Mitochondria-containing extracellular vesicles from mouse vs . human brain endothelial cells for ischemic stroke therapy.

Ischemic stroke-induced mitochondrial dysfunction in the blood-brain barrier-forming brain endothelial cells ( BECs ) results in long-term neurological dysfunction post-stroke. We previously reported that intravenous administration of human BEC ( hBEC )-derived mitochondria-containing extracellular vesicles ( EVs ) showed a potential efficacy signal in a mouse middle cerebral artery occlusion ( MCAo ) model of stroke. We hypothesized that EVs harvested from donor species homologous to the recipient species ( e.g., mouse) may improve therapeutic efficacy, and therefore, use of mouse BEC ( mBEC )-derived EVs may improve post-stroke outcomes in MCAo mice. We investigated if EVs derived from the same species as the recipient cell (mBEC-EVs and recipient mBECs or hBECs-EVs and recipient hBECs) show a greater EV mitochondria delivery efficiency than cross-species EVs and recipient cells (mBEC-EVs and recipient hBECs or vice versa ). Our results showed that mBEC-EVs outperformed hBEC-EVs in transferring EV mitochondria to the recipient ischemic mBECs, and improved mBEC mitochondrial function via increasing oxygen consumption rate. mBEC-EVs significantly reduced brain infarct volume and improved behavioral recovery compared to vehicle-injected MCAo mice. Our data suggests that mBEC-EVs show superior therapeutic efficacy in a mouse MCAo stroke model compared to hBEC-EVs-supporting the continued use of mBEC-EVs to optimize the therapeutic potential of mitochondria-containing EVs in preclinical studies.

Profiling DNA Cargos in Single Extracellular Vesicles via Hydrogel-Based Droplet Digital Multiple Displacement Amplification.

Due to the substantial heterogeneity among extracellular vesicle (EV) subpopulations, single-EV analysis has the potential to elucidate the mechanisms behind EV biogenesis and shed light on the myriad functions, leading to the development of novel diagnostics and therapeutics. While many studies have been devoted to reveal between-EV variations in surface proteins and RNAs, DNA cargos (EV-DNA) have received little attention. Here, we report a hydrogel-based droplet digital multiple displacement amplification approach for the comprehensive analysis of EV-DNA at the single-EV level. Single EVs are dispersed in thousands of hydrogel droplets and lysed for DNA amplification and identification. The droplet microfluidics strategy empowers the assay with single-molecule sensitivity and capability for absolute quantification of DNA-containing EVs. In particular, our findings indicate that 5-40% EVs are associated with DNA, depending on the cell of origin. Large EVs exhibit a higher proportion of DNA-containing EVs and a more substantial presence of intraluminal DNA, compared to small EVs. These DNA-containing EVs carry multiple DNA fragments on average. Furthermore, both double-stranded DNA and single-stranded DNA were able to be detected at the single-EV level. Utilizing this method, the abundance, distribution, and biophysical properties of EV-DNA in various EV populations are evaluated. The DNA level within EVs provides insight into the status of the originating cells and offers valuable information on the outcomes of anticancer treatments. The utilization of single-EV analysis for EV-DNA holds significant promise for early cancer detection and treatment response monitoring.

Clinical correlates and thyroid hormones of metabolic syndrome in first-episode and drug-naïve major depressive disorder outpatients with and without hyperglycemia: a comprehensive cross-sectional study.

Hyperglycemia and metabolic syndrome (MetS) are common in patients with major depressive disorder (MDD). This study aimed to explore the prevalence and clinical factors of MetS in first-episode and drug-naïve MDD (FEDND) patients with and without hyperglycemia. A total of 1,718 FEDND patients' symptoms were assessed using the Hamilton Depression Scale (HAMD), Hamilton Anxiety Scale (HAMA), and positive subscale of the Positive and Negative Syndrome Scale (PANSS). Blood glucose levels, metabolic index, and thyroid hormones were measured during fasting. The prevalence of MetS in FEDND patients with hyperglycemia was 35.67 times higher than in FEDND patients without hyperglycemia. FEDND patients with MetS were older, had later age of onset, and were predominantly married than those without MetS (p < 0.05). Among FEDND patients without hyperglycemia, suicide attempts, severe anxiety, HAMD, HAMA, PANSS subscale scores, thyroid stimulating hormone, antithyroglobulin, and total cholesterol levels were all higher in patients with MetS than those without MetS (all p < 0.05). In FEDND patients without hyperglycemia, the combination of age and TgAb distinguished those patients with and without MetS. Our results suggest a high prevalence of MetS in FEDND patients with hyperglycemia. Several clinical variables and thyroid function-related hormones impact MetS in patients with FEDND.

Comparative 6+studies of environmentally persistent free radicals on nano-sized coal dusts.

Coal dust is the major hazardous pollutant in the coal mining environment. Recently environmentally persistent free radicals (EPFRs) were identified as one of the key characteristics which could impart toxicity to the particulates released into the environment. The present study used Electron Paramagnetic Resonance (EPR) spectroscopy to analyze the characteristics of EPFRs present in different types of nano-size coal dust. Further, it analyzed the stability of the free radicals in the respirable nano-size coal dust and compared their characteristics in terms of EPR parameters (spin counts and g-values). It was found that free radicals in coal are remarkably stable (can remain intact for several months). Also, Most of the EPFRs in the coal dust particles are either oxygenated carbon centered or a mixture of carbon and oxygen-centered free radicals. EPFRs concentration in the coal dust was found to be proportional to the carbon content of coal. The characteristic g-values were found to be inversely related to the carbon content of coal dust. The spin concentrations in the lignite coal dust were between 3.819 and 7.089 µmol/g, whereas the g-values ranged from 2.00352 to 2.00363. The spin concentrations in the bituminous coal dust were between 11.614 and 25.562 µmol/g, whereas the g-values ranged from 2.00295 to 2.00319. The characteristics of EPFRs present in coal dust identified by this study are similar to the EPFRs, which were found in other environmental pollutants such as combustion-generated particulates, PM2.5, indoor dust, wildfires, biochar, haze etc., in some of the previous studies. Considering the toxicity analysis of environmental particulates containing EPFRs similar to those identified in the present study, it can be confidently hypothesized that the EPFRs in the coal dust might play a major role in modulating the coal dust toxicity. Hence, it is recommended that future studies should analyze the role of EPFR-loaded coal dust in mediating the inhalation toxicity of coal dust.

Nanoscale coordination polymers enabling antioxidants inhibition for enhanced chemodynamic therapy.

Reactive oxygen species (ROS) generation to induce cell death is an effective strategy for cancer therapy. In particular, chemodynamic therapy (CDT), using Fenton-type reactions to generate highly cytotoxic hydroxyl radical (•OH), is a promising treatment modality. However, the therapeutic efficacy of ROS-based cancer treatment is still limited by some critical challenges, such as overexpression of enzymatic and non-enzymatic antioxidants by tumor cells, as well as the low tumor targeting efficiency of therapeutic agents. To address those problems, biomimetic CuZn protoporphyrin IX nanoscale coordination polymers have been developed, which significantly amplify oxidative stress against tumors by simultaneously inhibiting enzymatic and non-enzymatic antioxidants and initiating the CDT. In this design, cancer cell membrane camouflaged nanoparticle exhibits an excellent homotypic targeting effect. After being endocytosed into tumor cells, the nanoparticles induce depletion of the main non-enzymatic antioxidant glutathione (GSH) by undergoing a redox reaction with GSH. Afterward, the redox reaction generated cuprous ion (Cu+) works as a CDT agent for •OH generation. Furthermore, the released Zn protoporphyrin IX strongly inhibits the activity of the typical enzymatic antioxidant heme oxygenase-1. This tetra-modal synergistic strategy endows the biomimetic nanoparticles with great capability for anticancer therapy, which has been demonstrated in both in vitro and in vivo studies.

Engineering Extracellular Vesicles to Modulate Their Innate Mitochondrial Load.

Introduction: Extracellular vesicles (EVs) are promising carriers for the delivery of biotherapeutic cargo such as RNA and proteins. We have previously demonstrated that the innate EV mitochondria in microvesicles (MVs), but not exosomes (EXOs) can be transferred to recipient BECs and mouse brain slice neurons. Here, we sought to determine if the innate EV mitochondrial load can be further increased via increasing mitochondrial biogenesis in the donor cells. We hypothesized that mitochondria-enriched EVs ("mito-EVs") may increase the recipient BEC ATP levels to a greater extent than naïve MVs. Methods: We treated NIH/3T3, a fibroblast cell line and hCMEC/D3, a human brain endothelial cell (BEC) line using resveratrol to activate peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), the central mediator of mitochondrial biogenesis. Naïve EVs and mito-EVs isolated from the non-activated and activated donor cells were characterized using transmission electron microscopy, dynamic light scattering and nanoparticle tracking analysis. The effect of mito-EVs on resulting ATP levels in the recipient BECs were determined using Cell Titer Glo ATP assay. The uptake of Mitotracker Red-stained EVs into recipient BECs and their colocalization with recipient BEC mitochondria were studied using flow cytometry and fluorescence microscopy. Results: Resveratrol treatment increased PGC-1α expression in the donor cells. Mito-MVs but not mito-EXOs showed increased expression of mitochondrial markers ATP5A and TOMM20 compared to naïve MVs. TEM images showed that a greater number of mito-MVs contained mitochondria compared to naïve MVs. Mito-MVs but not mito-EXOs showed a larger particle diameter compared to their naïve EV counterparts from the non-activated cells suggesting increased mitochondria incorporation. Mito-EVs were generated at higher particle concentrations compared to naïve EVs from non-activated cells. Mito-EVs increased the cellular ATP levels and transferred their mitochondrial load into the recipient BECs. Mito-MV mitochondria also colocalized with recipient BEC mitochondria. Conclusions: Our results suggest that the pharmacological modulation of mitochondrial biogenesis in the donor cells can change the mitochondrial load in the secreted MVs. Outcomes of physicochemical characterization studies and biological assays confirmed the superior effects of mito-MVs compared to naïve MVs-suggesting their potential to improve mitochondrial function in neurovascular and neurodegenerative diseases. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00738-8.

Single-Lens Light-Sheet Fluorescence Microscopy Based on Micro-Mirror Array.

Conventional light sheet fluorescence microscopy (LSFM) utilizes two perpendicularly arranged objective lenses for optical excitation and detection, respectively. Such a configuration often limits the use of high-numerical-aperture (NA) objectives or requires specially designed long-working-distance objectives. Here, a LSFM based on a micro-mirror array (MMA) to enable light sheet imaging with a single objective lens is reported. The planar fluorescent emission excited by the light sheet illumination is collected by the same objective, relayed onto an MMA and detected by a side-view camera. The proposed scheme makes LSFM compatible to single objective imaging system and shows promising candidacy for high spatiotemporal imaging.

Extruded small extracellular vesicles: splinters of circulating tumour cells may promote cancer metastasis?

We speculate ruptured circulating tumour cells (CTC) in capillaries could release a large number of small extracellular vesicle-like vesicles, namely mechanically extruded sEV (sEVme), which can encapsulate chromosomal DNA fragments. These sEVme have similar physicochemical properties compared to small extracellular vesicles spontaneously secreted by living cells (sEVss), and thus sEVme and sEVss cannot be effectively distinguished based on their size or membrane protein markers. Meanwhile, these sEVme derived from CTC inherit oncogenic payloads, deliver cargo through the bloodstream to recipient cells, and thus may promote cancer metastasis. The validation of this speculation could facilitate our understanding of EV biogenesis and cancer pathology. The potential finding will also provide a theoretical foundation for burgeoning liquid biopsy using DNA fragments derived from harvested sEV.

Affinity-Based Enrichment of Extracellular Vesicles with Lipid Nanoprobes.

Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles with sub-micrometer size that are released by various cells. EVs contain a tissue-specific signature wherein a variety of proteins and nucleic acids are selectively packaged. Growing evidence has shown important biological roles and clinical relevance of EVs in diseases. For EV-related studies to thrive, rapid efficient isolation of pure EVs is a prerequisite. However, lengthy procedure, low yield, low throughput, and high contaminants stemmed from existing isolation approaches hamper both basic research and large-scale clinical implementation. We have shown that lipid nanoprobes (LNP) enable spontaneous labeling and rapid isolation of EVs by coupling with magnetic enrichment. Recently, we further developed a one-step EV isolation platform that utilizes EV size-matched silica nanostructures and surface-conjugated LNPs with an integrated microfluidic mixer. EVs, derived from up to 2-ml clinical plasma, can be processed with this point-of-care device using optimized flow rate. Subsequently, contents of isolated EVs can be extracted on-chip and eluted from the device for downstream molecular analyses. The LNP-functionalized microfluidic device combined with state-of-the-art analysis platforms could have great potential in promoting EV-centered research and clinical use in the future.

Vertically Aligned Carbon Nanotubes as a Unique Material for Biomedical Applications.

Vertically aligned carbon nanotubes (VACNTs), a unique classification of CNT, highly oriented and normal to the respective substrate, have been heavily researched over the last two decades. Unlike randomly oriented CNT, VACNTs have demonstrated numerous advantages making it an extremely desirable nanomaterial for many biomedical applications. These advantages include better spatial uniformity, increased surface area, greater susceptibility to functionalization, improved electrocatalytic activity, faster electron transfer, higher resolution in sensing, and more. This Review discusses VACNT and its utilization in biomedical applications particularly for sensing, biomolecule filtration systems, cell stimulation, regenerative medicine, drug delivery, and bacteria inhibition. Furthermore, comparisons are made between VACNT and its traditionally nonaligned, randomly oriented counterpart. Thus, we aim to provide a better understanding of VACNT and its potential applications within the community and encourage its utilization in the future.

An Ultrafast One-Step Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Detection of SARS-CoV-2.

We developed an ultrafast one-step RT-qPCR assay for SARS-CoV-2 detection, which can be completed in only 30 min on benchtop Bio-Rad CFX96. The assay significantly reduces the running time of conventional RT-qPCR: reduced RT step from 10 to 1 min, and reduced the PCR cycle of denaturation from 10 to 1 s and extension from 30 to 1 s. A cohort of 60 nasopharyngeal swab samples testing showed that the assay had a clinical sensitivity of 100% and a clinical specificity of 100%.

Emerging methods in biomarker identification for extracellular vesicle-based liquid biopsy.

Extracellular vesicles (EVs) are released by many cell types and distributed within various biofluids. EVs have a lipid membrane-confined structure that allows for carrying unique molecular information originating from their parent cells. The species and quantity of EV cargo molecules, including nucleic acids, proteins, lipids, and metabolites, may vary largely owing to their parent cell types and the pathophysiologic status. Such heterogeneity in EV populations provides immense challenges to researchers, yet allows for the possibility to prognosticate the pathogenesis of a particular tissue from unique molecular signatures of dispersing EVs within biofluids. However, the inherent nature of EV's small size requires advanced methods for EV purification and evaluation from the complex biofluid. Recently, the interdisciplinary significance of EV research has attracted growing interests, and the EV analytical platforms for their diagnostic prospect have markedly progressed. This review summarizes the recent advances in these EV detection techniques and methods with the intention of translating an EV-based liquid biopsy into clinical practice. This article aims to present an overview of current EV assessment techniques, with a focus on their progress and limitations, as well as an outlook on the clinical translation of an EV-based liquid biopsy that may augment current paradigms for the diagnosis, prognosis, and monitoring the response to therapy in a variety of disease settings.

The Role of Extracellular Vesicles in the Pathogenesis and Treatment of Autoimmune Disorders.

Extracellular vesicles (EVs) are important players in autoimmune diseases, both in disease pathogenesis and as potential treatments. EVs can transport autoimmune triggers throughout the body, facilitating the process of antigen presentation. Understanding the link between cellular stress and EV biogenesis and intercellular trafficking will advance our understanding of autoimmune diseases. In addition, EVs can also be effective treatments for autoimmune diseases. The diversity of cell types that produce EVs leads to a wide range of molecules to be present in EVs, and thus EVs have a wide range of physiological effects. EVs derived from dendritic cells or mesenchymal stem cells have been shown to reduce inflammation. Since many autoimmune treatments are focused only on symptom management, EVs present a promising avenue for potential treatments. This review looks at the different roles EVs can play in autoimmune diseases, from disease pathology to diagnosis and treatment. We also overview various methodologies in isolating or generating EVs and look to the future for possible applications of EVs in autoimmune diseases.

Characterization of nano-to-micron sized respirable coal dust: Particle surface alteration and the health impact.

Chemical and physical properties of coal dust particles significantly influence the inhalation of respirable coal dust by miners, causing several lung diseases such as coal workers' pneumoconiosis (CWP) and silicosis. Multiple experimental techniques, including proximate/ultimate analyses, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), laser diffraction, and low-pressure CO2 and N2 adsorption, were used to investigate the chemical and physical properties of micron-/nano-coal particles comprehensively. Compared to the micron-scale coal dust, the nano-coal dust (prepared by cryogenic ballmill) shows the increase of carbon content and aromaticity and a decrease of oxygen content along with the reduction of oxygen-containing functional groups. Pore volume and surface area estimated by low-pressure CO2 and N2 adsorption have more than five-time increase for the nano-coal dust. The reduction of oxygen functional groups suggests the dropped wetting behavior of coal nanoparticles. The significantly increased pore volume and surface area in coal nanoparticles could be caused by the enhanced pore interconnectivity on the particle surface and the alteration of coal macromolecules. Weaker wettability and the highly enhanced surface area suggest potentially more significant toxicity of nano-coal dust inhaled by coal miners.

Extracellular Vesicles as Potential Biomarkers for Early Detection and Diagnosis of Pancreatic Cancer.

Pancreatic carcinoma (PC) is highly metastatic, and it tends to be detected at advanced stages. Identifying and developing biomarkers for early detection of PC is crucial for a potentially curative treatment. Extracellular vesicles (EVs) are bilayer lipid membrane-structured nanovesicles found in various human bodily fluids, and they play important roles in tumor biogenesis and metastasis. Cancer-derived EVs are enriched with DNA, RNA, protein, and lipid, and they have emerged as attractive diagnostic biomarkers for early detection of PC. In this article, we provided an overview of the cell biology of EVs and their isolation and analysis, and their roles in cancer pathogenesis and progression. Multiplatform analyses of plasma-based exosomes for genomic DNA, micro RNA, mRNA, circular RNA, and protein for diagnosis of PC were critically reviewed. Numerous lines of evidence demonstrate that liquid biopsy with analysis of EV-based biomarkers has variable performance for diagnosis of PC. Future investigation is indicated to optimize the methodology for isolating and analyzing EVs and to identify the combination of EV-based biomarkers and other clinical datasets, with the goal of improving the predictive value, sensitivity, and specificity of screening tests for early detection and diagnosis of PC.

Magnetically Driven Nanotransporter-Assisted Intracellular Delivery and Autonomous Release of Proteins.

Biofunctional proteins such as active enzymes and therapeutic proteins show tremendous promise in disease treatment. However, intracellular delivery of proteins is facing substantial challenges owing to their vulnerability to degradation and denaturation and the presence of various biological barriers such as their low membrane transport efficiency. Herein, we report a magnetically driven and redox-responsive nanotransporter (MRNT) for highly efficient intracellular delivery of biofunctional proteins. The MRNT has remarkably high cargo capacity, compared with that without nanoscale cargo compartments. We have demonstrated the directional and dynamic motion of the MRNT using both nanoparticle tracking analysis and magnetic driving evaluation. Moreover, the active MRNT can translocate into the cytosol and sense the reducing cytosolic environment to discharge protein cargoes autonomously. The internalization mechanism of the MRNT has been studied using endocytosis inhibitors. Under the magnetic drive, the MRNT can promote a protein transduction efficiency of over 95%, and the intracellular protein delivery by the active MRNT shows significantly higher (∼4 times) enzymatic activity and therapeutic efficiency than those achieved by the static ones. Our proof-of-concept study provides a valuable tool for intracellular protein transduction and contributes to biotechnology and protein therapeutics.

Microfluidics in Single-Cell Virology: Technologies and Applications.

Microfluidics has proven to be a powerful tool for probing biology at the single-cell level. However, it is only in the past 5 years that single-cell microfluidics has been used in the field of virology. An array of strategies based on microwells, microvalves, and droplets is now available for tracking viral infection dynamics, identifying cell subpopulations with particular phenotypes, as well as high-throughput screening. The insights into the virus-host interactions gained at the single-cell level are unprecedented and usually inaccessible by population-based experiments. Therefore, single-cell microfluidics, which opens new avenues for mechanism elucidation and development of antiviral therapeutics, would be a valuable tool for the study of viral pathogenesis.

Habenula lesions improve glucose metabolism in rats with type 2 diabetes by increasing insulin sensitivity and inhibiting gluconeogenesis.

INTRODUCTION: The habenular nucleus (Hb), a famous relay station in the midbrain, is vital for controlling many physiological functions of vertebrates. The role of Hb in the pathogenesis of depression has been thoroughly studied, but whether it functions in the pathogenesis of diabetes remains unknown. In this study, we found that Hb lesions could improve glucose metabolism in type 2 diabetes mellitus (T2DM) by inhibiting the peripheral sympathetic nervous system and hepatic glucose production. RESEARCH DESIGN AND METHODS: T2DM rats were induced by a high-carbohydrate and fat diet combined with streptozotocin. Electrical lesion method was applied to suppress the function of Hb. Serum and tissue samples of rats in the control group, T2DM group, sham group, and Hb lesion group were detected by ELISA, western blotting, and biochemical methods. RESULTS: Compared with the sham group, the expression levels of AMPK phosphorylation and insulin receptor (IR) were significantly increased, whereas glucose-6-phosphatase and phosphoenolpyruvate carboxylated kinase were reduced in the liver of the Hb lesion group. In the glucose tolerance test and pyruvate tolerance test, the lesion group showed stronger glucose tolerance and lower hepatic gluconeogenesis than the sham. These results suggest that Hb lesions not only effectively increase insulin sensitivity and improve insulin resistance but also inhibit gluconeogenesis in T2DM rats. Moreover, Hb lesions increase the expression of brain-derived neurotrophic factor, tropomyosin receptor kinase B, glucocorticoid receptor, and IR in the hippocampus. In this study, we also found that Hb lesions increase the content of acetylcholine in the adrenal glands and reduce the content of epinephrine in both the adrenal glands and the liver, which may be the main reason for the Hb lesions to regulate glucose metabolism in the liver. CONCLUSION: Hb is an important neuroanatomical target for the regulation of glucose metabolism in the central nervous system of diabetic rats.