embedded in a Hyflon AD 60 polymer and spin-coated as a 100 nm thick layer onto an ITO glass serving as the DMF ground electrode. This DMF-integrated oxygen sensing film was found to cause no negative effects to the droplet manipulation or cell growth on the chip. The developed DMF platform was used to monitor the DO consumption during Escherichia coli(E. coli) growth caused by cellular respiration. A rapid and reliable twofold dilution procedure was developed and performed, and the AST with E. coli ATCC 25922 in the presence of ampicillin, chloramphenicol, and tetracycline at different concentrations from 0.5 to 8 µg mL-1 was investigated. All sample dispensation, dilution, and mixing were performed automatically on the chip within 10 min. The minimum inhibitory concentration values measured from the DMF chip were consistent with those from the standard broth microdilution method but requiring only minimal sample handling and working with much smaller sample volumes.

Three-component radical homo Mannich reaction.

Aliphatic amine, especially tertiary aliphatic amine, is one of the most popular functionalities found in pharmaceutical agents. The Mannich reaction is a classical and widely used transformation for the synthesis of ß-amino-carbonyl products. Due to an ionic nature of the mechanism, the Mannich reaction can only use non-enolizable aldehydes as substrates, which significantly limits the further applications of this powerful approach. Here we show, by employing a radical process, we are able to utilize enolizable aldehydes as substrates and develop the three-component radical homo Mannich reaction for the streamlined synthesis of γ-amino-carbonyl compounds. The electrophilic radicals are generated from thiols via the desulfurization process facilitated by visible-light, and then add to the electron-rich double bonds of the in-situ formed enamines to provide the products in a single step. The broad scope, mild conditions, high functional group tolerance, and modularity of this metal-free approach for the synthesis of complex tertiary amine scaffolds will likely be of great utility to chemists in both academia and industry.

Composite Films of CsPbBr3 Perovskite Nanocrystals in a Hydrophobic Fluoropolymer for Temperature Imaging in Digital Microfluidics.

A composite film material that combines CsPbBr3 perovskite nanocrystals with a Hyflon AD 60 fluoropolymer was developed and utilized for high-resolution optical temperature imaging. It exhibited bright luminescence and, most importantly, long-term stability in an aqueous medium. CsPbBr3 nanocrystal-Hyflon films immersed in aqueous solutions showed stable luminescence over at least 4 months and exhibited a fully reversible pronounced temperature sensitivity of 1.2% K-1 between 20 and 80 °C. They were incorporated into a digital microfluidic (electrowetting on dielectric) platform and were used for spatially resolved temperature measurements during droplet movements. Thermal mapping with a CsPbBr3 nanocrystal-Hyflon sensing layer in a room temperature environment (22.0 °C) revealed an increase in local temperatures of up to 40.2 °C upon voltage-driven droplet manipulations in a digital microfluidic system, corresponding to a local temperature change of up to 18.2 °C.

Protective Effect of Folic Acid on Oxidative DNA Damage: A Randomized, Double-Blind, and Placebo Controlled Clinical Trial.

Although previous reports have linked DNA damage with both transmissions across generations as well as our own survival, it is unknown how to reverse the lesion. Based on the data from a Randomized, Double-blind, Placebo Controlled Clinical Trial, this study aimed to assess the efficacy of folic acid supplementation (FAS) on DNA oxidative damage reversal.In this randomized clinical trial (RCT), a total of 450 participants were enrolled and randomly assigned to 3 groups to receive folic acid (FA) 0.4 mg/day (low-FA), 0.8 mg/day (high-FA), or placebo (control) for 8 weeks. The urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and creatinine (Cr) concentration at pre- and post-FAS were measured with modified enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), respectively. A multivariate general linear model was applied to assess the individual effects of FAS and the joint effects between FAS and hypercholesterolemia on oxidative DNA damage improvement. This clinical trial was registered with ClinicalTrials.gov, number NCT02235948.Of the 438 subjects that received FA fortification or placebo, the median (first quartile, third quartile) of urinary 8-OHdG/Cr for placebo, low-FA, and high-FA groups were 58.19 (43.90, 82.26), 53.51 (38.97, 72.74), 54.73 (39.58, 76.63) ng/mg at baseline and 57.77 (44.35, 81.33), 51.73 (38.20, 71.30), and 50.65 (37.64, 76.17) ng/mg at the 56th day, respectively. A significant decrease of urinary 8-OHdG was observed after 56 days FA fortification (P < 0.001). Compared with the placebo, after adjusting for some potential confounding factors, including the baseline urinary 8-OHdG/Cr, the urinary 8-OHdG/Cr concentration significantly decreased after 56 days FAS [ß (95% confidence interval) = -0.88 (-1.62, -0.14) and P = 0.020 for low-FA; and ß (95% confidence interval) = -2.68 (-3.42, -1.94) and P < 0.001 for high-FA] in a dose-response fashion (Ptrend < 0.001). Test of interaction between hypercholesterolemia and FA supplementation on urinary 8-OHdG reduction was significant (P = 0.001).The present study demonstrates that FA fortification is independently linked to the reduction of urinary 8-OHdG/Cr in a dose-related pattern, which suggests that FA is beneficial to protect against oxidative damage to DNA. This effect is apparently stronger in those with hypercholesterolemia. The authors provide a new insight into the prevention and reversal of oxidative DNA damage.

Arsenic responsive microRNAs in vivo and their potential involvement in arsenic-induced oxidative stress.

Arsenic exposure is postulated to modify microRNA (miRNA) expression, leading to changes of gene expression and toxicities, but studies relating the responses of miRNAs to arsenic exposure are lacking, especially with respect to in vivo studies. We utilized high-throughput sequencing technology and generated miRNA expression profiles of liver tissues from Sprague Dawley (SD) rats exposed to various concentrations of sodium arsenite (0, 0.1, 1, 10 and 100mg/L) for 60days. Unsupervised hierarchical clustering analysis of the miRNA expression profiles clustered the SD rats into different groups based on the arsenic exposure status, indicating a highly significant association between arsenic exposure and cluster membership (p-value of 0.0012). Multiple miRNA expressions were altered by arsenic in an exposure concentration-dependent manner. Among the identified arsenic-responsive miRNAs, several are predicted to target Nfe2l2-regulated antioxidant genes, including glutamate-cysteine ligase (GCL) catalytic subunit (GCLC) and modifier subunit (GCLM) which are involved in glutathione (GSH) synthesis. Exposure to low concentrations of arsenic increased mRNA expression for Gclc and Gclm, while high concentrations significantly reduced their expression, which were correlated to changes in hepatic GCL activity and GSH level. Moreover, our data suggested that other mechanisms, e.g., miRNAs, rather than Nfe2l2-signaling pathway, could be involved in the regulation of mRNA expression of Gclc and Gclm post-arsenic exposure in vivo. Together, our findings show that arsenic exposure disrupts the genome-wide expression of miRNAs in vivo, which could lead to the biological consequence, such as an altered balance of antioxidant defense and oxidative stress.

[Association between dyslipidemia and 8-OHdG/Cr among a population exposed to chronic arsenic].

OBJECTIVE: To explore the association between dyslipidemia and the level of 8-OHdG/Cr in urine among a population exposed to chronic arsenic. METHODS: Four hundred and seven subjects were randomly selected in an arsenic-affected area in Inner Mongolia. After blood biochemical examination, all the subjects were divided into 4 groups based on the results of total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C)and low density lipoprotein cholesterol (LDL-C). The groups consisted of hypercholesterolemia, HDL-C ratio anomaly, combined hypercholesterolemia and HDL-C ratio anomaly, as well as a normal lipid group. Urine samples were collected and 8-OHdG/Cr was measured using the ELISA method. A generalized linear mixed model was used to analyze the association between dyslipidemia and 8-OHdG/Cr. RESULTS: The levels of 8-OHdG/Cr as 55.73 (39.90-79.94) ng/mg, 58.08 (44.94-69.91)ng/mg, 65.28 (49.29-92.95) ng/mg and 51.43 (36.86-68.57) ng/mgin the HDL-C ratio anomaly, hypercholesterolemia, combined hypercholesterolemia and HDL-C ratio anomaly groups and the control group, respectively, which showed significant differences on the levels of 8-OHdG/Cr in the four groups(P = 0.006). From the linear regression analysis results showed that the 8-OHdG/Cr level in combined hypercholesterolemia and HDL-C ratio anomaly group was higher (4.25 ± 0.55 ng/mg) than in the control group (3.96 ± 0.55 ng/mg) (P = 0.018). After adjusting for important covariates, there was a linear trend between the levels of 8-OHdG/Cr and dyslipidemia (P = 0.016). CONCLUSION: Data from our study showed a linear relation between hypercholesterolemia, HDL-C ratio anomaly and the 8-OHdG/Cr level, suggesting that dyslipidemia was associated with oxidative DNA damage among those .

A cutoff point for arterial stiffness using the cardio-ankle vascular index based on carotid arteriosclerosis.

The cardio-ankle vascular index (CAVI) has been widely accepted as a good indicator of arteriosclerosis. However, the lack of a reliable diagnostic criterion for CAVI hampers the proper clinical screening for arteriosclerosis using CAVI and impedes the prompt treatment of cardiovascular disease (CVD). There is an urgent need to determine a criterion for CAVI in arteriosclerosis prevention. We conducted a cross-sectional study to determine this criterion based on receiver operating characteristic (ROC) analyses in a Chinese population consisting of 328 participants. CAVI was measured in duplicate, and carotid ultrasound detection was performed in a quiet environment by well-trained physicians. After multivariate adjustment, CAVI was positively associated with the risk of carotid arteriosclerosis. Compared with participants in the lowest tertile of CAVI (5.15-7.40), those in the medium (7.41-8.65) and highest (8.66-13.60) tertiles had odds ratios (95% confidence interval) of 2.2 (1.0, 4.9) and 4.4 (1.5, 13.3), respectively, for developing carotid arteriosclerosis (P trend=0.007). The areas under the ROC curve (AUC) of the male, female and pooled populations were 0.789, 0.897 and 0.856, respectively. The cutoff point of CAVI≥8.0 resulted in the largest sensitivity and specificity. Furthermore, CAVI and age acted synergistically to increase the risk of carotid arteriosclerosis. CAVI≥8.0 may be an optimal cutoff point for carotid arteriosclerosis prediction. The older population with higher CAVI scores had a higher risk of carotid arteriosclerosis. Additional large prospective studies are needed to confirm our findings.

Relationship between long-term exposure to low-level arsenic in drinking water and the prevalence of abnormal blood pressure.

Arsenic increases the risk and incidence of cardiovascular disease. To explore the impact of long-term exposure to low-level arsenic in drinking water on blood pressure including pulse pressure (PP) and mean arterial blood pressure (MAP), a cross-sectional study was conducted in 2010 in which the blood pressure of 405 villagers was measured, who had been drinking water with an inorganic arsenic content <50 µg/L. A multivariate logistic regression model was used to estimate odds ratios and 95% confidence intervals. After adjusting for age, gender, Body Mass Index (BMI), alcohol consumption and smoking, the odds ratios showed a 1.45-fold (95%CI: 0.63-3.35) increase in the group with >30-50 years of arsenic exposure and a 2.95-fold (95%CI: 1.31-6.67) increase in the group with >50 years exposure. Furthermore, the odds ratio for prevalence of abnormal PP and MAP were 1.06 (95%CI: 0.24-4.66) and 0.87 (95%CI: 0.36-2.14) in the group with >30-50 years of exposure, and were 2.46 (95%CI: 0.87-6.97) and 3.75 (95%CI: 1.61-8.71) for the group with >50 years exposure, compared to the group with arsenic exposure ≤ 30 years respectively. Significant trends for Hypertension (p<0.0001), PP (p<0.0001) and MAP (p=0.0016) were found. The prevalence of hypertension and abnormal PP as well as MAP is marked among a low-level arsenic exposure population, and significantly increases with the duration of arsenic exposure.