Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50–200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex “work-in-progress” MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.     

Mesorhizobium sp. J8 can establish symbiosis with Glycyrrhiza uralensis,increasing glycyrrhizin production

Licorice (Glycyrrhiza uralensis) is a medicinal plant that contains glycyrrhizin (GL), which has various pharmacological activities. Because licorice is a legume, it can establish a symbiotic relationship with nitrogen-fixing rhizobial bacteria. However, the effect of this symbiosis on GL production is unknown. Rhizobia were isolated from root nodules of Glycyrrhiza glabra, and a rhizobium that can form root nodules in G. uralensis was selected. Whole-genome analysis revealed a single circular chromosome of 6.7 Mbp. This rhizobium was classified as Mesorhizobium by phylogenetic analysis and was designated Mesorhizobium sp. J8. When G. uralensis plants grown from cuttings were inoculated with J8, root nodules formed. Shoot biomass and SPAD values of inoculated plants were significantly higher than those of uninoculated controls, and the GL content of the roots was 3.2 times that of controls. Because uninoculated plants from cuttings showed slight nodule formation, we grew plants from seeds in plant boxes filled with sterilized vermiculite, inoculated half of the seedlings with J8, and grew them with or without 100 µM KNO₃. The SPAD values of inoculated plants were significantly higher than those of uninoculated plants. Furthermore, the expression level of the CYP88D6 gene, which is a marker of GL synthesis, was 2.5 times higher than in inoculated plants. These results indicate that rhizobial symbiosis promotes both biomass and GL production in G. uralensis.     

Analysis of the acrolein-modified sites of apolipoprotein B-100 in LDL

We have reported that acrolein-conjugated low-density lipoprotein (Acro-LDL) uptake by scavenger receptor class A type 1 (SR-A1) can mediate macrophage foam cell formation. The purpose of this study was to determine which amino acid residues of apoB protein in LDL are conjugated with acrolein. Acro-apoB was prepared by incubation of LDL with acrolein (10 to 60 μM) at 37 °C for 7 days. Identification of acrolein-conjugated amino acid residues in apoB was performed using LC-MS/MS. The levels of acrolein-conjugated amino acid residues of apoB as well as crosslinking apoB increased in proportion to acrolein concentration. The level of LDL uptake by macrophages was parallel with the acrolein-conjugated monomer apoB. Acrolein-conjugated amino acid residues in apoB were C212, K327, K742, K949, K1087, H1923, K2634, K3237 and K3846. The NH₂-teriminal four amino acid residues (C212, K327, K742 and K949) were located at the scavenger receptor SR-A1 recognition site, suggesting that these four acrolein-conjugated amino acids are involved in the rapid uptake of Acro-LDL by macrophages. It is proposed that the rapid uptake of LDL by macrophages is dependent on acrolein conjugation of four amino acids residues at the scavenger receptor recognition site of apoB in LDL.     

Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis

In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3–activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.     

Cellular Auxin Homeostasis under High Temperature Is Regulated through a SORTING NEXIN1–Dependent Endosomal Trafficking Pathway

High-temperature-mediated adaptation in plant architecture is linked to the increased synthesis of the phytohormone auxin, which alters cellular auxin homeostasis. The auxin gradient, modulated by cellular auxin homeostasis, plays an important role in regulating the developmental fate of plant organs. Although the signaling mechanism that integrates auxin and high temperature is relatively well understood, the cellular auxin homeostasis mechanism under high temperature is largely unknown. Using the Arabidopsis thaliana root as a model, we demonstrate that under high temperature, roots counterbalance the elevated level of intracellular auxin by promoting shootward auxin efflux in a PIN-FORMED2 (PIN2)-dependent manner. Further analyses revealed that high temperature selectively promotes the retrieval of PIN2 from late endosomes and sorts them to the plasma membrane through an endosomal trafficking pathway dependent on SORTING NEXIN1. Our results demonstrate that recycling endosomal pathway plays an important role in facilitating plants adaptation to increased temperature.     

Oxysterols As Indices of Oxidative Stress in Man After Paraquat Ingestion

The aim of this study is to evaluate oxidative stress in man after paraquat ingestion by analyzing 7 &#102 - and 7 &#103 -hydroperoxycholest-5-en-3 &#103 -ol (7 &#102 - and 7 &#103 -OOH) as well as oxysterols, cholesterol oxidation products, as indices of lipid peroxidation. Lung, kidney, and liver were collected at autopsy from seven patients with paraquat poisoning and seven controls matched for age and sex. We identified for the first time 7-ketocholesterol (7-keto) and 7-hydroxycholesterol (7 &#102 -OH and 7 &#103 -OH) in human kidney by LC-MS. Next, we quantified 7 &#102 -OOH and 7 &#103 -OOH by HPLC with postcolumn chemiluminescence as well as oxysterols by HPLC-UV. Both 7 &#102 -OOH and 7 &#103 -OOH detected in lung and kidney from the controls were as low as the paraquat group. In contrast, we found both 7-keto and 7 &#103 -OH in lung and 7-keto in kidney from the paraquat group were significantly higher than from the controls. This is the first report on accumulated oxysterols in lung and kidney from human paraquat poisoning. It seems to reflect greater oxidative stress in the pathology of paraquat intoxication.     

Detection of Cryptomeria japonica roots with ground penetrating radar

Abstract

Coarse tree roots, which are responsible for most root carbon storage, are usually measured by destructive methods such as excavation and coring. Ground penetrating radar (GPR) is a non-destructive tool that could be used to detect coarse roots in forest soils. In this study, we examined whether the roots of Cryptomeria japonica, a major plantation species in Japan, can be detected with GPR. We also looked for factors that impact the analysis and detection of roots. Roots and wooden dowels of C. japonica were buried 30 cm deep in sandy granite soil. From GPR measurements with a 900 MHz antenna, the distribution and diameter of samples in several transects were recorded. The buried roots were detected clearly and could be distinguished at diameters of 1.1–5.2 cm. There were significant positive relationships between root diameter and parameters extracted from the resultant GPR waveform. The difference in water content between roots and soil is a crucial factor impacting the ability to detect roots with GPR. We conclude that GPR can be used as a non-destructive tool, but further investigation is needed to determine optimal conditions (e.g. water content) and analytical methods for using GPR to examine roots in forest sites.     

Oxidative stress and aging in Caenorhabditis elegans

Much attention has been focused on the hypothesis that oxidative damage plays in cellular and organismal aging. A mev-1 (kn1) mutant of Caenorhabditis elegans, isolated on the basis of its methyl viologen (paraquat) hypersensitivity, is also hypersensitive to elevated oxygen levels. Unlike the wild type, its life span decreases dramatically as oxygen concentrations are increased from 1% to 60%. Strains, which bear this mutation, accumulate fluorescent materials and protein carbonyl groups, markers of aging, at faster rates than the wild type. We have cloned mev-1 gene by transformation rescue and found that it is, in fact, the previously sequenced gene (cyt-1) that encodes succinate dehydrogenase cytochrome b. A missense mutation abolishes complex II activity in the mitochondrial membrane but not succinate dehydrogenase enzyme activity per se. These data suggest that CYT-1 directly participates in electron transport from FADH₂ to coenzyme Q. Moreover, mutational inactivation of this process renders animals susceptible to oxidative stress and, as a result, leads to premature aging.     

Global overexpression of divalent metal transporter 1 delays crocidolite-induced mesothelial carcinogenesis in male mice

Abstract

Exposure to asbestos fiber is central to mesothelial carcinogenesis, for which iron overload in or near mesothelial cells is a key pathogenic mechanism. Alternatively, iron chelation therapy with deferasirox or regular phlebotomy was significantly preventive against crocidolite-induced mesothelial carcinogenesis in rats. However, the role of iron transporters during asbestos-induced carcinogenesis remains elusive. Here, we studied the role of divalent metal transporter 1 (DMT1; Slc11a2), which is a Fe(II) transporter, that is present not only on the apical plasma membrane of duodenal cells but also on the lysosomal membrane of every cell, in crocidolite-induced mesothelial carcinogenesis using DMT1 transgenic (DMT1Tg) mice. DMT1Tg mice show mucosal block of iron absorption without cancer susceptibility under normal diet. We unexpectedly found that superoxide production was significantly decreased upon stimulation with crocidolite both in neutrophils and macrophages of DMT1Tg mice, and the macrophage surface revealed higher iron content 1?h after contact with crocidolite. Intraperitoneal injection of 3?mg crocidolite ultimately induced malignant mesothelioma in ～50% of both wild-type and DMT1Tg mice (23/47 and 14/28, respectively); this effect was marginally (p?=?0.069) delayed in DMT1Tg mice, promoting survival. The promotional effect of nitrilotriacetic acid was limited, and the liver showed significantly higher iron content both in DMT1Tg mice and after crocidolite exposure. The results indicate that global DMT1 overexpression causes decreased superoxide generation upon stimulation in inflammatory cells, which presumably delayed the promotional stage of crocidolite-induced mesothelial carcinogenesis. DMT1Tg mice with low-stamina inflammatory cells may be helpful to evaluate the involvement of inflammation in various pathologies.