Circulating MicroRNA-125b Predicts the Presence and Progression of Uremic Vascular Calcification.

OBJECTIVE: Vascular calcification (VC) is a major cause of mortality in patients with end-stage renal diseases. Biomarkers to predict the progression of VC early are in urgent demand. APPROACH AND RESULTS: We identified circulating, cell-free microRNAs as potential biomarkers using in vitro VC models in which both rat and human aortic vascular smooth muscle cells were treated with high levels of phosphate to mimic uremic hyperphosphatemia. Using an Affymetrix microRNA array, we found that miR-125b and miR-382 expression levels declined significantly as biomineralization progressed, but this decline was only observed for miR-125b in the culture medium. A time-dependent decrease in aortic tissue and serum miR-125b levels was also found in both ex vivo and in vivo renal failure models. We examined the levels of circulating, cell-free miR-125b in sera from patients with end-stage renal diseases (n=88) and found an inverse association between the severity of VC and the circulating miR-125b level, irrespective of age or mineral-related hormones (odds ratio, 0.71; P=0.03). Furthermore, serum miR-125b levels on enrollment can predict VC progression years later (for high versus low, odds ratio, 0.14; P<0.01; for the highest versus lowest tertile and middle versus lowest tertile, odds ratio, 0.55 and 0.13; P=0.3 and <0.01, respectively). The uremic VC prediction efficacy using circulating miR-125b levels was also observed in an independent cohort (n=135). CONCLUSIONS: The results suggest that serum miR-125b levels are associated with VC severity and serve as a novel predictive marker for the risk of uremia-associated calcification progression.

MicroRNA signature predicts survival and relapse in lung cancer.

We investigated whether microRNA expression profiles can predict clinical outcome of NSCLC patients. Using real-time RT-PCR, we obtained microRNA expressions in 112 NSCLC patients, which were divided into the training and testing sets. Using Cox regression and risk-score analysis, we identified a five-microRNA signature for the prediction of treatment outcome of NSCLC in the training set. This microRNA signature was validated by the testing set and an independent cohort. Patients with high-risk scores in their microRNA signatures had poor overall and disease-free survivals compared to the low-risk-score patients. This microRNA signature is an independent predictor of the cancer relapse and survival of NSCLC patients.

Safety assessment of a proprietary fermented soybean solution, Symbiota®, as an ingredient for use in foods and dietary supplements: Non-clinical studies and a randomized trial.

A safety evaluation was performed of Symbiota®, which is made by a proprietary anaerobic fermentation process of soybean with multistrains of probiotics and a yeast. The battery of genotoxicity studies showed that Symbiota® has no genotoxic effects. Safety and tolerability were further assessed by acute or repeated dose 28- and 90-day rodent studies, and no alterations in clinical observations, ophthalmological examination, blood chemistry, urinalysis, or hematology were observed between the control group and the different dosing groups (1.5, 5, and 15 mL/kg/day). There were no adverse effects on specific tissues or organs in terms of weight and histopathology. Importantly, the Symbiota® treatment did not perturb hormones and other endocrine-related endpoints. Of note, the No-Observed-Adverse-Effect-Level was determined to be 15 mL/kg/day in rats. Moreover, a randomized, double-blind, placebo-controlled clinical trial was recently conducted with healthy volunteers who consumed 8 mL/day of placebo or Symbiota® for 8 weeks. Only mild adverse events were reported in both groups, and the blood chemistry and blood cell profiles were also similar between the two groups. In summary, this study concluded that the oral consumption of Symbiota® at 8 mL/day by the general population does not pose any human health concerns.

MS-20 enhances the gut microbiota-associated antitumor effects of anti-PD1 antibody.

Cancer immunotherapy has been regarded as a promising strategy for cancer therapy by blocking immune checkpoints and evoking immunity to fight cancer, but its efficacy seems to be heterogeneous among patients. Manipulating the gut microbiota is a potential strategy for enhancing the efficacy of immunotherapy. Here, we report that MS-20, also known as "Symbiota®", a postbiotic that comprises abundant microbial metabolites generated from a soybean-based medium fermented with multiple strains of probiotics and yeast, inhibited colon and lung cancer growth in combination with an anti-programmed cell death 1 (PD1) antibody in xenograft mouse models. Mechanistically, MS-20 remodeled the immunological tumor microenvironment by increasing effector CD8+ T cells and downregulating PD1 expression, which were mediated by the gut microbiota. Fecal microbiota transplantation (FMT) from mice receiving MS-20 treatment to recipient mice increased CD8+ T-cell infiltration into the tumor microenvironment and significantly improved antitumor activity when combined with anti-PD1 therapy. Notably, the abundance of Ruminococcus bromii, which increased following MS-20 treatment, was positively associated with a reduced tumor burden and CD8+ T-cell infiltration in vivo. Furthermore, an ex vivo study revealed that MS-20 could alter the composition of the microbiota in cancer patients, resulting in distinct metabolic pathways associated with favorable responses to immunotherapy. Overall, MS-20 could act as a promising adjuvant agent for enhancing the efficacy of immune checkpoint-mediated antitumor therapy.

Resident Willingness to Pay for Ecosystem Services in Hillside Forests.

This study investigated the willingness of residents to pay for ecosystem services in a hillside forest in the Lanyang River Basin, which is among the most vulnerable watersheds in Taiwan. The economic value of provisioning, regulating, cultural, and supporting ecosystem services was evaluated. The Contingent Valuation Method (CVM) was applied for economic analysis of public welfare. The determinants of the economic values were identified. A total of 444 respondents completed the questionnaire. The results revealed that the four ecosystem services had high economic value, indicating that conserving hillside forests can ensure the welfare of nearby residents. The findings of this study can serve as reference for regional land planning and social and economic system development policies. In addition, this study addressed policy implementation from the perspective of ecological economics to contribute to an improved Anthropocene.

New Horizons of Macrophage Immunomodulation in the Healing of Diabetic Foot Ulcers.

Diabetic foot ulcers (DFUs) are one of the most costly and troublesome complications of diabetes mellitus. The wound chronicity of DFUs remains the main challenge in the current and future treatment of this condition. Persistent inflammation results in chronic wounds characterized by dysregulation of immune cells, such as M1 macrophages, and impairs the polarization of M2 macrophages and the subsequent healing process of DFUs. The interactive regulation of M1 and M2 macrophages during DFU healing is critical and seems manageable. This review details how cytokines and signalling pathways are co-ordinately regulated to control the functions of M1 and M2 macrophages in normal wound repair. DFUs are defective in the M1-to-M2 transition, which halts the whole wound-healing machinery. Many pre-clinical and clinical innovative approaches, including the application of topical insulin, CCL chemokines, micro RNAs, stem cells, stem-cell-derived exosomes, skin substitutes, antioxidants, and the most recent Phase III-approved ON101 topical cream, have been shown to modulate the activity of M1 and M2 macrophages in DFUs. ON101, the newest clinically approved product in this setting, is designed specifically to down-regulate M1 macrophages and further modulate the wound microenvironment to favour M2 emergence and expansion. Finally, the recent evolution of macrophage modulation therapies and techniques will improve the effectiveness of the treatment of diverse DFUs.

Restoring Prohealing/Remodeling-Associated M2a/c Macrophages Using ON101 Accelerates Diabetic Wound Healing.

Diabetic wounds exhibit chronic inflammation and delayed tissue proliferation or remodeling, mainly owing to prolonged proinflammatory (M1) macrophage activity and defects in transition to prohealing/proremodeling (M2a/M2c; CD206+ and/or CD163+) macrophages. We found that topical treatment with ON101, a plant-based potential therapeutic for diabetic foot ulcers, increased M2c-like (CD163+ and CD206+) cells and suppressed M1-like cells, altering the inflammatory gene profile in a diabetic mouse model compared with that in the controls. An in vitro macrophage-polarizing model revealed that ON101 directly suppressed CD80+ and CD86+ M1-macrophage polarization and M1-associated proinflammatory cytokines at both protein and transcriptional levels. Notably, conditioned medium collected from ON101-treated M1 macrophages reversed the M1-conditioned medium‒mediated suppression of CD206+ macrophages. Furthermore, conditioned medium from ON101-treated adipocyte progenitor cells significantly promoted CD206+ and CD163+ macrophages but strongly inhibited M1-like cells. ON101 treatment also stimulated the expression of GCSF and CXCL3 genes in human adipocyte progenitor cells. Interestingly, treatment with recombinant GCSF protein enhanced both CD206+ and CD163+ M2 markers, whereas CXCL3 treatment only stimulated CD163+ M2 macrophages. Depletion of cutaneous M2 macrophages inhibited ON101-induced diabetic wound healing. Thus, ON101 directly suppressed M1 macrophages and facilitated the GCSF- and CXCL3-mediated transition from M1 to M2 macrophages, lowering inflammation and leading to faster diabetic wound healing.

Tumor microenvironment-based screening repurposes drugs targeting cancer stem cells and cancer-associated fibroblasts.

The tumorous niche may drive the plasticity of heterogeneity and cancer stemness, leading to drug resistance and metastasis, which is the main reason of treatment failure in most cancer patients. The aim of this study was to establish a tumor microenvironment (TME)-based screening to identify drugs that can specifically target cancer stem cells (CSCs) and cancer-associated fibroblasts (CAFs) in the TME. Methods: Lung cancer patient-derived cancer cell and CAFs were utilized to mimic the TME and reproduce the stemness properties of CSCs in vitro and develop a high-throughput drug screening platform with phenotypical parameters. Limiting dilution assay, sphere-forming and ALDH activity assay were utilized to measure the cancer stemness characteristics. In vivo patient-derived xenograft (PDX) models and single-cell RNA sequencing were used to evaluate the mechanisms of the compounds in CSCs and CAFs. Results: The TME-based drug screening platform could comprehensively evaluate the response of cancer cells, CSCs and CAFs to different treatments. Among the 1,524 compounds tested, several drugs were identified to have anti-CAFs, anticancer and anti-CSCs activities. Aloe-emodin and digoxin both show anticancer and anti-CSCs activity in vitro and in vivo, which was further confirmed in the lung cancer PDX model. The combination of digoxin and chemotherapy improved therapeutic efficacy. The single-cell transcriptomics analysis revealed that digoxin could suppress the CSCs subpopulation in CAFs-cocultured cancer cells and cytokine production in CAFs. Conclusions: The TME-based drug screening platform provides a tool to identify and repurpose compounds targeting cancer cells, CSCs and CAFs, which may accelerate drug development and therapeutic application for lung cancer patients.

Hinokitiol induces DNA damage and autophagy followed by cell cycle arrest and senescence in gefitinib-resistant lung adenocarcinoma cells.

Despite good initial responses, drug resistance and disease recurrence remain major issues for lung adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutations taking EGFR-tyrosine kinase inhibitors (TKI). To discover new strategies to overcome this issue, we investigated 40 essential oils from plants indigenous to Taiwan as alternative treatments for a wide range of illnesses. Here, we found that hinokitiol, a natural monoterpenoid from the heartwood of Calocedrus formosana, exhibited potent anticancer effects. In this study, we demonstrated that hinokitiol inhibited the proliferation and colony formation ability of lung adenocarcinoma cells as well as the EGFR-TKI-resistant lines PC9-IR and H1975. Transcriptomic analysis and pathway prediction algorithms indicated that the main implicated pathways included DNA damage, autophagy, and cell cycle. Further investigations confirmed that in lung cancer cells, hinokitiol inhibited cell proliferation by inducing the p53-independent DNA damage response, autophagy (not apoptosis), S-phase cell cycle arrest, and senescence. Furthermore, hinokitiol inhibited the growth of xenograft tumors in association with DNA damage and autophagy but exhibited fewer effects on lung stromal fibroblasts. In summary, we demonstrated novel mechanisms by which hinokitiol, an essential oil extract, acted as a promising anticancer agent to overcome EGFR-TKI resistance in lung cancer cells via inducing DNA damage, autophagy, cell cycle arrest, and senescence in vitro and in vivo.

Cancer-associated fibroblasts regulate the plasticity of lung cancer stemness via paracrine signalling.

Cancer stem cells (CSCs) are a promising target for treating cancer, yet how CSC plasticity is maintained in vivo is unclear and is difficult to study in vitro. Here we establish a sustainable primary culture of Oct3/4(+)/Nanog(+) lung CSCs fed with CD90(+) cancer-associated fibroblasts (CAFs) to further advance our knowledge of preserving stem cells in the tumour microenvironment. Using transcriptomics we identify the paracrine network by which CAFs enrich CSCs through de-differentiation and reacquisition of stem cell-like properties. Specifically, we find that IGF1R signalling activation in cancer cells in the presence of CAFs expressing IGF-II can induce Nanog expression and promote stemness. Moreover, this paracrine signalling predicts overall and relapse-free survival in stage I non-small cell lung cancer (NSCLC) patients. IGF-II/IGF1R signalling blockade inhibits Nanog expression and attenuates cancer stem cell features. Our data demonstrate that CAFs constitute a supporting niche for cancer stemness, and targeting this paracrine signalling may present a new therapeutic strategy for NSCLC.

Curcumin inhibits lung cancer cell invasion and metastasis through the tumor suppressor HLJ1.

Curcumin (diferuloylmethane) is an active component of the spice turmeric and has a diversity of antitumor activities. In this study, we found that curcumin can inhibit cancer cell invasion and metastasis through activation of the tumor suppressor DnaJ-like heat shock protein 40 (HLJ1). Human lung adenocarcinoma cells (CL1-5) treated with curcumin (1-20 mumol/L) showed a concentration-dependent reduction in cell migration, invasion, and metastatic ability, and this was associated with increased HLJ1 expression. Knockdown of HLJ1 expression by siRNA was able to reverse the curcumin-induced anti-invasive and antimetastasis effects in vitro and in vivo. The HLJ1 promoter and enhancer in a luciferase reporter assay revealed that curcumin transcriptionally up-regulates HLJ1 expression through an activator protein (AP-1) site within the HLJ1 enhancer. JunD, one of the AP-1 components, was significantly up-regulated by curcumin (1-20 mumol/L) in a concentration- and time-dependent manner. Knockdown of JunD expression could partially reduce the curcumin-induced HLJ1 activation and diminish the anti-invasive effect of curcumin, indicating that JunD would seem to be involved in curcumin-induced HLJ1 expression. Curcumin was able to induce c-Jun NH(2)-kinase (JNK) phosphorylation, whereas the JNK inhibitor (SP-600125) could attenuate curcumin-induced JunD and HLJ1 expression. Activation of HLJ1 by curcumin further leads to up-regulation of E-cadherin and a suppression of cancer cell invasion. Our results show that curcumin induces HLJ1, through activation of the JNK/JunD pathway, and inhibits lung cancer cell invasion and metastasis by modulating E-cadherin expression. This is a novel mechanism and supports the application of curcumin in anti-cancer metastasis therapy.