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Purpose: To clarify the effects of cyclin E1 suppression on antitumor efficacy of sorafenib in hepatocellular carcinoma cells and to explore the potential of combining sorafenib with cyclin-dependent kinase (CDK) inhibition in therapy.Experimental Design: The effects of cyclin E1 suppression on sorafenib-induced apoptosis were tested in both sorafenib-sensitive (Huh-7 and HepG2, IC50 5–6 μmol/L) and sorafenib-resistant (Huh-7R and HepG2R, IC50 14–15 μmol/L) hepatocellular carcinoma cells. The activity of pertinent signaling pathways and the expression of cell cycle and apoptosis-related proteins were measured using Western blotting. Efficacy of sorafenib combined with the pan-CDK inhibitor flavopiridol was tested both in vitro and in xenograft experiments. The pertinent downstream mediators of antitumor efficacy were tested in transient transfection and RNA interference experiments.Results: Cyclin E1 mRNA and protein expressions were suppressed after sorafenib treatment in sorafenib-sensitive but not in sorafenib-resistant hepatocellular carcinoma cells. Changes in cyclin E2 or D1 were not correlated with sorafenib sensitivity. The knockdown of cyclin E1 expression reversed the resistance of hepatocellular carcinoma cells to sorafenib in terms of cell growth and apoptosis induction, whereas the overexpression of cyclin E1 increased the resistance to sorafenib. The growth-inhibitory and apoptosis-inducing effects of sorafenib were enhanced by flavopiridol, and Mcl-1 suppression was determined to play a critical role in mediating this enhancing effect.Conclusions: The cyclin E1 suppression in hepatocellular carcinoma cells may serve as a pharmacodynamic biomarker for predicting sorafenib efficacy. The combination of sorafenib and CDK inhibitors may improve the efficacy of sorafenib in hepatocellular carcinoma. Clin Cancer Res; 22(10); 2555–64. ©2015 AACR.

Supplement Figure S1: The six E2Fs binding sites in cyclin E1 promoter; Supplement Figure S2: Suppression of cyclin E1 by sorafenib correlated with sorafenib sensitivity; Supplement Figure S3: Effects of sorafenib and the Raf kinase inhibitor on cyclin E1 suppression in HCC cells; Supplement Figure S4: Knockdown of cyclin E1, but not cyclin E2, significantly increased the sensitivity to sorafenib in HCC cells; Supplement Figure S5: Effects of cyclin E1 or Mcl-1 inhibition on sorafenib-induced apoptosis; Supplement Figure S6: Effects of E2F1 suppression on cyclin E1 expression; Supplement Figure S7: Effects of flavopiridol on cyclin E1 and Rb protein levels in HCC cells; Supplement Figure S8: Synergistic anti-tumor activity between sorafenib and the CDK inhibitor flavopiridol; Supplement Figure S9: Differential effect between sorafenib and the CDK inhibitor flavopiridol on cell-cycle progression of HCC cells; Supplement Figure S10: The apoptosis-related proteins measured by the Human Apoptosis Array kit; Supplement Figure S11: In vivo evidence of toxicity between sorafenib and flavopiridol in HCC cells; Supplement Figure S12: Flavopiridol enhanced the anti-tumor efficacy of sorafenib in vivo; and Supplement Table S1: Primers list and Supplementary materials and methods.

Introduction: Injection of induced pluripotent stem cell-derived cardiomyocytes has been reported as a promising approach to regenerate loss myocardium and restore heart function after ischemic injury. However, immaturity of the transplanted cardiomyocytes and their poor survival rates caused by limited blood supply remain as major hurdles for clinical translation. Hypothesis: We tested the hypothesis that co-culture of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) with hiPSC-derived endothelial cells (ECs) promotes CM maturation in vitro, and that co-transplantation of both hiPSC-CMs and hiPSC-ECs facilitates hiPSC-CM muscularization in myocardial ischemic injured mice and non-human primates. Methods and Results: We examined the therapeutic effect of co-transplantation of hiPSC-CMs and hiPSC-ECs in NOD-SCID mice undergoing myocardial infarction (N = 14 / group). Mice receiving co-transplantation had an improvement in ejection fraction compared to control (4.2 ± 1.2 % vs -8.4 ± 0.9 %, P < 0.0001), and even those receiving high-dose (-0.3 ± 0.9 %, P = 0.052) and low-dose (-2.4 ± 1.1 %, P = 0.001) hiPSC-CMs alone treatment. Moreover, less arrhythmic events were observed in co-transplantation using three-lead electrogram. To be more clinically relevant, we first showed in healthy non-human primates (N = 4) that hiPSC-CM engraftment, maturation, and integration was achieved when co-transplanted with hiPSC-ECs. Furthermore, we then examined the therapeutic effect of co-transplantation of hiPSC-CMs and hiPSC-ECs in rhesus macaques undergoing ischemia-reperfusion surgery (N = 3 / group). Consistent with the mouse model, co-transplantation in rhesus macaques significantly improved the ejection fraction (10 ± 1.3 % vs -1.8 ± 2.2 %, P = 0.010), accompanied by a reduced infarct size compared to control (16 ± 1.1 % vs 23 ± 3.3 %, P = 0.091). Conclusions: This study demonstrates the beneficial effects of co-transplantation of hiPSC-CMs with hiPSC-ECs, promoting hiPSC-CM maturation, enhancing neovascularization, and improving cardiac function in both mouse and non-human primate hearts. Delivery of this combined cell therapy holds promise for future clinical translation.

Introduction: The gut microbiota has been implicated in various heart diseases by producing metabolites that modulate host immunity and metabolism. In our previous study, we found that mice with dysbiosis suffered more from cardiac rupture, resulting in higher mortality than mice possessing commensal gut flora after myocardial infarction. This suggests that there are unexplored routes to cardiovascular health through the gut microbiota. Hypothesis: We hypothesize that a community of symbiotic gut microbiota is required for heart mechanical modifications during adaptive cardiac remodeling under stress. Methods and Results: Pressure-overload stress was induced by a transverse aortic constriction (TAC) surgery and dysbiosis was induced by antibiotic treatment (ABX) in mice. Under echocardiography, ABX-TAC mice showed worse cardiac outcomes versus controls (ejection fraction = 59% ± 2% vs 67% ± 2%, P < 0.001, N ≥ 12/group). Under microscopic examination of the extracellular matrix and tensile tests, ABX-TAC mice had larger fibrotic areas (9.4% ± 0.4% vs 5.7% ± 0.4%, P < 0.0001) and collagen disarray, accompanied by more severe ventricular stiffening (Young’s moduli = 360 ± 10 kPa vs 280 ± 43 kPa, P < 0.01, N ≥ 6/group). When establishing normal gut flora before surgery, germ-free mice had heart malfunctioning rescued (change of myocardial performance index = 15% ± 13% vs 120% ± 32% untreated, P < 0.05, N ≥ 5/group). The normal gut flora was profiled by third-generation 16S sequencing to acquire bacterial information with high accuracy, followed by PICRUSt analysis revealing that the microbes favored the production of short-chain fatty acids (SCFAs) (N ≥ 8/group). When supplemented with SCFA before surgery, ABX mice gained better cardiac outcomes (ejection fraction = 65% ± 2% vs 53% ± 3% untreated, P < 0.01, N ≥ 5/group). Cardiac fibroblasts treated with SCFA were less susceptible to TGF-β1-triggered fibrogenesis (COL1A1/GAPDH = 0.8 ± 0.3 vs 2.3 ± 0.3 untreated, P < 0.01, n ≥ 4/group). Conclusions: In conclusion, our study demonstrates firstly that gut microbiota-derived SCFAs manipulate heart mechanical functioning under stress potentially acting via cardiac fibroblasts. This provides new insights into the management of heart diseases through the gut microbiota.

In December 2019, the novel coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spread around the globe resulting in ~435 million confirmed cases and ~6 million related deaths as of March 2022, according to the World Health Organization. To combat COVID-19 quickly, there have been many attempts to repurpose current FDA-approved drugs or to revive old drugs with anti-viral properties. However, aside from the biological stress imposed by the virus, many of the current treatment options have been known to cause adverse drug reactions. We established a population-based human induced pluripotent stem cell drug screening platform to assess the toxicity of the first line of anti-COVID-19 drugs and to understand viral infection of cardiomyocytes and neurons. We found that iPSC-derived cardiomyocytes express the ACE2 receptor which correlated with a higher infection of the SARS-CoV-2 virus (r=0.86). However, ACE2 expression was undetectable in neurons which correlated with low infection of neurons. We then assessed the toxicity of anti-COVID-19 drugs and identified two cardiotoxic compounds (remdesivir and arbidol) and 4 neurotoxic compounds (arbidol, remdesivir, hydroxychloroquine, and chloroquine) which were validated by dose-response curves. These data show that this platform can quickly and easily be employed to further our understanding of cell-specific infection and identify drug toxicity of potential treatment options helping clinicians better decide on treatment options.

Since December 2019, the novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected ~435 million people and caused ~6 million related deaths as of March 2022. To combat COVID-19, there have been many attempts to repurpose FDA-approved drugs or revive old drugs. However, many of the current treatment options have been known to cause adverse drug reactions. We employed a population-based drug screening platform using 13 human leukocyte antigen (HLA) homozygous human induced pluripotent cell (iPSC) lines to assess the cardiotoxicity and neurotoxicity of the first line of anti-COVID-19 drugs. We also infected iPSC-derived cells to understand the viral infection of cardiomyocytes and neurons. We found that iPSC-derived cardiomyocytes express the ACE2 receptor which correlated with a higher infection of the SARS-CoV-2 virus (r = 0.86). However, we were unable to detect ACE2 expression in neurons which correlated with a low infection rate. We then assessed the toxicity of anti-COVID-19 drugs and identified two cardiotoxic compounds (remdesivir and arbidol) and four neurotoxic compounds (arbidol, remdesivir, hydroxychloroquine, and chloroquine). These data show that this platform can quickly and easily be employed to further our understanding of cell-specific infection and identify drug toxicity of potential treatment options helping clinicians better decide on treatment options.

The advent of induced pluripotent stem cells (iPSCs) has advanced our understanding of the molecular mechanisms of human disease, drug discovery, and regenerative medicine. As such, the use of iPSCs in drug development and validation has shown a sharp increase in the past 15 years. Furthermore, many labs have been successful in reproducing many disease phenotypes, often difficult or impossible to capture, in commonly used cell lines or animal models. However, there still remain limitations such as the variability between iPSC lines as well as their maturity. Here, we aim to discuss the strategies in generating iPSC-derived cardiomyocytes and neurons for use in disease modeling, drug development and their use in cell therapy.

Surface-enhanced Raman scattering (SERS) spectroscopy is a rapid and non-destructive optical detection method that has been applied in various applications. Recently, three-dimensional (3D) substrate-based silicon nanostructures have been widely used as SERS substrates due to their high detection sensitivity, repeatability, and reusability. This paper uses a simple and low-cost electroless etching deposition process to generate silver nanoparticle-decorated porous silicon (Ag-PS) substrates. We propose a contact deposition process to generate localized Ag-PS (LocAg-PS) for SERS analysis. Due to the hydrophilic LocAg-PS pad on the hydrophobic PS background, the sample droplets self-aligned to the predefined LocAg-PS pads and condensed into a higher local concentration for high sensitivity SERS detection without extensive search for the hot spot. The effects of critical fabrication parameters and SERS analysis on the LocAg-PS surface were evaluated.

B-cell translocation gene 3 (BTG3) is a member of the antiproliferative BTG gene family and is a downstream target of p53. Here, we show that senescence triggered by BTG3 depletion was accompanied by a secretome enriched with cytokines, growth factors, and matrix-remodeling enzymes, which could promote angiogenesis and cell scattering in vitro. We present evidence that at least part of these activities can be explained by elevated HIF-1α activity. Mechanistically, the BTG3 C-terminal domain competes with the coactivator p300 for binding the HIF-1α transactivation domain. The angiogenic promoting effect of BTG3 knockdown was largely diminished upon co-depletion of HIF-1α, indicating that HIF-1α is a major downstream target of BTG3 in the control of angiogenesis. In vivo, ectopic expression of BTG3 suppresses angiogenesis in xenograft tumors; and syngenic tumor growth and metastasis were enhanced in Btg3-null mice. Moreover, analysis of clinical datasets revealed that a higher BTG3/VEGFA expression ratio correlates with improved patient survival in a number of cancer types. Taken together, our findings highlight the non-autonomous regulation of tumor microenvironment by BTG3 while suppressing tumor progression.

Little is known about genes that induce stem cells differentiation into astrocytes. We previously described that heat shock protein 27 (HSP27) downregulation is directly related to neural differentiation under chemical induction in placenta-derived multipotent stem cells (PDMCs). Using this neural differentiation cell model, we cross-compared transcriptomic and proteomic data and selected 26 candidate genes with the same expression trends in both omics analyses. Those genes were further compared with a transcriptomic database derived from Alzheimer’s disease (AD). Eighteen out of 26 candidates showed opposite expression trends between our data and the AD database. The mRNA and protein expression levels of those candidates showed downregulation of HSP27, S100 calcium-binding protein A16 (S100A16) and two other genes in our neural differentiation cell model. Silencing these four genes with various combinations showed that co-silencing HSP27 and S100A16 has stronger effects than other combinations for astrocyte differentiation. The induced astrocyte showed typical astrocytic star-shape and developed with ramified, stringy and filamentous processes as well as differentiated endfoot structures. Also, some of them connected with each other and formed continuous network. Immunofluorescence quantification of various neural markers indicated that HSP27 and S100A16 downregulation mainly drive PDMCs differentiation into astrocytes. Immunofluorescence and confocal microscopic images showed the classical star-like shape morphology and co-expression of crucial astrocyte markers in induced astrocytes, while electrophysiology and Ca2+ influx examination further confirmed their functional characteristics. In conclusion, co-silencing of S100A16 and HSP27 without chemical induction leads to PDMCs differentiation into functional astrocytes. Graphical abstract

In vitro culture of endothelial cells to form capillary-like networks is essential in tissue engineering. Vascular endothelial growth factor (VEGF) is one of the primary signal proteins stimulating blood vessel formation. This growth factor can be soluble in the medium or protein-bound to the substrate. However, less attention has been paid to distinguishing the specific stimulations by soluble and bound VEGF. We conducted a series of experiments to explore the respective effects of these two VEGF forms. An in-house synthesized biogel comprising a definite concentration of collagen and fibronectin was designed to cultivate human umbilical vein endothelial cells to form the capillary-like network. Collagen served as the primary substrate for cell attachment. Fibronectin provided the surface to bind soluble VEGF in the culture medium to create the bound VEGF. The experiment of adding VEGF-blocking-peptide was conducted to prevent the formation of VEGF bound to the fibronectin domains, to distinguish the respective effects of the soluble and bound VEGF. With the in-house biogel of definite components, we were able to clarify the different roles of soluble and bound VEGF. The results indicated that the soluble VEGF promptly induced the cells to change from round to elongated shape, which contributed to forming network cords. Simultaneously, the bound VEGF provided long-term stimulation, causing the cells to migrate and differentiate into the final capillary-like network.

In bladder cancer, urothelial carcinoma is the most common histologic subtype, accounting for more than 90% of cases. Pathogenic effects due to the dysbiosis of gut microbiota are localized not only in the colon, but also in regulating bladder cancer distally. Butyrate, a short-chain fatty acid produced by gut microbial metabolism, is mainly studied in colon diseases. Therefore, the resolution of the anti-cancer effects of butyrate-producing microbes on bladder urothelial cells and knowledge of the butyrate-responsive molecules must have clinical significance. Here, we demonstrate a correlation between urothelial cancer of the bladder and Butyricicoccus pullicaecorum. This butyrate-producing microbe or their metabolite, butyrate, mediated anti-cancer effects on bladder urothelial cells by regulating cell cycle, cell growth, apoptosis, and gene expression. For example, a tumor suppressor against urothelial cancer of the bladder, bladder cancer-associated protein, was induced in butyrate-treated HT1376 cells, a human urinary bladder cancer cell line. In conclusion, urothelial cancer of the bladder is a significant health problem. To improve the health of bladder urothelial cells, supplementation of B. pullicaecorum may be necessary and can further regulate butyrate-responsive molecular signatures.

Background Myocardial infarction (MI) is a life-threatening disease, often leading to heart failure. Defining therapeutic targets at an early time point is important to prevent heart failure. Methods MicroRNA screening was performed at early time points after MI using paired samples isolated from the infarcted and remote myocardium of pigs. We also examined the microRNA expression in plasma of MI patients and pigs. For mechanistic studies, AAV9-mediated microRNA knockdown and overexpression were administrated in mice undergoing MI. Findings MicroRNAs let-7a and let-7f were significantly downregulated in the infarct area within 24 h post-MI in pigs. We also observed a reduction of let-7a and let-7f in plasma of MI patients and pigs. Inhibition of let-7 exacerbated cardiomyocyte apoptosis, induced a cardiac hypertrophic phenotype, and resulted in worsened left ventricular ejection fraction. In contrast, ectopic let-7 overexpression significantly reduced those phenotypes and improved heart function. We then identified TGFBR3 as a target of let-7, and found that induction of Tgfbr3 in cardiomyocytes caused apoptosis, likely through p38 MAPK activation. Finally, we showed that the plasma TGFBR3 level was elevated after MI in plasma of MI patients and pigs. Interpretation Together, we conclude that the let-7-Tgfbr3-p38 MAPK signalling plays an important role in cardiomyocyte apoptosis after MI. Furthermore, microRNA let-7 and Tgfbr3 may serve as therapeutic targets and biomarkers for myocardial damage. Fund Ministry of Science and Technology, National Health Research Institutes, Academia Sinica Program for Translational Innovation of Biopharmaceutical Development-Technology Supporting Platform Axis, Thematic Research Program and the Summit Research Program, Taiwan.

Background: Myocardial infarction (MI) is a life-threatening disease, often leading to heart failure. Defining therapeutic targets at an early time point is important to prevent heart failure. Methods: MicroRNA screening was performed at early time points after MI using paired samples isolated from the infarcted and remote myocardium of pigs. We also examined the microRNA expression in plasma of MI patients and pigs. For mechanistic studies, AAV9-mediated microRNA knockdown and overexpression were administrated in mice undergoing MI. Findings: MicroRNAs let-7a and let-7f were significantly downregulated in the infarct area within 24 h post-MI in pigs. We also observed a reduction of let-7a and let-7f in plasma of MI patients and pigs. Inhibition of let-7 exacerbated cardiomyocyte apoptosis, induced a cardiac hypertrophic phenotype, and resulted in worsened left ventricular ejection fraction. In contrast, ectopic let-7 overexpression significantly reduced those phenotypes and improved heart function. We then identified TGFBR3 as a target of let-7, and found that induction of Tgfbr3 in cardiomyocytes caused apoptosis, likely through p38 MAPK activation. Finally, we showed that the plasma TGFBR3 level was elevated after MI in plasma of MI patients and pigs. Interpretation: Together, we conclude that the let-7-Tgfbr3-p38 MAPK signalling plays an important role in cardiomyocyte apoptosis after MI. Furthermore, microRNA let-7 and Tgfbr3 may serve as therapeutic targets and biomarkers for myocardial damage. Fund: Ministry of Science and Technology, National Health Research Institutes, Academia Sinica Program for Translational Innovation of Biopharmaceutical Development-Technology Supporting Platform Axis, Thematic Research Program and the Summit Research Program, Taiwan. Keywords: MicroRNA let-7, TGFBR3, Gene therapy, Biomarkers

In this study, we establish a population-based human induced pluripotent stem cell (hiPSC) drug screening platform for toxicity assessment. After recruiting 1,000 healthy donors and screening for high-frequency human leukocyte antigen (HLA) haplotypes, we identify 13 HLA-homozygous "super donors" to represent the population. These "super donors" are also expected to represent at least 477,611,135 of the global population. By differentiating these representative hiPSCs into cardiomyocytes and neurons we show their utility in a high-throughput toxicity screen. To validate hit compounds, we demonstrate dose-dependent toxicity of the hit compounds and assess functional modulation. We also show reproducible in vivo drug toxicity results using mouse models with select hit compounds. This study shows the feasibility of using a population-based hiPSC drug screening platform to assess cytotoxicity, which can be used as an innovative tool to study inter-population differences in drug toxicity and adverse drug reactions in drug discovery applications.

We tested the osteoblastic differentiation effects caused by physical stimulation such as hydrostatic pressure using placenta-derived multipotent cells.The placenta-derived multipotent cells (PDMCs) were treated with osteogenic medium to induce PDMCs differentiation into osteoblast-like cells. The induced PDMCs were stimulated using hydrostatic pressure at a magnitude of 30 kPa for 1 h/day for up to 12 days. The calcium deposition monitored by Alizarin Red staining and the calcium content of each experimental group were quantified.The results demonstrated both the calcium deposition and concentration were elevated through hydrostatic pressure stimulation. Moreover, in order to indicate of PDMC osteodifferentiation, RT-qPCR analysis were performed and mRNA expression of osteoblast differentiation markers (type I collagen, alkaline phosphatase, RUNX2, and BGLAP), the bone morphogenetic protein family (BMP1-7) and BMP receptors (BMPR1A, BMPR1B, and BMPR2) were examined. Among them, the mRNA levels of RUNX2, COL1A1, BMP1, BMP3, and BMPR1A increased significantly in the hydrostatic-pressure-stimulated groups, whereas BGLAP, ALP, BMP2, BMP6, BMPR1B, and BMPR2 exhibited a slight upregulation between the control and experimental groups, indicating the specific signal route induced by hydrostatic pressure on PDMCs.Our results revealed the beneficial effects of stem cells stimulated using hydrostatic pressure, which could enhance calcium deposition considerably and facilitate osteodifferentiation, and the results may be applied to tissue regeneration in the near future.

Composite electrospun fibers were fabricated to develop drug loaded scaffolds to promote bone tissue regeneration. Multi-wall carbon nanotubes (MWCNTs) were incorporated to polylactic acid (PLA) to strengthen electrospun nanofibers. To modulate drug release behavior, different ratios of hydrophilic polyethylene glycol (PEG) were added to composite fibers. Glass transition temperature (Tg) can be reduced by the incorporated PEG to enhance the ductility of the nanofibers. The SEM images and the MTT results demonstrated that composite fibers are suitable scaffolds for cell adhesion and proliferation. Dexamethasone (DEX), an osteogenic inducer, was loaded to PLA/MWCNT/PEG fibers. The surface element analysis performed by XPS showed that fluorine of DEX in pristine PLA fibers was much higher than those of the MWCNT-containing fibers, suggesting that the pristine PLA fibers mainly load DEX on their surfaces, whereas MWCNTs can adsorb DEX with evenly distribution in nanofibers. Drug release experiments demonstrated that the release profiles of DEX were manipulated by the ratio of PEG, and that the more PEG in the nanofibers, the faster DEX was released. When rat bone marrow stromal cells (rBMSCs) were seeded on these nanofibers, the Alizarin Red S staining and calcium quantification results demonstrated that loaded DEX were released to promote osteogenic differentiation of rBMSCs and facilitate mineralized tissue formation. These results indicated that the DEX-loaded PLA/MWCNT/PEG nanofibers not only enhanced mechanical strength, but also promoted osteogenesis of stem cells via the continuous release of DEX. The nanofibers should be a potential scaffold for bone tissue engineering application.

Many proteins involved in the DNA damage pathway shuttle between the cytoplasm and nucleus, and their localizations are important for functions. In that regard, immunofluorescence microscopy has been widely used to delineate the temporal and spatial regulation of proteins. Here, we describe an unconventional method for studying the cellular localization of CHK1, a cell cycle checkpoint kinase that undergoes shuttling from the cytoplasm to the nucleus in response to genotoxic stress. In this study, we included an acid extraction step to better reveal the nuclear localization of CHK1.

Restenosis and destructive vascular remodeling are the main reasons for treatment failure in patients undergoing percutaneous coronary intervention (PCI). In this study, we explored the efficacy of magnolol (a traditional Chinese medicine) in the treatment of restenosis. The results of this study showed that the activities of thrombin and PAR-1 (protease-activated receptor 1) were significantly decreased by the treatment of magnolol. Based on protein docking analysis, magnolol exhibits its potential to bind to the PAR-1 active site. In addition, thrombin-induced connective tissue growth factor (CTGF) expression and the upstream of CTGF such as JNK-1 (but not JNK-2), c-Jun, and AP-1 were distinctly inhibited by magnolol (50 μM) in vascular smooth muscle cells (VSMC). For the functional assay, magnolol (50 μM) significantly inhibited the migration of VSMC, and rats treated with magnolol (13 mg/kg/day) after balloon angioplasty has observed a significant reduction in the formation of common arterial neointima. In conclusion, we identified a novel mechanism by which magnolol acts as the thrombin activity inhibitor and may be the PAR-1 antagonist. In accordance with these functions, magnolol could decrease thrombin-induced CTGF expression in VSMCs via PAR-1/JNK-1/AP-1 signaling.

Malignant cancer may contain highly heterogeneous populations of cells, including stem-like cells which were resistant to chemotherapy agents, radiation, mechanical stress, and immune surveillance. The characterization of these specific subpopulations might be critical to develop novel strategy to remove malignant tumors. We selected and enriched small population of human melanoma A2058 cells by repetitive selection cycles (selection, restoration, and amplification). These subpopulation of melanoma cells persisted the characteristics of slower cell proliferation, enhanced drug-resistance, elevated percentage of side population as analyzed by Hoechst33342 exclusion, in vitro sphere formation, and in vivo xenograft tumor formation by small amount of tumor cells. The selected populations would be melanoma stem-like cells with high expression of stem cell markers and altered kinase activation. Microarray and bioinformatics analysis highlighted the high expression of angiopoietin-like 4 protein in drug-selected melanoma stem-like cells. Further validation by specific shRNA demonstrated the role of angiopoietin-like 4 protein in drug-selected subpopulation associated with enhanced drug-resistance, sphere formation, reduced kinase activation, in vitro tube-forming ability correlated with heparan-sulfate proteoglycans. Our finding would be applicable to explore the mechanism of melanoma stemness and use angiopoietin-like 4 as potential biomarkers to identify melanoma stem-like cells.

Thyroid cancer is the most commonly diagnosed endocrine cancer. Anaplastic thyroid cancer (ATC) is the most aggressive type of thyroid cancer and has a poor prognosis. Loss of p53 function has been reported to lead to poorly differentiated thyroid tumors; therefore, mutant p53 protein can be considered a crucial therapeutic target in patients with ATC. Sorafenib, a multi-kinase inhibitor, has been approved for the treatment of metastatic and differentiated thyroid cancer. Combined targeted therapy, including sorafenib, may be clinically significant for patients with ATC harboring p53 mutations. In the present study, CP-31398, a p53-restoring agent, was used to improve the therapeutic efficacy of sorafenib in SW579 cells, an ATC cell line harboring p53 mutations. The molecular function of CP-31398 was evaluated using western blot analysis and a luciferase reporter assay. The decreased viability of SW579 cells, following CP-31398 treatment, was augmented by sorafenib, and CP-31398 enhanced the antimitogenic effect of sorafenib; thus, sorafenib and CP-31398 synergistically inhibited the growth of SW579 cells. These results indicate a potential clinical application of CP-31398 for patients with ATC harboring p53 abnormalities, since these individuals generally respond poorly to sorafenib alone.

Tolerance and associated hyperalgesia induced by long‑term morphine administration substantially restrict the clinical use of morphine in pain treatment. Melatonin, a neurohormone released by the pineal gland, has been demonstrated to attenuate anti‑nociceptive morphine tolerance. The present study investigates differentially expressed genes in the process of morphine tolerance and altered gene expression subsequent to melatonin treatment in chronic morphine‑infused ratspinal cords. Morphine tolerance was induced in male Wistar rats by intrathecal morphine infusion (the MO group). Melatonin (the MOMa group) was administered to overcome the effects derived by morphine. The mRNA collected from L5‑S3 of the spinal cord was extracted and analysed by rat expression microarray. Principal component analysis and clustering analysis revealed that the overall gene profiles were different in morphine and melatonin treatments. Subsequent to Gene Ontology analysis, the biological processes of differentially expressed genes of MO and MOMa compared with the control group were constructed. Furthermore, a panel of genes exclusively expressed following melatonin treatment and another panel of genes with inverse expression between the MO and MOMa group were also established. Subsequent to PANTHER pathway analysis, a group of genes with inverse expression following melatonin administrated compared with morphine alone were identified. The expression levels of genes of interest were also confirmed using a reverse transcription‑quantitative polymerase chain reaction. The gene panel that was constructed suggests a potential signaling pathway in morphine tolerance development and is valuable for investigating the mechanism of morphine tolerance and the regulatory gene profiles of melatonin treatment. These results may contribute to the discovery of potential drug targets in morphine tolerance treatments in the future.

The received signal strengths (RSSs) of wireless radios are susceptible to environmental dynamics, such as weather conditions, which can severely degrade the performance of RSS-based distance estimators. This paper proposes an enhanced RSS-based distance estimator that accounts for precipitation. The rain attenuation effect is incorporated into the path loss model to derive an analytical expression for maximum-likelihood-based distance estimation by using an iterative form of Newton’s method. In addition, the Cramer–Rao lower bound for the proposed distance estimator is proposed. This study performed experiments on the mobile networks at a campus, sensing the actual RSS using smartphones and deploying local weather stations to record transmission conditions. This is a pioneer work to overcome the rain attenuation problems and real measurements verify the effectiveness of an RSS-based distance estimator. Simulation and experimental results indicated that the proposed algorithm provides robust distance estimation during the periods of precipitation. The parameters of radio propagation and rain rate models can be extracted offline. With these parameters and the real-time measured RSSs and rain rate, our proposed distance estimation method achieves an 85%–90% error reduction rate over the conventional approach.

Checkpoint kinase 1 (CHK1), a Ser/Thr protein kinase, is modified by the K63-linked ubiquitin chain in response to genotoxic stress, which promotes its nuclear localization, chromatin association, and activation. Interestingly, this bulky modification is linked to a critical residue, K132, at the kinase active site. It is unclear how this modification affects the kinase activity and how it is removed to enable the release of CHK1 from chromatin. Herein, we show that the K63-linked ubiquitin chain at CHK1's K132 residue has an inhibitory effect on the kinase activity. Furthermore, we demonstrate that this modification can be removed by ubiquitin-specific protease 3 (USP3), a deubiquitinating enzyme that targets K63-linked ubiquitin chains. Wild-type USP3, but not the catalytically defective or nuclear localization sequence-deficient mutants, reduced CHK1 K63-linked ubiquitination. Conversely, USP3 knockdown elevated K63-linked ubiquitination of the kinase, leading to prolonged CHK1 chromatin association and phosphorylation. Paradoxically, by removing the bulky ubiquitin chain at the active site, USP3 also increased the accessibility of CHK1 to its substrates. Thus, our findings on the dual roles of USP3 (namely, one to release CHK1 from the chromatin and the other to open up the active site) provide further insights into the regulation of CHK1 following DNA damage.

Leucine rich repeat kinase (LRRK2) is the most prevalent genetic cause for Parkinson's disease. LRRK2 p.G2385R is an Asian specific genetic risk factor for sporadic Parkinson's disease. We generated two induced pluripotent stem cells (iPSCs), IBMS-iPSC-018-09 and IBMS-iPSC-020-01, from the peripheral blood mononuclear cells of two patients carrying LRRK2 p.G2385R variant by using the Sendai-virus delivery system. These iPSCs had a normal karyotype and exhibited pluripotency, such as an embryonic stem cell-like morphology, expression of pluripotent markers, and capacity to differentiate into three germ layers. This cellular model will provide a platform for pathophysiological studies of neurodegeneration in Parkinson's disease.

Mitochondrial defects are associated with clinical manifestations from common diseases to rare genetic disorders. Myoclonus epilepsy associated with ragged-red fibers (MERRF) syndrome results from an A to G transition at nucleotide position 8344 in the tRNALys gene of mitochondrial DNA (mtDNA) and is characterized by myoclonus, myopathy and severe neurological symptoms. In this study, Sendai reprogramming method was used to generate an iPS cell line carrying the A8344G mutation of mtDNA from a MERRF patient. This patient-specific iPSC line expressed pluripotent stem cell markers, possessed normal karyotype, and displayed the capability to differentiate into mature cells in three germ layers.

Current chemotherapy and immunotherapy treatments followed by transurethral resection for urinary bladder urothelial carcinoma (UC) usually suffer from poor prognosis and high recurrence rate. Design and modification of current formulation with the novel adjuvants are needed. A recombinant protein derived from Ganoderma microsporum named as Ganoderma microsporum immunomodulatory protein (GMIP) was used to treat UC cells. We found GMIP elicits a dose-dependent and time-dependent anti-UC cell proliferation effect, with a half-maximal inhibition concentration (IC50 ) comparable to mitomycin C (MMC), a commonly used chemotherapy agent. After GMIP treatment, UC cells showed apoptotic phenomenon including cell cycle arrest in the G1 phase, elevated sub-G1 population, mitochondrial membrane potential loss, up-regulated p21 expression, p21 nuclear translocation, caspase activation, and PARP cleavage in a p53-independent but p21-mediated pathways. Unlike lung cancer cells, GMIP treated UC cells showed no autophagic scheme including Beclin-1, an autophagy to apoptosis switch marker, was not cleaved by caspase 3 and slight LC3B-II accumulation. Also, the classic autophagic inhibitor, chloroquine had no effect in GMIP-mediated cell death made us conclude that GMIP induced apoptosis through caspase activation but not autophagy in UC cells. Additionally, GMIP showed synergistic effects with MMC in killing UC cells and thus decreased the concentration of MMC usage to reach the comparable apoptotic effects. Our results delineate novel strategies for treatment of UC by GMIP alone or in combination with MMC application and provide a promising therapeutic cocktail for better treatment of urinary bladder urothelial carcinoma.

In bladder cancer, urothelial carcinoma is the most common histologic subtype, accounting for more than 90% of cases. Pathogenic effects due to the dysbiosis of gut microbiota are localized not only in the colon, but also in regulating bladder cancer distally. Butyrate, a short-chain fatty acid produced by gut microbial metabolism, is mainly studied in colon diseases. Therefore, the resolution of the anti-cancer effects of butyrate-producing microbes on bladder urothelial cells and knowledge of the butyrate-responsive molecules must have clinical significance. Here, we demonstrate a correlation between urothelial cancer of the bladder and Butyricicoccus pullicaecorum. This butyrate-producing microbe or their metabolite, butyrate, mediated anti-cancer effects on bladder urothelial cells by regulating cell cycle, cell growth, apoptosis, and gene expression. For example, a tumor suppressor against urothelial cancer of the bladder, bladder cancer-associated protein, was induced in butyrate-treated HT1376 cells, a human urinary bladder cancer cell line. In conclusion, urothelial cancer of the bladder is a significant health problem. To improve the health of bladder urothelial cells, supplementation of B. pullicaecorum may be necessary and can further regulate butyrate-responsive molecular signatures.

A novel triazole-cored, star-shaped, conjugated molecule (TDGTPA) has been synthesized for use as an electron-selective interlayer in inverted polymer solar cells (PSCs). This star-shaped molecule comprised a triazole unit as the central core, 2,5-thienyl diketopyrrolopyrrole units as π-conjugated bridges, and tert-butyl-substituted triphenylamine units as both end groups and donor units. The inverted PSC had the device structure indium tin oxide/ZnO/TDGTPA/poly(3-hexylthiophene) (P3HT)/fullerene derivative (PC71BM)/MoO3/Ag. Inserting TDGTPA as the electron-selective layer enhanced the compatibility of the ZnO-based electron transport layer and the P3HT:PC71BM blend-based photoactive layer. The low energy of the lowest unoccupied molecular orbital (−3.98 eV) of TDGTPA was favorable for electron transfer from the photoactive layer to the ZnO layer, thereby enhancing the photovoltaic performance of the PSC. The photo-conversion efficiency of the device incorporating TDGTPA as the electron-selective layer was 15.8% greater than that of the corresponding device prepared without it.