Stability of Plant Leaf-Derived Extracellular Vesicles According to Preservative and Storage Temperature.

Plant-derived extracellular vesicles (EVs) are capable of efficiency delivering mRNAs, miRNAs, bioactive lipids, and proteins to mammalian cells. Plant-derived EVs critically contribute to the ability of plants to defend against pathogen attacks at the plant cell surface. They also represent a novel candidate natural substance that shows potential to be developed for food, cosmetic, and pharmaceutical products. However, although plant-derived EVs are acknowledged as having potential for various industrial applications, little is known about how their stability is affected by storage conditions. In this study, we evaluated the stability of Dendropanax morbifera leaf-derived extracellular vesicles (LEVs) alone or combined with the preservatives, 1,3-butylene glycol (to yield LEVs-1,3-BG) or TMO (LEVs-TMO). We stored these formulations at -20, 4, 25, and 45 °C for up to 4 weeks, and compared the stability of fresh and stored LEVs. We also assessed the effect of freeze-thawing cycles on the quantity and morphology of the LEVs. We found that different storage temperatures and number of freeze-thawing cycles altered the stability, size distribution, protein content, surface charge, and cellular uptake of LEVs compared to those of freshly isolated LEVs. LEVs-TMO showed higher stability when stored at 4 °C, compared to LEVs and LEVs-1,3-BG. Our study provides comprehensive information on how storage conditions affect LEVs and suggests that the potential industrial applications of plant-derived EVs may be broadened by the use of preservatives.

Cyclic tangential flow filtration system for isolation of extracellular vesicles.

Size-based filtration techniques have been developed for high-throughput isolation of extracellular vesicles (EVs). Conventional direct filtration systems have limitations in that large particles generally not only block the pores of the membrane but also damage the particles because of the high fluid pressure. Here, we propose a cyclic tangential flow filtration (TFF) system that includes two membranes with pore sizes of 200 and 30 nm, connected to a peristaltic pump that feeds the stream flowing to the membrane for continuous circulation. The cyclic TFF system is better able to isolate the specific 30-200 nm size range in one step through dual cyclic filtration compared with direct filtration (DF) and single cyclic TFF (scTFF). We further introduced a buffer-exchange process to the dcTFF system after filtration to remove contaminants for more efficient purification. As a result of comparative evaluation of dcTFF and ExoQuick, EVs isolated by dcTFF had more abundant exosome markers and active EVs. The cyclic TFF system not only has great potential to separate EVs with high selectivity and separation efficiency in small volumes of samples but can also be used in clinical applications, including medical diagnostic procedures.

Cancer-Associated Fibroblasts Differentiated by Exosomes Isolated from Cancer Cells Promote Cancer Cell Invasion.

Cancer-associated fibroblasts (CAFs) in the cancer microenvironment play an essential role in metastasis. Differentiation of endothelial cells into CAFs is induced by cancer cell-derived exosomes secreted from cancer cells that transfer molecular signals to surrounding cells. Differentiated CAFs facilitate migration of cancer cells to different regions through promoting extracellular matrix (ECM) modifications. However, in vitro models in which endothelial cells exposed to cancer cell-derived exosomes secreted from various cancer cell types differentiate into CAFs or a microenvironmentally controlled model for investigating cancer cell invasion by CAFs have not yet been studied. In this study, we propose a three-dimensional in vitro cancer cell invasion model for real-time monitoring of the process of forming a cancer invasion site through CAFs induced by exosomes isolated from three types of cancer cell lines. The invasiveness of cancer cells with CAFs induced by cancer cell-derived exosomes (eCAFs) was significantly higher than that of CAFs induced by cancer cells (cCAFs) through physiological and genetic manner. In addition, different genetic tendencies of the invasion process were observed in the process of invading cancer cells according to CAFs. Our 3D microfluidic platform helps to identify specific interactions among multiple factors within the cancer microenvironment and provides a model for cancer drug development.

Anti-Metastatic Effects of Plant Sap-Derived Extracellular Vesicles in a 3D Microfluidic Cancer Metastasis Model.

Natural medicinal plants have attracted considerable research attention for their potential as effective drugs. The roots, leaves and stems of the plant, Dendropanax morbifera, which is endemic to southern regions of Asia, have long been used as a folk medicine to treat variety of diseases. However, the sap of this plant has not been widely studied and its bioactive properties have yet to be clearly elucidated. Here, we isolated extracellular vesicles from D. morbifera sap with the goal of improving the intracellular delivery efficiency and clinical effectiveness of bioactive compounds in D. morbifera sap. We further investigated the anti-metastatic effects of D. morbifera sap-derived extracellular vesicles (DMS-EVs) using a cancer metastasis model based on 3D microfluidic system that closely mimics the in vivo tumor environment. We found that DMS-EVs exerted a concentration-dependent suppressive effect on cancer-associated fibroblasts (CAFs), which are important mediators of cancer metastasis. DMS-EVs also altered expression level of genes, especially growth factor and extracellular matrix (ECM)-related genes, including integrin and collagen. Our findings suggest that DMS-EVs can act as anti-CAF agents to reduce CAFs in the tumor microenvironment. They further indicate the utility of our 3D microfluidic model for various drug-screening assays as a potential alternative to animal testing for use in validating therapeutic effects on cancer metastasis.

Cytotoxic Effects of Plant Sap-Derived Extracellular Vesicles on Various Tumor Cell Types.

Edible plants have been widely used in traditional therapeutics because of the biological activities of their natural ingredients, including anticancer, antioxidant, and anti-inflammatory properties. Plant sap contains such medicinal substances and their secondary metabolites provide unique chemical structures that contribute to their therapeutic efficacy. Plant extracts are known to contain a variety of extracellular vesicles (EVs) but the effects of such EVs on various cancers have not been investigated. Here, we extracted EVs from four plants-Dendropanax morbifera, Pinus densiflora, Thuja occidentalis, and Chamaecyparis obtusa-that are known to have cytotoxic effects. We evaluated the cytotoxic effects of these EVs by assessing their ability to selectively reduce the viability of various tumor cell types compared with normal cells and low metastatic cells. EVs from D. morbifera and P. densiflora sap showed strong cytotoxic effects on tumor cells, whereas those from T. occidentalis and C. obtusa had no significant effect on any tumor cell types. We also identified synergistic effect of EVs from D. morbifera and P. densiflora saps on breast and skin tumor cells and established optimized treatment concentrations. Our findings suggest these EVs from plant sap as new candidates for cancer treatment.

The Effectiveness of Virtual Reality in Patients With Spinal Pain: A Systematic Review and Meta-Analysis.

BACKGROUND: Virtual reality (VR) technologies have been shown to be beneficial in various areas of health care; to date, there are no systematic reviews examining the effectiveness of VR technology for the treatment of spinal pain. PURPOSE: To investigate the effectiveness of VR technology in the management of individuals with acute, subacute, and chronic spinal pain. METHODS: Six electronic databases were searched until November 2019. Randomized controlled trials (RCTs) assessing the effectiveness of VR were eligible for inclusion. Two independent reviewers extracted the data and assessed the risk of bias for each study and the overall quality of evidence. Mean differences of outcomes were pooled as appropriate using random-effects models. RESULTS: Seven RCTs with high risk of bias met review criteria. Quality of evidence ranged from very low to low quality. In patients with chronic neck pain, VR improved global perceived effect (GPE), satisfaction, and general health at short-term follow-up, as well as general health and balance at intermediate-term follow-up compared to kinematic training. VR improved pain intensity and disability at short-term and long-term follow-up compared to conventional proprioceptive training in patients with chronic neck pain. In patients with either subacute or chronic low back pain (LBP), VR improved pain, disability, and fear of movement compared to lumbar stabilization exercises and improved pain compared to conventional physical therapy (at short-term follow-up). In patients with chronic LBP, VR improved pain compared to lumbar stabilization exercises and improved fear of movement compared to conventional physical therapy (at short-term follow-up). CONCLUSION: VR's potential for improvement in outcomes for spinal pain that demonstrated statistical and/or clinical significance (pain intensity, disability, fear of movement, GPE, patient satisfaction, general health status, and balance) highlights the need for more focused, higher-quality research on the efficacy and effectiveness of VR for treatment of patients with spinal pain.

Anti-melanogenic effects of extracellular vesicles derived from plant leaves and stems in mouse melanoma cells and human healthy skin.

Consumer interest in cosmetic industry products that produce whitening effects has increased demand for agents that decrease melanin production. Many such anti-melanogenic agents are associated with side effects, such as contact dermatitis and high toxicity, and also exhibit poor skin penetration. Considerable recent research has focused on plant-derived products as alternatives to chemotherapeutic agents that possess fewer side effects. In the current study, we investigated the anti-melanogenic effects of extracellular vesicles (EVs) extracted from leaves and stems of Dendropanax morbifera. Using spectrophotometric and biochemical approaches, we found that leaf-derived extracellular vesicles (LEVs) and stem-derived extracellular vesicles (SEVs) reduced melanin content and tyrosinase (TYR) activity in the B16BL6 mouse melanoma cell line in a concentration-dependent manner. An electron microscopy analysis further confirmed that LEVs and SEVs induce a concentration-dependent decrease in melanin content in melanoma cells. Both LEVs and SEVs exerted a greater whitening effect on melanoma cells than arbutin, used as a positive control, with LEVs producing the greater effect. Notably, neither LEVs nor SEVs induced significant cytotoxicity. We also examined the effects of plant-derived EVs on the expression of tyrosinase-related proteins (TRPs) in melanoma cells. LEVs inhibited expression of melanogenesis-related genes and proteins, including microphthalmia-associated transcription factor (MITF), TYR, TRP-1 and TRP-2. In a human epidermis model, LEVs exerted a stronger inhibitory effect on melanin production than arbutin. Collectively, our data suggest that LEVs from D. morbifera may be a novel candidate natural substance for use as an anti-melanogenic agent in cosmeceutical formulations.

Cancer-derived exosomes trigger endothelial to mesenchymal transition followed by the induction of cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) play a pivotal role in tumor growth, but very little has been known about its characteristics and origin. Recently, cancer-derived exosome has been suggested to transdifferentiate CAFs, by a new mechanism of endothelial to mesenchymal transition (EndMT), initiating angiogenic processes and triggering metastatic evolution. However, an enabling tool in vitro is yet to be developed to investigate complicated procedures of the EndMT and the transdifferentiation under reconstituted tumor microenvironment. Here we proposed an in vitro microfluidic model which enables to monitor a synergetic effect of complex tumor microenvironment in situ, including extracellular matrix (ECM), interstitial flow and environmental exosomes. The number of CAFs differentiated from human umbilical vein endothelial cells (HUVECs) increased with melanoma-derived exosomes, presenting apparent morphological and molecular changes with pronounced motility. Mesenchymal stem cell (MSC)-derived exosomes were found to suppress EndMT, induce angiogenesis and maintain vascular homeostasis, while cancer-derived exosomes promoted EndMT. Capabilities of the new microfluidic model exist in precise regulation of the complex tumor microenvironment and therefore successful reconstitution of 3D microvasculature niches, enabling in situ investigation of EndMT procedure between various cell types. STATEMENT OF SIGNIFICANCE: This study presents an in vitro 3D EndMT model to understand the progress of the CAF generation by recapitulating the 3D tumor microenvironment in a microfluidic device. Both cancer-derived exosomes and interstitial fluid flow synergetically played a pivotal role in the EndMT and consequent formation of CAFs through a collagen-based ECM. Our approach also enabled the demonstration of a homeostatic capability of MSC-derived exosomes, ultimately leading to the recovery of CAFs back to endothelial cells. The in vitro 3D EndMT model can serve as a powerful tool to validate exosomal components that could be further developed to anti-cancer drugs.

Numerical verification of bounce-harmonic resonances in neoclassical toroidal viscosity for tokamaks.

This Letter presents the first numerical verification for the bounce-harmonic (BH) resonance phenomena of the neoclassical transport in a tokamak perturbed by nonaxisymmetric magnetic fields. The BH resonances were predicted by analytic theories of neoclassical toroidal viscosity (NTV), as the parallel and perpendicular drift motions can be resonant and result in a great enhancement of the radial momentum transport. A new drift-kinetic δf guiding-center particle code, POCA, clearly verified that the perpendicular drift motions can reduce the transport by phase-mixing, but in the BH resonances the motions can form closed orbits and particles radially drift out fast. The POCA calculations on resulting NTV torque are largely consistent with analytic calculations, and show that the BH resonances can easily dominate the NTV torque when a plasma rotates in the perturbed tokamak and therefore, is a critical physics for predicting the rotation and stability in the International Thermonuclear Experimental Reactor.

Effectiveness of mirtazapine for nausea and insomnia in cancer patients with depression.

AIMS: The purpose of the present paper was to evaluate the effectiveness of mirtazapine orally disintegrating tablets for nausea and sleep disturbance, which are common and distressing symptoms of cancer. METHODS: This was a 4-week, prospective, open-labeled study of cancer patients. Assessments were performed at baseline and on days 1, 3, 5, 7, 14, and 28. Primary outcome measures were the Clinical Global Impression scale for nausea/vomiting and the Chonnam National University Hospital-Leeds Sleep Evaluation Questionnaire (C-LSEQ) including total amount of night sleep time. The secondary outcome measures consisted of pain items in the 36-item Short Form Health Survey, the Montgomery-Asberg Depression Rating Scale (MADRS), and the EuroQoL (EQ)-5D. Forty-two cancer patients were enrolled. RESULTS: Those with nausea (n = 28) improved significantly from day 1. The total night sleep time and each item on the C-LSEQ improved from days 1-5. The scores on the MADRS and the depression/anxiety dimension and visual analog scale of EQ-5D improved significantly from the first week. Pain measures also improved from day 1. Exacerbation of sleepiness developed in approximately one-third of subjects during the initial few days, but disappeared gradually. CONCLUSION: In the present study mirtazapine rapidly improved nausea, sleep disturbance, pain and quality of life, as well as depression in cancer patients. Mirtazapine may be an effective treatment option in managing cancer patients with multiple distressing symptoms, including nausea and sleep disturbance.

Use of long-acting injectable risperidone before and throughout pregnancy in schizophrenia.

Data on the use of long-acting injectable (LAI) risperidone, the first atypical depot antipsychotic, during pregnancy are limited. A 35-year-old woman with schizophrenia was given LAI risperidone before and throughout her pregnancy. She gave birth to a female infant weighing 2230 g at 36 weeks and 6 days of pregnancy, following premature rupture of the membranes. The baby had no congenital malformation and was healthy 8 months postnatal. To our knowledge, this is the first reported use of LAI risperidone throughout an entire pregnancy. In this paper, we discuss the rationale and problems of LAI risperidone use in pregnancy, based on a literature review.