Size-Dependent Internalization of Microplastics and Nanoplastics Using In Vitro Model of the Human Intestine-Contribution of Each Cell in the Tri-Culture Models.

Pollution by microplastics and nanoplastics (MNPs) raises concerns, not only regarding their environmental effects, but also their potential impact on human health by internalization via the small intestine. However, the detailed pathways of MNP internalization and their toxicities to the human intestine have not sufficiently been understood, thus, further investigations are required. This work aimed to understand the behavior of MNPs, using in vitro human intestine models, tri-culture models composed of enterocyte Caco-2 cells, goblet-like HT29-MTX-E12 cells, and microfold cells (M cells) induced by the lymphoblast cell line Raji B. Three sizes (50, 100, and 500 nm) of polystyrene (PS) particles were exposed as MNPs on the culture model, and size-dependent translocation of the MNPs and the contributions of each cell were clarified, emphasizing the significance of the tri-culture model. In addition, potential concerns of MNPs were suggested when they invaded the circulatory system of the human body.

Modulation of hepatic cellular tight junctions via coculture with cholangiocytes enables non-destructive bile recovery.

Estimation of the biliary clearance of drugs and their metabolites in humans is crucial for characterizing hepatobiliary disposition and potential drug-drug interactions. Sandwich-cultured hepatocytes, while useful for in vitro bile analysis, require cell destruction for bile recovery, limiting long-term or repeated dose drug effect evaluations. To overcome this limitation, we investigated the feasibility of coculturing a human hepatic carcinoma cell line (HepG2-NIAS cells) and a human cholangiocarcinoma cell line (TFK-1 cells) using the collagen vitrigel membrane in a variety of coculture configurations. The coculture configuration with physiological bile flow increased the permeability of fluorescein-labeled bile acids (CLF) across the HepG2-NIAS cell layer by approximately 1.2-fold compared to the HepG2-NIAS monoculture. This enhancement was caused by paracellular leakage due to the loosened tight junctions of HepG2-NIAS, confirmed by the use of an inhibitor for bile acid transporters, the increase of permeability of dextran, and the decrease of the transepithelial electrical resistance (TEER) value. Based on the results of loosening hepatic tight junctions via coculture with TFK-1 in the CLF permeability assay, we next attempted to collect the CLF accumulated in the bile canaliculi of HepG2-NIAS. The recovery of the CLF accumulated in the bile canaliculi was increased 1.4 times without disrupting hepatic tight junctions by the coculture of HepG2-NIAS cells and TFK-1 cells compared to the monoculture of HepG2-NIAS cells. This non-destructive bile recovery has the potential as a tool for estimating the biliary metabolite and provides valuable insights to improve in vitro bile analysis.

Ethyl acetate fraction of Osmanthus fragrans var. aurantiacus and its triterpenoids suppress proliferation and survival of colorectal cancer cells by inhibiting NF-κB and COX2.

ETHNOPHARMACOLOGICAL RELEVANCE: Colorectal cancer (CRC) remains a significant global health concern, and targeting inflammation has emerged as a promising approach for its prevention and treatment. Medicinal plants and phytochemicals have garnered attention for their potential efficacy against inflammation with minimal toxicity. Osmanthus fragrans var. aurantiacus Makino (O. fragrans) has a history of traditional use in Korea and China in treating various inflammation-related conditions, but its potential use for CRC has not been uncovered. AIM OF THE STUDY: This study aims to explore the potential anti-proliferative and pro-apoptotic properties of O. fragrans, focusing on its impact on CRC treatment. By investigating O. fragrans, we aim to uncover its anti-proliferative and apoptotic effects in human CRC cells, potentially paving the way for effective and well-tolerated therapeutic strategies for CRC patients. MATERIALS AND METHODS: Ethanol (EtOH) extracts of O. fragrans leaf and flower, along with specific fractions (n-hexane, ethyl acetate (EtOAc), n-butanol, and the aqueous residue) were evaluated for their anti-proliferative effects in human CRC cells using MTT assays, and compared to normal colon cells. Mechanistic insights and chemical profiling were obtained through flow cytometry, colorimetric assays, western blotting, and molecular docking, and high-performance liquid chromatography (HPLC) system. RESULTS: Both flower and leaf EtOH extracts of O. fragrans exhibited significant anti-proliferative effects in human CRC cells, with the leaf extract demonstrating higher potency. The EtOAc fraction from the leaf extract displayed the strongest anti-CRC cell proliferative effects while no cytotoxic effects in normal colon cells. Chemical profiling of these fractions identified triterpenoids as significant components in the EtOAc fractions. The leaf EtOAc fraction caused cell cycle arrest and apoptosis, accompanied by elevating intracellular reactive oxygen species and mitochondrial dysfunction in CRC cells. Additionally, it inhibited NF-κB and ERK1/2 signaling, leading to reduced COX2 expression. Notably, two triterpenoids isolated from the leaf EtOAc fraction, maslinic acid and corosolic acid, displayed potent anti-cancer activity in CRC cells without affecting normal colon cells. Corosolic acid exhibited a strong binding affinity to COX2 and reduced its expression, supporting its role in the anti-inflammatory and anti-cancer effects. CONCLUSIONS: Our findings suggest that O. fragrans, particularly its triterpenoid-rich EtOAc fraction, holds promise as a novel therapeutic agent for CRC prevention and therapy. These results provide valuable insights into the potential application of O. fragrans and its bioactive compounds in combating CRC.

Anti-inflammatory and antioxidant activity of elemental sulfur-derived sustainable sulfur quantum dots.

Sulfur quantum dots (SQDs) are attracting increasing attention in the biomedical field due to their unique properties, such as antibacterial activity, free radical scavenging potential, optical properties, biocompatibility, and non-toxicity. Ethylenediamine passivated SQDs (ED-SQDs) were synthesized using a hydrothermal method. Cytotoxicity evaluation of ED-SQDs on RAW264.7 cells showed more than 90% cell viability even at 500 µg/mL of ED-SQDs, with an established IC50 value of 880.9 µg/mL. In addition, ED-SQDs showed potent antioxidant activity in vitro, effectively scavenging ABTS and DPPH free radicals at concentrations below 100 µg/mL, comparable to ascorbic acid. ED-SQD reduced lipopolysaccharide (LPS)-induced nitric oxide and reactive oxygen species in macrophages, lowered pro-inflammatory cytokines, and inactivated LPS-activated STAT3. In addition, ED-SQD increased nuclear NRF2 and the expression of genes encoding antioxidant enzymes in LPS-stimulated cells. These results reveal the antioxidant and anti-inflammatory potential of ED-SQDs at non-toxic concentrations, providing evidence for their potential anti-inflammatory applications.

Effectiveness and Safety of Progressive Loading-Motion Style Acupuncture Treatment for Acute Low Back Pain after Traffic Accidents: A Randomized Controlled Trial.

BACKGROUND: Traffic injuries include acute low back pain (LBP) needing active treatment to prevent chronicity. This two-armed, parallel, assessor-blinded, randomized controlled trial evaluated the effectiveness and safety of progressive loading-motion style acupuncture treatment (PL-MSAT) for acute LBP following traffic accidents. METHODS: Based on an effect size of 1.03, 104 participants were recruited and divided in a 1:1 ratio into PL-MAST and control groups using block randomization. Both groups underwent integrative Korean medicine treatment (IKMT) daily; only the PL-MSAT group underwent three PL-MSAT sessions. The outcomes were assessed before and after the treatment sessions and at 1 and 3 months post-discharge. The primary outcome was the difference in the numeric rating scale (NRS) for LBP. The secondary outcomes included a visual analog scale for LBP, leg pain status, the Oswestry disability index, lumbar active range of motion (ROM), quality of life, Patient Global Impression of Change, and Post-Traumatic Stress Disorder Checklist adverse events. RESULTS: In the modified intention-to-treat analysis, 50 and 51 participants were included in the PL-MSAT and control groups. On Day 4, the mean LBP NRS score was 3.67 (3.44-3.90) in the PL-MSAT group, indicating a significantly lower NRS 0.77 (0.44-1.11) compared to 4.44 (4.20-4.68) for the control group (p < 0.001). The PL-MSAT group exhibited greater ROM flexion (-5.31; -8.15 to -2.48) and extension (-2.09; -3.39 to -0.80). No significant differences were found for the secondary outcomes and follow-ups. CONCLUSIONS: Compared with IKMT alone, PL-MSAT plus IKMT showed significantly better outcomes for reducing pain and increasing the ROM in acute LBP.

Antioxidant and Anti-Inflammatory Activities of High-Glucosinolate-Synthesis Lines of Brassica rapa.

Excessive oxidative stress and inflammatory responses are associated with the development of various diseases, including cancer. Glucosinolates (GSLs) are phytochemicals known for their antioxidant properties, and doubled haploid lines (DHLs) of Brassica rapa with high GSL contents (HGSL) were intentionally developed from two edible subspecies of Brassica rapa: B. rapa subsp. trilocularis and B. rapa subsp. chinensis. The purpose of the present study is to assess the capacity of HGSL DHLs to mitigate oxidative stress and inflammation in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, compared to pak choi as a parental control. Our findings demonstrate that HGSL DH lines effectively suppressed the expression of inducible nitric oxide synthase, leading to the reduced levels of nitric oxide at non-toxic concentrations. Additionally, these lines exhibited scavenging activity against reactive oxygen species and free radicals. The enhanced antioxidant capacity of HGSL DHLs was mechanistically attributed to the upregulation of antioxidant enzymes, such as NADPH quinone oxidoreductase 1 (NQO1), the glutamate-cysteine ligase catalytic subunit (GCLC), and heme oxygenase-1 (HMOX1). Furthermore, we confirmed that these effects were mediated through the nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway via p38 phosphorylation. Moreover, HGSL DHLs demonstrated inhibitory effects on pro-inflammatory cytokines and signal transducers and activators of transcription 3 (STAT3) phosphorylation. Collectively, our results indicate that HGSL DHLs possess better antioxidant and anti-inflammatory properties compared to the parental control pak choi in LPS-stimulated RAW264.7 cells, suggesting that HGSL DHLs of Brassica rapa could be considered as a beneficial daily vegetable for reducing the risk of inflammation-associated diseases.

Inflammatory liver tissue formation using oxygen permeable membrane based culture platform.

During chronic liver injury, inflammation leads to liver fibrosis, particularly due to the activation of hepatic stellate cells (HSCs). The involvement of inflammatory cytokines in HSC activation and the interplay among different liver cells are elaborated. To examine their interactions in vitro, many cultured liver tissue models are performed in organoid or spheroid culture with random 3D structure. Herein, we demonstrated the hierarchical coculture of primary rat hepatocytes with non-parenchymal cells such as the human-derived HSC line (LX-2) and liver sinusoidal endothelial cell line (TMNK-1). The cocultured tissue had high usability with simple operation of separating solid and liquid phases with improved liver functions such as albumin production and hepatic cytochrome P450 3A4 activity. We also studied the effects of stimulation by both oxygen tension and the key pro-fibrogenic cytokine, transforming growth factor beta (TGF-ß), on HSC activation. Gene expression of collagen type I and alpha-smooth muscle actin were enhanced in the hierarchical coculture under lower oxygen tension and TGF-ß1 stimulation. Therefore, this hierarchical in vitro cocultured liver tissue could provide a useful platform as a disease model for elucidating the interactions of various liver cell types and biochemical signals in future liver fibrogenesis studies.

Optimization of physical microenvironment to maintain the quiescence of human induced pluripotent stem cell-derived hepatic stellate cells.

Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis by producing excessive extracellular matrix (ECM) following chronic inflammation. However, studying HSC function has been challenging due to the limited availability of primary human quiescent HSCs (qHSCs) in vitro, and the fact that primary qHSCs quickly activate when cultured on plastic plates. Advances in stem cell technology have allowed for the generation of qHSCs from human induced pluripotent stem cells (hiPSCs) with the potential to provide an unlimited source of cells. However, differentiated quiescent-like HSCs (iqHSCs) also activate spontaneously on conventional plastic plates. In this study, we generated iqHSCs from hiPSCs and developed a culture method to maintain such iqHSCs in a lowly activated state for up to 5 days by optimizing their physical culture microenvironment. We observed that three-dimensional (3D) culture of iqHSCs in soft type 1 collagen hydrogels significantly inhibited their spontaneous activation in vitro while maintaining their ability to convert to activated state. Activation of iqHSC was successfully modeled by stimulating them with the fibrotic cytokine TGFß1. Hence, our culture method can be used to generate HSCs with functions comparable to those in a healthy liver, facilitating the development of accurate in vitro liver models for identifying novel therapeutic agents.

Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) pathway is a powerful tool to combat tumor growth and progression; however, drug resistance frequently emerges. We identify CD5L (CD5 antigen-like precursor) as an important gene upregulated in response to antiangiogenic therapy leading to the emergence of adaptive resistance. By using both an RNA-aptamer and a monoclonal antibody targeting CD5L, we are able to abate the pro-angiogenic effects of CD5L overexpression in both in vitro and in vivo settings. In addition, we find that increased expression of vascular CD5L in cancer patients is associated with bevacizumab resistance and worse overall survival. These findings implicate CD5L as an important factor in adaptive resistance to antiangiogenic therapy and suggest that modalities to target CD5L have potentially important clinical utility.

Anticancer Effects of High Glucosinolate Synthesis Lines of Brassica rapa on Colorectal Cancer Cells.

Chemoprevention is a method of health control in modern industrialized societies. Traditional breeding (hybridization) has been widely used to produce new (sub)species with beneficial phenotypes. Previously, we produced a number of doubled haploid (DH) lines of Brassica rapa with a high glucosinolate (GSL) content. In this study, we evaluated the anticancer activities of extracts from three selected high-GSL (HGSL)-containing DH lines (DHLs) of Brassica rapa in human colorectal cancer (CRC) cells. The three HGSL DHL extracts showed anti-proliferative activities in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay and pro-apoptotic activities in the cell cycle or annexin V analysis with the induction of pro-apoptotic protein expression in CRC cells. Mechanistically, HGSL DHL extracts inhibited the NF-κB and ERK pathways, leading to a reduction in the nuclear localization of NF-κB p65. In addition, reactive oxygen species were induced by HGSL DHL extract treatment in CRC cells. In conclusion, our data suggest that the newly developed HGSL DHLs possess enhanced anticancer activities and are potentially helpful as a daily vegetable supplement with chemopreventive activities.

Deephos: predicted spectral database search for TMT-labeled phosphopeptides and its false discovery rate estimation.

MOTIVATION: Tandem mass tag (TMT)-based tandem mass spectrometry (MS/MS) has become the method of choice for the quantification of post-translational modifications in complex mixtures. Many cancer proteogenomic studies have highlighted the importance of large-scale phosphopeptide quantification coupled with TMT labeling. Herein, we propose a predicted Spectral DataBase (pSDB) search strategy called Deephos that can improve both sensitivity and specificity in identifying MS/MS spectra of TMT-labeled phosphopeptides. RESULTS: With deep learning-based fragment ion prediction, we compiled a pSDB of TMT-labeled phosphopeptides generated from ∼8000 human phosphoproteins annotated in UniProt. Deep learning could successfully recognize the fragmentation patterns altered by both TMT labeling and phosphorylation. In addition, we discuss the decoy spectra for false discovery rate (FDR) estimation in the pSDB search. We show that FDR could be inaccurately estimated by the existing decoy spectra generation methods and propose an innovative method to generate decoy spectra for more accurate FDR estimation. The utilities of Deephos were demonstrated in multi-stage analyses (coupled with database searches) of glioblastoma, acute myeloid leukemia and breast cancer phosphoproteomes. AVAILABILITY AND IMPLEMENTATION: Deephos pSDB and the search software are available at https://github.com/seungjinna/deephos.

Efficacy of Geumguesingi-hwan (jinkuishenqi-wan, kinkijinki-gan) in decreasing blood glucose levels in patients with uncomplicated type 2 diabetes: A protocol for systematic review and meta-analysis.

BACKGROUND: The purpose of a systematic review and meta-analysis is to verify the clinical efficacy and safety of Geumguesingihwan for patients with uncomplicated type 2 diabetes. METHODS: The systematic review and meta-analysis will be performed following the guidelines of the National Evidence-based Healthcare Collaborating Agency. We will conduct a systematic search of randomized controlled trials in 8 electronic databases until August 31, 2021. RESULTS: This study will provide evidence regarding the clinical efficacy of Geumguesingi-hwan from the following 3 perspectives: improving blood glucose level, insulin resistance, and ß-cell function. Additionally, we will examine the safety of Geumguesingi-hwan by evaluating the adverse effects. CONCLUSIONS: This study will verify the antidiabetic efficacy and safety of Geumguesingi-hwan in patients with uncomplicated type 2 diabetes.

Effects of added cereal fibers on the quality characteristics of black pudding prepared with duck blood.

We investigated the physicochemical and rheological properties of black pudding prepared with duck blood using various combinations of cereal fiber sources: oat, buckwheat, quinoa, amaranth, and sorghum. The processing yield of black pudding made with duck blood and the cereals was higher than that of the control (without cereals) in all cases (P < 0.05). The moisture content of the black pudding was the highest in the buckwheat and amaranth groups (P < 0.05). The water activity, pH, and yellowness of the black pudding combined with duck blood and cereals were lower than that of the control (P < 0.05). The hardness of the black pudding with duck blood and cereals was higher than that of the control (P < 0.05), except for the amaranth group. The cohesiveness, gumminess, and chewiness of the black pudding with duck blood and cereals were higher than that of the control (P < 0.05). Differential scanning colorimetry showed distinct peak points according to treatment at the same temperature, and all treatments exhibited 2 peak temperatures, except for sorghum. The viscosities of all samples, including the control, decreased as the shear rate increased, and the viscosity of the black pudding with oat was slightly lower than that of the other samples. Thus, black pudding prepared with duck blood and cereal fibers showed excellent physicochemical and rheological properties, suggesting an improved processing method. These findings can further the development of products using duck blood as a valuable nutritional source rather than being lost as a by-product during slaughter.

Efficient High-Density hiPSCs Expansion in Simple Dialysis Device.

iPSCs are potential cell types that can be used for regenerative medicine. Suspension culture is the main approach to produce a sufficient amount of required cells to realize the application of hiPSCs for the transplantation of the large organ. The dialysis culture system holds the potential to reduce the cost by utilizing endogenous growth factors and recycle the remaining exogenous growth factors at the same time. However, the current large scale dialysis culture system was not optimized for expanding the high-density culture. Several problems such as the requirement of the large volume and technical complexity make the optimization of this system remain challenging. Also, the interference of mechanical stress in the dynamic suspension culture may reduce cellular viability, pluripotency, and differentiation capacity. Here, we describe the simple miniaturized dialysis platform to evaluate the feasibility of high-density hiPSCs culture by combining the utilization of endogenous growth factors supported by a continuous exchange of nutrition and toxic metabolic product in a low mechanical stress culture environment in a viscoelastic medium.

How can we improve patients' access to new drugs under uncertainties? : South Korea's experience with risk sharing arrangements.

BACKGROUND: New drugs including cancer drugs and orphan drugs are becoming increasingly more expensive. Risk sharing arrangements (RSAs) could manage the risk based on both financial impact and the health outcome of new drugs if reimbursed. To improve patients' access to new drugs under uncertainties, many developed countries have adopted RSAs. In this study, we aimed to understand the effects of RSAs in South Korea on patients' access. METHODS: We reviewed current status of RSA drugs in South Korea. The number of appraisals and time gap between market approval and reimbursement per RSA drug were considered to quantify improvement of patients' access as they showed how rapidly decisions on reimbursement of RSA drugs were derived. Then, we applied a comparative analysis to determine whether the RSA drugs in South Korea were reimbursed in the UK, Italy, and Australia. Most data for this study were obtained from websites of the governmental department/agencies responsible for appraisal of drug reimbursement in each country. And literatures related to RSAs were investigated as well. RESULTS: The eligibility for Korean RSAs had two key components - drugs for cancer and rare diseases and not having other alternative treatments. As of the first half of 2019, there were 39 RSA drugs reimbursed in South Korea, the majority of which were financial-based schemes. Refund and expenditure cap were the representative types (89.7%). After introduction of RSAs, the time gap and number of appraisals were decreased. Based on the indications of RSA drugs, the level of drug coverage in South Korea was found lower than Italy, similar to the UK, and higher than Australia. CONCLUSIONS: RSAs in South Korea significantly enhanced patients' access to new drugs and led to the alleviation of patients' out-of-pocket expenses. The drug coverage of South Korea had a level comparable to that of other countries. This study provides implications for countries that have a dual mission of containing pharmaceutical expenditure and improving access to new drugs.

Differentiation of Human-Induced Pluripotent Stem Cell-Derived Endocrine Progenitors to Islet-like Cells Using a Dialysis Suspension Culture System.

The production of functional islet-like cells from human-induced pluripotent stem cells (hiPSCs) is a promising strategy for the therapeutic use and disease modeling for type 1 diabetes. However, the production cost of islet-like cells is extremely high due to the use of expensive growth factors for differentiation. In a conventional culture method, growth factors and beneficial autocrine factors remaining in the culture medium are removed along with toxic metabolites during the medium change, and it limits the efficient utilization of those factors. In this study, we demonstrated that the dialysis suspension culture system is possible to reduce the usage of growth factors to one-third in the differentiation of hiPSC-derived endocrine progenitor cells to islet-like cells by reducing the medium change frequency with the refinement of the culture medium. Furthermore, the expression levels of hormone-secretion-related genes and the efficiency of differentiation were improved with the dialysis suspension culture system, possibly due to the retaining of autocrine factors. In addition, we confirmed several improvements required for the further study of the dialysis culture system. These findings showed the promising possibility of the dialysis suspension culture system for the low-cost production of islet-like cells.

The hidden role of paxillin: localization to nucleus promotes tumor angiogenesis.

Paxillin (PXN), a key component of the focal adhesion complex, has been associated with cancer progression, but the underlying mechanisms are poorly understood. The purpose of this study was to elucidate mechanisms by which PXN affects cancer growth and progression, which we addressed using cancer patient data, cell lines, and orthotopic mouse models. We demonstrated a previously unrecognized mechanism whereby nuclear PXN enhances angiogenesis by transcriptionally regulating SRC expression. SRC, in turn, increases PLAT expression through NF-ĸB activation; PLAT promotes angiogenesis via LRP1 in endothelial cells. PXN silencing in ovarian cancer mouse models reduced angiogenesis, tumor growth, and metastasis. These findings provide a new understanding of the role of PXN in regulating tumor angiogenesis and growth.

Triplet chemotherapy vs doublet chemotherapy plus bevacizumab in metastatic, recurrent, and persistent cervical cancer.

OBJECTIVE: The effectiveness of paclitaxel-cisplatin-ifosfamide triplet regimen (TIP) was reported to be superior to that of paclitaxel-cisplatin doublet. However, the efficacy of paclitaxel-cisplatin-bevacizumab triplet (TPA) and TIP has not been compared. Here, we compared the efficacy and safety of TIP and TPA in patients with metastatic, recurrent, or persistent cervical cancer. METHODS: We retrospectively reviewed the medical records of patients with recurrent, persistent, or metastatic cervical cancer who were at the Samsung Medical Center, Seoul, Korea between January 2005 and September 2018. Of the 161 patients included in the study, 92 had received TIP and 71 had received TPA. For the study, we compared the response rates, progression-free survival (PFS), overall survival (OS), and safety in the 2 treatment groups. RESULTS: The response rates of patients who received TIP and TPA were comparable (64.1% vs 73.2%, P = 0.239). Histology (squamous vs nonsquamous) was the only prognostic factor that affected the response to therapy (odds ratio, 0.259; 95% confidence interval [CI], 0.119-0.562; P = 0.001). The PFS after TIP and TPA treatment was similar: 12.0 months (95%CI, 10.26-13.74) vs 11.5 months (95%CI, 10.18-12.83), respectively. In a Cox proportional hazard model, objective response to therapies was the only independent prognostic factor for both PFS and OS. However, different types of therapy (TIP vs TPA) did not affect the oncological outcomes for either PFS or OS. Although hematologic toxicity was significantly higher in the TIP-treated group than in the TPA-treated group, both regimens were safe and well-tolerated. CONCLUSIONS: The effectiveness and safety of TIP was comparable to TPA in terms of response rates, survival, and adverse effects. TIP could be an effective alternative in the treatment of cervical cancer when TPA is contraindicated or unaffordable.