Flubendazole exposure disrupts neural development and function of zebrafish embryos (Danio rerio).

Flubendazole (FBZ) is a benzimidazole anthelmintic drug widely used for treating parasitic infections by disrupting microtubule formation and function through tubulin binding. Recently, its use has extended to include anticancer applications, leading to increased environmental exposure to benzimidazole drugs. However, the impact of FBZ on neural development in aquatic organisms, particularly in aquatic vertebrates, remains poorly understood. This study aimed to investigate the potential developmental toxicity of FBZ during neural development using zebrafish model. Various assessments, including analysis of overall developmental changes, morphological abnormalities, apoptosis, gene expression alterations, axon length measurements, and electrophysiological neural function, were performed. FBZ exposure resulted in concentration-dependent effects on survival rate, hatching rate, heartbeat, and the occurrence of developmental abnormalities. Notably, FBZ-induced changes included reductions in body length, head size, and eye size, as well as the detection of apoptotic cells in the central nervous system. Gene expression analysis revealed upregulation of apoptosis-related genes (p53, casp3, and casp8), downregulation of neural differentiation-related genes (shha, nrd, ngn1, and elavl3), and alterations in neural maturation and axon growth-related genes (gap43, mbp, and syn2a). Additionally, shortened motor neuron axon length and impaired electrophysiological neural function were observed. These findings provide novel insights into the potential risks of FBZ on the neural development of zebrafish embryos, emphasizing the need for risk prevention strategies and therapeutic approaches to address the environmental toxicity of benzimidazole anthelmintics.

Generation of human iPSCs derived heart organoids structurally and functionally similar to heart.

Currently, due to the increasing demand for 3D culture, various organoids that mimic organs are being actively studied. Despite active reports, information on heart organoids (HOs), which are the first functional organs, is still insufficient. Parameters for reproducing hearts are: chamber formation, organization with cardiac cells, vascularization, and simulation of electrophysiological signals. In particular, since the heart reflects complex factors, it is necessary to develop HOs that can be simulated in depth. In this study, we have created self-organized HOs using human iPSCs, and validated mimicry of cardiac structures such as chamber and epicardium/myocardium and atrium/ventricle-similar areas. Furthermore, mechanical/electrophysiological features were verified through multiple analyzes after inhibition of ion channels. More importantly, the HOs function, due to the cardiovascular characteristics of HOs, was maintained through vascularization after in vivo transplantation. In conclusion, this study has the advantage of being able to easily and closely recapitulate morphological/functional aspects of the heart.

Immunomodulation of Pluripotent Stem Cell-Derived Mesenchymal Stem Cells in Rotator Cuff Tears Model.

BACKGROUND: Rotator cuff tears (RCTs) induce chronic muscle weakness and shoulder pain. Treatment of RCT using surgery or drugs causes lipid infiltration and fibrosis, which hampers tissue regeneration and complete recovery. The pluripotent stem cell-derived multipotent mesenchymal stem cells (M-MSCs) represent potential candidate next-generation therapies for RCT. METHODS: The difference between M-MSCs and adult-MSCs was compared and analyzed using next-generation sequencing (NGS). In addition, using a rat model of RCT, the muscle recovery ability of M-MSCs and adult-MSCs was evaluated by conducting a histological analysis and monitoring the cytokine expression level. RESULTS: Using NGS, it was confirmed that M-MSC was suitable for transplantation because of its excellent ability to regulate inflammation that promotes tissue repair and reduced apoptosis and rejection during transplantation. In addition, while M-MSCs persisted for up to 8 weeks in vivo, they significantly reduced inflammation and adipogenesis-related cytokine levels in rat muscle. Significant differences were also confirmed in histopathological remission. CONCLUSIONS: M-MSCs remain in the body longer to modulate immune responses in RCTs and have a greater potential to improve muscle recovery by alleviating acute inflammatory responses. This indicates that M-MSCs could be used in potential next-generation RCT therapies.

Albendazole exerts antiproliferative effects on prostate cancer cells by inducing reactive oxygen species generation.

Benzimidazole derivatives are used for their antihelmintic properties, but have also been reported to exert anticancer effects. In the present study, the anticancer effects of albendazole on prostate cancer cells were assessed using proliferation, clonogenic and migration assays. To investigate the anticancer mechanisms of albendazole, reactive oxygen species (ROS) levels were measured, and the expression of genes associated with oxidative stress and Wnt/ß-catenin signaling was confirmed by reverse transcription-quantitative PCR and western blotting. Albendazole selectively inhibited the proliferation of the PC3, DU145, LNCaP and AT2 prostate cancer cell lines at concentrations that did not affect the proliferation of a normal prostate cell line (RWPE-1). Albendazole also inhibited the colony formation and migration of PC3 and DU145 cells, as well as inducing ROS production. Diphenyleneiodonium chloride, an inhibitor of NADPH oxidase (NOX), one of the sources of ROS, decreased basal ROS levels in the PC3 and DU145 cells, but did not reduce albendazole-associated ROS production, suggesting that ROS production following albendazole treatment was NOX-independent. The anticancer effect was decreased when albendazole-induced ROS was reduced by treatment with antioxidants (glutathione and N-acetylcysteine). Furthermore, albendazole decreased the mRNA expression of CDGSH iron sulfur domain 2, which regulates antioxidant activity against ROS, as well as the antioxidant enzymes catalase, and glutathione peroxidase 1 and 3. Albendazole also decreased the mRNA expression of catenin ß1 and transcription factor 4, which regulate Wnt/ß-catenin signaling and its associated targets, Twist family BHLH transcription factor 1 and BCL2. The albendazole-related decrease in the expression levels of oxidative stress-related genes and Wnt/ß-catenin signaling proteins was thought to be associated with ROS production. These results suggest that the antihelmintic drug, albendazole, has inhibitory effects against prostate cancer cells in vitro. Therefore, albendazole may potentially be used as a novel anticancer agent for prostate cancer.

In vivo tracking of 14C thymidine labeled mesenchymal stem cells using ultra-sensitive accelerator mass spectrometry.

Despite the tremendous advancements made in cell tracking, in vivo imaging and volumetric analysis, it remains difficult to accurately quantify the number of infused cells following stem cell therapy, especially at the single cell level, mainly due to the sensitivity of cells. In this study, we demonstrate the utility of both liquid scintillator counter (LSC) and accelerator mass spectrometry (AMS) in investigating the distribution and quantification of radioisotope labeled adipocyte derived mesenchymal stem cells (AD-MSCs) at the single cell level after intravenous (IV) transplantation. We first show the incorporation of 14C-thymidine (5 nCi/ml, 24.2 ng/ml) into AD-MSCs without affecting key biological characteristics. These cells were then utilized to track and quantify the distribution of AD-MSCs delivered through the tail vein by AMS, revealing the number of AD-MSCs existing within different organs per mg and per organ at different time points. Notably, the results show that this highly sensitive approach can quantify one cell per mg which effectively means that AD-MSCs can be detected in various tissues at the single cell level. While the significance of these cells is yet to be elucidated, we show that it is possible to accurately depict the pattern of distribution and quantify AD-MSCs in living tissue. This approach can serve to incrementally build profiles of biodistribution for stem cells such as MSCs which is essential for both research and therapeutic purposes.

Vitrification for cryopreservation of 2D and 3D stem cells culture using high concentration of cryoprotective agents.

BACKGROUND: Vitrification is the most promising technology for successful cryopreservation of living organisms without ice crystal formation. However, high concentrations (up to ~ 6-8 M) of cryoprotective agents (CPAs) used in stem cell induce osmotic and metabolic injuries. Moreover, the application of conventional slow-freezing methods to cultures of 3-D organoids of stem cells in various studies, is limited by their size. RESULTS: In this study, we evaluated the effect of high concentrations of CPAs including cytotoxicity and characterized human mesenchymal stem cell (MSC) at single cell level. The cell viability, cellular damage, and apoptotic mechanisms as well as the proliferation capacity and multipotency of cells subjected to vitrification were similar to those in the slow-freezing group. Furthermore, we identified the possibility of vitrification of size-controlled 3-D spheroids for cryopreservation of organoid with high survivability. CONCLUSIONS: Our results demonstrate successful vitrification of both single cell and spheroid using high concentration of CPAs in vitro without cytotoxicity.

Loss of glutathione peroxidase 3 induces ROS and contributes to prostatic hyperplasia in Nkx3.1 knockout mice.

BACKGROUND: Glutathione peroxidase 3 (Gpx3) protects cells from oxidative stress, and its reduced expression in human prostate cancer has been reported. OBJECTIVES: We hypothesized that Gpx3 might play an important role in the development of prostatic intraepithelial neoplasia (PIN), a pre-cancerous state of the prostate, and aimed to highlight the underlying molecular mechanism. MATERIALS AND METHODS: The following double-knockout mice Nkx3.1-/-; Gpx3+/+, Nkx3.1-/-; Gpx3+/-, Nkx3.1-/-; Gpx3-/- were produced. Randomly divided animals were weighed, and their genitourinary tract (GUT) weights were determined after euthanasia at 4, 8, and 12 months. The mRNA expression of the genes involved in oxidative stress and Wnt signaling was analyzed in the prostate. Histopathology, ROS, and superoxide dismutase (SOD) activities were also measured. RESULTS: Loss of Gpx3 did not affect body weight and GUT weight in Nkx3.1 knockout mice. The mRNA expression of SOD3, iNOS, Hmox, and CISD2, which are associated with oxidative stress, was increased in Nkx3.1-/-; Gpx3-/- mice at 4 months but decreased at 8 and 12 months. There was no change in ß-catenin and its targets associated with Wnt signaling. Increased ROS and decreased SOD activity were observed in Nkx3.1-/-; Gpx3-/- mice at 12 months of age. The histopathologic score and epithelium thickness were increased, and lumen area was decreased in Gpx3 knockout mice. DISCUSSION AND CONCLUSIONS: Gpx3 loss increased the hyperplasia of PIN in the pre-cancerous stage of the prostate. Loss of Gpx3 induced oxidative stress. Histopathologically, no invasive carcinoma was identified, and Gpx3 loss did not increase Wnt/ß-catenin signaling. Further research on the role of GPX3 in the transition of PIN to invasive carcinoma is needed. We show, for the first time, that the antioxidant enzyme GPX3 plays a vital role in inhibiting hyperplasia in the PIN stage of the prostate gland in vivo.

Improved human hematopoietic reconstitution in HepaRG co-transplanted humanized NSG mice.

Several humanized mouse models are being used to study humanspecific immune responses and diseases. However, the pivotal needs of fetal tissues for the humanized mice model have been huddled because of the demand for ethical and medical approval. Thus, we have verified the hematopoietic and immunomodulatory function of HepaRG and developed a new and easy humanized mouse model to replace the use of fetal liver tissue. HepaRG co-transplanted Hu-NSG mice significantly increased CD45+ lymphocytes and CD19+ B cells and CD3+ T cells than normal Hu-NSG, suggesting enhanced reconstitution of the human immune system. These results have improved the applicability of humanized mice by developing new models easily accessible. [BMB Reports 2020; 53(9): 466-471].

Molecular Design of Highly Efficient Heavy-Atom-Free Triplet BODIPY Derivatives for Photodynamic Therapy and Bioimaging.

Novel BODIPY photosensitizers were developed for imaging-guided photodynamic therapy. The introduction of a strong electron donor to the BODIPY core through a phenyl linker combined with the twisted arrangement between the donor and the BODIPY acceptor is essential for reducing the energy gap between the lowest singlet excited state and the lowest triplet state (ΔEST ), leading to a significant enhancement in the intersystem crossing (ISC) of the BODIPYs. Remarkably, the BDP-5 with the smallest ΔEST (ca. 0.44 eV) exhibited excellent singlet oxygen generation capabilities in both organic and aqueous solutions. BDP-5 also displayed bright emission in the far-red/near-infrared region in the condensed states. More importantly, both in vitro and in vivo studies demonstrated that BDP-5 NPs displayed a high potential for photodynamic cancer therapy and bioimaging.

Phloretin Inhibits the Human Prostate Cancer Cells Through the Generation of Reactive Oxygen Species.

Phloretin is a flavonoid with known anticancer activities. However, we do not fully understand how phloretin mitigates prostate cancer on the molecular level. In the present study, we examined changes in proliferation, colony formation, and migration after phloretin treatment in human prostate cancer cells PC3 and DU145. We measured reactive oxygen species (ROS) and gene expression. Phloretin increased ROS and suppressed cell proliferation, migration, and colony formation in both cell lines. Additionally, phloretin treatment increased oxidative stress, as demonstrated through lower antioxidant enzymes (catalase, SOD2, Gpx1, Gpx3). In addition, their regulator CISD2 decreased in expression. We also found that increased ROS significantly downregulated multiple components of the Wnt/ß-catenin signaling pathway (ß-catenin, TCF4, FoxA2, c-Myc) and Twist1. Thus, anticancer activity of phloretin against human prostate cancer cells occurs through generating ROS to influence Wnt/ß-catenin signaling. The results of this study suggest that phloretin has a therapeutic effect on prostate cancer in vitro, inhibiting the proliferation and migration of cancer cell lines PC3 and DU145. The mechanism of phloretin appears to be increasing ROS production. We thus recommend phloretin as a promising anticancer therapeutic agent.

Correction to: Phloretin Inhibits the Human Prostate Cancer Cells through the Generation of Reactive Oxygen Species.

AbstractThe original version of this article unfortunately contained an error in Figs. 1, 5 and 6. The asterisks and bars indicating statistical significance were missing in the figures.

Phenotyping analysis of p53 knockout mice produced by gene editing and comparison with conventional p53 knockout mice.

BACKGROUND: Knockout (KO) mice developed by homologous recombination (HR) have become useful tools to elucidate gene function. However, HR has low KO efficiency and is time-consuming, labor-intensive, and expensive. 'Gene editing' has received much attention for efficient genetic manipulation. OBJECTIVE: As generation of KO mice is simplified, KO mice produced by HR can be feasibly reproduced using gene editing. However, phenotyping analysis and comparison between KO mice produced by these two techniques is necessary. METHODS: We generated p53 KO mice through gene editing and compared their phenotype with the already reported HR-mediated p53 KO mice. RESULTS: Tumors occurred in 36 (73%) of 49 homozygous KO mice and the mean age of occurrence was 23 weeks, with lymphoma (64%) and sarcoma (23%) being the most common. Tumors were also developed in 12 heterozygous mice and the mean age of occurrence was 40 weeks, with sarcoma (54%) and lymphoma (46%) in high proportion. Homozygotes had a mean life span of 157 ± 52 days and developmental abnormalities were found in females compared to in males (P < 0.05, P < 0.001). CONCLUSION: We analyzed the basic phenotype of p53 KO mice and observed no significant difference from the conventional HR-mediated p53 KO mice.

Pimozide Inhibits the Human Prostate Cancer Cells Through the Generation of Reactive Oxygen Species.

The United States Food and Drug Administration-approved antipsychotic drug, pimozide, has anticancer activities. However, the role of reactive oxygen species (ROS) in its effect on prostate cancer is not well-known. We examined cell proliferation, colony formation, migration, ROS production, and the expression of antioxidant-related genes after treatment of human prostate cancer PC3 and DU145 cells with pimozide. In addition, histopathology, ROS production, and superoxide dismutase (SOD) activity were analyzed after administering pimozide to TRAMP, a transgenic mouse with prostate cancer. Pimozide increased the generation of ROS in both cell lines and inhibited cell proliferation, migration, and colony formation. Oxidative stress induced by pimozide caused changes in the expression of antioxidant enzymes (SOD1, peroxiredoxin 6, and glutathione peroxidase 2) and CISD2. Co-treatment with glutathione, an antioxidant, reduced pimozide-induced ROS levels, and counteracted the inhibition of cell proliferation. Administration of pimozide to TRAMP mice reduced the progression of prostate cancer with increased ROS generation and decreased SOD activity. These results suggest that the antipsychotic drug, pimozide, has beneficial effects in prostate cancer in vivo and in vitro. The mechanism of pimozide may be related to augmenting ROS generation. We recommend pimozide as a promising anticancer agent.

Mesenchymal stem cell-driven activatable photosensitizers for precision photodynamic oncotherapy.

Precise targeting with minimal side effects is of particular interest for personalized medicine, although it remains a challenge. Herein, we demonstrate precision photodynamic therapy (PDT) utilizing human mesenchymal stem cells (MSCs) as cellular vehicles to deliver a new activatable photosensitizer (PcS). In vivo real-time optical imaging tests indicated that PcS-loaded MSCs possess excellent tumor-homing properties. More importantly, dye transfer assays confirm that MSCs precisely transfer PcS into human colon cancer cells (HCT116) via the "bystander effect." Upon localized light irradiation, the growth of intraperitoneal xenograft tumors was significantly inhibited by the photodynamic effect. These findings represent a promising strategy for precise oncotherapy.

The Pharmacological Inhibition of ERK5 Enhances Apoptosis in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs. Extracellular signal-regulated kinase 5 (ERK5) plays a novel role in chemoresistance in some cancer cells and this pathway is a central mediator of cell survival and apoptotic regulation. The aim of this study was to investigate the effect of ERK5 inhibitor, XMD8-92, on proliferation and apoptosis in AML cell lines. Findings showed that XMD8-92 inhibited the activation of ERK5 by G-CSF and decreased the expression of c-Myc and Cyclin D1. The treatment of XMD8-92 reduced the phosphorylation of ERK5 leading to a distinct inhibition of cell proliferation and increased apoptosis in Kasumi-1 and HL-60 cells. Taken together, our study suggests that the inhibition of ERK5 by XMD8-92 can trigger apoptosis and inhibit proliferation in AMLs. Therefore, the inhibition of ERK5 may be an effective adjuvant in AML chemotherapy.

An outbreak of toxoplasmosis in squirrel monkeys (Saimiri sciureus) in South Korea.

BACKGROUND: Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite that can infect warm-blooded animals including humans. New World monkeys, such as squirrel monkeys, are more susceptible to T. gondii than Old World monkeys, often developing fatal disease. METHODS: In this study, seven of thirteen dead squirrel monkeys at Seoul Grand Park were tested to find the cause of sudden death. RESULTS: The main histopathological findings included interstitial pneumonia, necrotizing hepatitis, and splenitis. Periodic acid-Schiff staining of liver, spleen, and lung revealed cyst structures consistent with bradyzoites. Amplification of the B1 gene was detected in the liver or spleen of all monkeys. Additionally, a restriction fragment length polymorphism assay and phylogenetic analysis of the GRA6 amplicon revealed a consistent clustering with the type II strain of T. gondii. CONCLUSIONS: This study is the first report of T. gondii infection of squirrel monkeys in Korea, and the first report of type II T. gondii based on GRA6 analysis in Korea.

The Bromodomain Inhibitor JQ1 Enhances the Responses to All-trans Retinoic Acid in HL-60 and MV4-11 Leukemia Cells.

All-trans retinoic acid (ATRA) is a highly effective treatment for acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML). However, ATRA-based treatment is not effective in other subtypes of AML. In non-APL AML, ATRA signaling pathway is impaired or downmodulated, and consequently fails to respond to pharmacological doses of ATRA. Therefore, complementary treatment strategies are needed to improve ATRA responsiveness in non-APL AML. In this study, we investigated the combined effect of ATRA and bromodomain inhibitor JQ1, proven to have potent anti-cancer activity mainly through inhibition of c-Myc. We showed that the combination of ATRA with JQ1 synergistically inhibited proliferation of AML cells. The synergistic growth inhibition was resulted from differentiation or apoptosis depending on the kind of AML cells. Concomitantly, the combined treatment of ATRA and JQ1 caused greater depletion of c-Myc and hTERT expression than each agent alone in AML cells. Taken together, these findings support the rationale for the use of the combination of ATRA and JQ1 as a therapeutic strategy for the treatment of AML.

Immunomodulatory effect of CD200-positive human placenta-derived stem cells in the early phase of stroke.

Human placenta amniotic membrane-derived mesenchymal stem cells (AMSCs) regulate immune responses, and this property can be exploited to treat stroke patients via cell therapy. We investigated the expression profile of AMSCs cultured under hypoxic conditions and observed interesting expression changes in various genes involved in immune regulation. CD200, an anti-inflammatory factor and positive regulator of TGF-ß, was more highly expressed under hypoxic conditions than normoxic conditions. Furthermore, AMSCs exhibited inhibition of pro-inflammatory cytokine expression in co-cultures with LPS-primed BV2 microglia, and this effect was decreased in CD200-silenced AMSCs. The AMSCs transplanted into the ischemic rat model of stroke dramatically inhibited the expression of pro-inflammatory cytokines and up-regulated CD200, as compared with the levels in the sham-treated group. Moreover, decreased microglia activation in the boundary region and improvements in behavior were confirmed in AMSC-treated ischemic rats. The results suggested that the highly expressed CD200 from the AMSCs in a hypoxic environment modulates levels of inflammatory cytokines and microglial activation, thus increasing the therapeutic recovery potential after hypoxic-ischemic brain injury, and further demonstrated the immunomodulatory function of AMSCs in a stroke model.

Development of an alternative zebrafish model for drug-induced intestinal toxicity.

An evaluation of intestinal toxicity is important because the mucosal lining of the gastrointestinal tract is the first barrier for oral xenobiotics. Until now, a rat model has been recommended as the standard intestinal toxicity model and the Caco-2 cell line, originated from a human colon adenocarcinoma, has been used as an alternative to this model, but there are limitations regarding cost-effectiveness and the need for mimicry of the human system. In this study, we investigated whether zebrafish could be a valid alternative to rats and Caco-2 cells as an intestinal toxicity model. We focused on intestinal gene expression of cytochrome P450 3A65, oxidative stress, apoptosis, inflammation, and intestinal function. Reverse transcription-quantitative polymerase chain reaction analysis was conducted using three models: zebrafish, Sprague-Dawley rats and Caco-2 cells, and the transcript levels and patterns of indicator genes were analyzed in conjunction with histopathological changes. Our results suggested that representative intestinal toxicants, indomethacin, diclofenac and methotrexate, induced significant transcript level changes in marker genes such as CYP3A, inducible nitric oxide synthase, heme oxygenase 1, superoxide dismutase 1, glutathione peroxidase 1, BCL2 associated X, B-cell lymphoma 2, caspase 9, tumor protein p53, nuclear factor-κB, interleukin-1ß, tumor necrosis factor-alphaα and toll-like receptor 2 in the zebrafish model as in the rat and Caco-2 cells models. These results suggest that zebrafish model is sufficiently worth developing as an intestinal toxicity model that can replace or compensate the rat model or Caco-2 cell model.

Nanostructured Phthalocyanine Assemblies with Protein-Driven Switchable Photoactivities for Biophotonic Imaging and Therapy.

Switchable phototheranostic nanomaterials are of particular interest for specific biosensing, high-quality imaging, and targeted therapy in the field of precision nanomedicine. Here, we develop a "one-for-all" nanomaterial that self-assembles from flexible and versatile phthalocyanine building blocks. The nanostructured phthalocyanine assemblies (NanoPcTBs) display intrinsically unique photothermal and photoacoustic properties. Fluorescence and reactive oxygen species generation can be triggered depending on a targeted, protein-induced, partial disassembly mechanism, which creates opportunities for low-background fluorescence imaging and activatable photodynamic therapy. In vitro evaluations indicate that NanoPcTB has a high selectivity for biotin receptor-positive cancer cells (e.g., A549) compared to biotin receptor-negative cells (e.g., WI38-VA13) and permits a combined photodynamic and photothermal therapeutic effect. Following systemic administration, the NanoPcTBs accumulate in A549 tumors of xenograft-bearing mice, and laser irradiation clearly induces the inhibition of tumor growth.