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.     

Analysis of methylparaben in cosmetics based on a chemiluminescence H2O2−NaIO4−CNQDs system

In this article, a simple, effective chemiluminescence (CL) method for the detection of methylparaben (MP) in cosmetic samples was developed based on an IO₄^?–H₂O₂–carbon nitrogen quantum dots (CNQDs) system without a separation process. The results indicated that the redox reaction between periodate and hydrogen peroxide released hydroxide radicals and superoxide radical anions in the presence of bicarbonate. These two radicals were responsible for the formation of excited luminophor CNQD* with a maximum wavelength at 480 nm. Due to the competitive reaction with hydroxide radicals, CL intensity was markedly diminished in the presence of MP. The relative standard deviation in the intraday assay was below 5.5% (n = 9), and the detection limit was as low as 0.50 μmol/L. The proposed method allowed for the successful, selective determination of MP in cosmetics.     

Knockout of Akt1/2 suppresses the metastasis of human prostate cancer cells CWR22rv1 in vitro and in vivo

Although primary androgen deprivation therapy resulted in tumour regression, unfortunately, majority of prostate cancer progress to a lethal castration-resistant prostate cancer, finally die to metastasis. The mutual feedback between AKT and AR pathways plays a vital role in the progression and metastasis of prostate cancer. Therefore, the treatment of a single factor will eventually inevitably lead to failure. Therefore, better understanding of the molecular mechanisms underlying metastasis is critical to the development of new and more effective therapeutic agents. In this study, we created prostate cancer CWR22rv1 cells with the double knockout of Akt1 and Akt2 genes through CRISPR/Cas9 method to investigate the effect of Akt in metastasis of prostate cancer. It was found that knockout of Akt1/2 resulted in markedly reduced metastasis in vitro and in vivo, and appeared to interfere AR nuclear translocation through regulating downstream regulatory factor, FOXO proteins. It suggests that some downstream regulatory factors in the AKT and AR interaction network play a vital role in prostate cancer metastasis and are potential targeting molecules for prostate cancer metastasis treatment.     

Prostaglandin E3 attenuates macrophage-associated inflammation and prostate tumour growth by modulating polarization

Alternative polarization of macrophages regulates multiple biological processes. While M1-polarized macrophages generally mediate rapid immune responses, M2-polarized macrophages induce chronic and mild immune responses. In either case, polyunsaturated fatty acid (PUFA)-derived lipid mediators act as both products and regulators of macrophages. Prostaglandin E₃ (PGE₃) is an eicosanoid derived from eicosapentaenoic acid, which is converted by cyclooxygenase, followed by prostaglandin E synthase successively. We found that PGE₃ played an anti-inflammatory role by inhibiting LPS and interferon-γ-induced M1 polarization and promoting interleukin-4-mediated M2 polarization (M2a). Further, we found that although PGE₃ had no direct effect on the growth of prostate cancer cells in vitro, PGE₃ could inhibit prostate cancer in vivo in a nude mouse model of neoplasia. Notably, we found that PGE₃ significantly inhibited prostate cancer cell growth in a cancer cell-macrophage co-culture system. Experimental results showed that PGE₃ inhibited the polarization of tumour-associated M2 macrophages (TAM), consequently producing indirect anti-tumour activity. Mechanistically, we identified that PGE₃ regulated the expression and activation of protein kinase A, which is critical for macrophage polarization. In summary, this study indicates that PGE₃ can selectively promote M2a polarization, while inhibiting M1 and TAM polarization, thus exerting an anti-inflammatory effect and anti-tumour effect in prostate cancer.     

The detrimental effect of iron on OA chondrocytes: Importance of pro-inflammatory cytokines induced iron influx and oxidative stress

Iron overload is common in elderly people which is implicated in the disease progression of osteoarthritis (OA), however, how iron homeostasis is regulated during the onset and progression of OA and how it contributes to the pathological transition of articular chondrocytes remain unknown. In the present study, we developed an in vitro approach to investigate the roles of iron homeostasis and iron overload mediated oxidative stress in chondrocytes under an inflammatory environment. We found that pro-inflammatory cytokines could disrupt chondrocytes iron homeostasis via upregulating iron influx transporter TfR1 and downregulating iron efflux transporter FPN, thus leading to chondrocytes iron overload. Iron overload would promote the expression of chondrocytes catabolic markers, MMP3 and MMP13 expression. In addition, we found that oxidative stress and mitochondrial dysfunction played important roles in iron overload-induced cartilage degeneration, reducing iron concentration using iron chelator or antioxidant drugs could inhibit iron overload-induced OA-related catabolic markers and mitochondrial dysfunction. Our results suggest that pro-inflammatory cytokines could disrupt chondrocytes iron homeostasis and promote iron influx, iron overload-induced oxidative stress and mitochondrial dysfunction play important roles in iron overload-induced cartilage degeneration.     

Down-regulated LINC00115 inhibits prostate cancer cell proliferation and invasion via targeting miR-212-5p/FZD5/Wnt/β-catenin axis

Prostate cancer is the second most frequent malignancy in men worldwide, and its incidence is increasing. Therefore, it is urgently required to clarify the underlying mechanisms of prostate cancer. Although the long non-coding RNA LINC00115 was identified as an oncogene in several cancers, the expression and function of LINC00115 in prostate cancer have not been explored. Our results showed that LINC00115 was significantly up-regulated in prostate cancer tissues, which was significantly associated with a poor prognosis for prostate cancer patients. Functional studies showed that knockdown LINC00115 inhibited cell proliferation and invasion. In addition, LINC00115 served as a competing endogenous RNA (ceRNA) through sponging miR-212-5p to release Frizzled Family Receptor 5 (FZD5) expression. The expression of miR-212-5p was noticeably low in tumour tissues, and FZD5 expression level was down-regulated with the knockdown of LINC00115. Knockdown LINC00115 inhibited the Wnt/β‑catenin signalling pathway by inhibiting the expression of FZD5. Rescue experiments further showed that LINC00115 inhibits prostate cancer cell proliferation and invasion via targeting miR-212-5p/ FZD5/ Wnt/β-catenin axis. The present study provided clues that LINC00115 may be a promising novel therapeutic target for prostate cancer patients.     

Eremophilane Sesquiterpenes and Benzene Derivatives from the Endophyte Microdiplodia sp. WGHS5

Three new eremophilane sesquiterpenes phomadecalins G−I ( 1 – 3 ) and two new benzene derivatives microdiplzenes A and B ( 12 and 13 ), together with nine known eremophilane sesquiterpenes ( 4 – 11 and 14 ) were isolated from an endophytic fungus, Microdiplodia sp. WGHS5. Their structures were elucidated by the interpretation of HR-ESI-MS and NMR data; meanwhile, the absolute configurations of new compounds were determined on the base of ECD calculations. All compounds were evaluated for the antimicrobial activities and antiproliferative effect on human gastric cancer cell lines (BGC-823).