Water-soluble Tetrazolium Salt Research Articles

Bone morphogenetic protein 2-enhanced osteogenic differentiation of stem cell spheres by regulation of Runx2 expression.

Bone morphogenetic protein 2 (BMP-2) is a growth factor that is used to induce osteogenic differentiation in stem cells. The present study assessed the effects of BMP-2 on stem cell spheroid morphology, viability and osteogenic differentiation. Stem cell spheres were constructed and treated with BMP-2 at predetermined concentrations (0-100 ng/ml) using concave microwells. Cell viability was qualitatively and quantitatively analyzed via microscopy and a water-soluble tetrazolium salt assay kit, respectively. Alkaline phosphatase activity was assessed and an anthraquinone dye for calcium deposit evaluation was performed to determine osteogenic differentiation. The expressions of (runt-related transcription factor 2) and collagen 1 were also determined via quantitative PCR. Spherical shapes were formed using concave microwells on day 1, which were maintained up to day 21. On day 1, the relative cell viability of 0, 10 and 100 ng/ml BMP-2 treated cells was 100.0±1.9, 97.3±4.4 and 101.3±2.6%, respectively. Significantly higher values for alkaline phosphatase activity were determined in the 100 ng/ml treated group when compared with the control group. Additionally, Runx2 mRNA levels were significantly higher in the 100 ng/ml BMP-2 group compared with the control group, as determined via quantitative PCR. The results of the present study indicated that BMP-2 enhanced the differentiation of stem cell spheres, which was demonstrated by increased alkaline phosphatase activity and Runx2 expression.

The role of the globular heads of the C1q receptor in TcdA-induced human colonic epithelial cell apoptosis via a mitochondria-dependent pathway

BackgroundClostridioides (formerly Clostridium) difficile infection is the leading cause of antibiotic-associated colitis. Studies have demonstrated that C. difficile toxin A (TcdA) can cause apoptosis of many human cell types. The purpose of this study was to investigate the relationships among exposure to TcdA, the role of the receptor for the globular heads of C1q (gC1qR) gene and the underlying intracellular apoptotic mechanism in human colonic epithelial cells (NCM 460). In this study, gC1qR expression was examined using real-time polymerase chain reaction (PCR), western blotting and immunohistochemical staining. Cell viability was assessed by the water-soluble tetrazolium salt (WST-1) assay, and cell apoptosis was assessed by flow cytometry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Mitochondrial function was assessed based on reactive oxygen species (ROS) generation, changes in the mitochondrial membrane potential (ΔΨm) and the content of ATP.ResultsOur study demonstrated that increasing the concentration of TcdA from 10 ng/ml to 20 ng/ml inhibited cell viability and induced cell apoptosis (p < 0.01). Moreover, the TcdA-induced gC1qR expression and enhanced expression of gC1qR caused mitochondrial dysfunction (including production of ROS and decreases in the ΔΨm and the content of ATP) and cell apoptosis. However, silencing of the gC1qR gene reversed TcdA-induced cell apoptosis and mitochondrial dysfunction.ConclusionThese data support a mechanism by which gC1qR plays a crucial role in TcdA-induced apoptosis of human colonic epithelial cells in a mitochondria-dependent manner.

Highly Absorbent Antibacterial and Biofilm-Disrupting Hydrogels from Cellulose for Wound Dressing Applications.

In this study, a carboxyl-modified cellulosic hydrogel was developed as the base material for wound dressings. ε-poly-l-lysine, a natural polyamide, was then covalently linked to the hydrogel through a bioconjugation reaction, which was confirmed by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR). The antibacterial efficacy of the hydrogel was tested against two model bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, two of the most commonly found bacteria in wound infections. Bacterial viability and biofilm formation after exposure of bacteria to the hydrogels were used as efficacy indicators. Live/Dead assay was used to measure the number of compromised bacteria using a confocal laser scanning microscope. The results show that the antibacterial hydrogel was able to kill approximately 99% of the exposed bacteria after 3 h of exposure. In addition, NIH/3T3 fibroblasts were used to study the biocompatibility of the developed hydrogels. Water-soluble tetrazolium salt (WST)-1 assay was used to measure the metabolic activity of the cells and Live/Dead assay was used to measure the viability of the cells after 24, 48, and 72 h. The developed antibacterial hydrogels are light weight, have a high water-uptake capacity, and show high biocompatibility with the model mammalian cells, which make them a promising candidate to be used for wound dressing applications.

Abstract 1830: Evaluation of anti-tumor effects of VER-155008, a functional inhibitor of HSP70, through macroautophagy in mesothelioma cells

Abstract Pleural mesothelioma is a poor prognostic malignancy because the effect of therapies remains limited; and thus, it is urgent to elucidate the pathophysiology leading to newer treatment. Our previous investigation showed that growth of mesothelioma cells was inhibited by suppression of dishevelled-3 on Wnt signaling pathway, accompanied with reduction of heat shock protein 70 (HSP70), which works as a cell-protective molecular chaperon against heat stress, oxidative stress, heavy metals and others, by refolding proteins. Furthermore, HSP70 has been showed to affect cell signaling such as Akt/mTOR pathway, which negatively regulates macroautophagy. This study evaluated effects of VER-155008, an adenosine-derived functional inhibitor of HSP70, on cell growth and macroautophagy of mesothelioma cell lines. Mesothelioma cell lines including MSTO-211H (211H), NCI-H2452 (H2452) and NCI-H28 (H28) were cultured with 0.5-40 µM VER-155008 for cell viability analysis by water-soluble tetrazolium salt-8, colony formation assay, cell cycle analysis and the following analyses. Western blotting confirmed the expression of HSP70, Akt, phosphorylated (p-) Akt, JNK, p-JNK, light chain 3A (LC3A) and lysosome-associated membrane protein 2A (LAMP2A). For evaluation of macroautophagy, a plasmid, pMRX-IP-GFP-LC3-RFP-LC3ΔG, was transfected to mesothelioma cells. The transfected cells generate GFP-LC3-RFP-LC3ΔG, and it was divided into GFP-LC3 and RFP-LC3ΔG by inherent ATG4. GFP-LC3 decreases by macroautophagy activation, while RFP-LC3ΔG serves as an internal control. VER-155008 suppressed cell viability dose-dependently, and colony formation of 211H, H2452 and H28. In 211H and H28 cells, the proportions of cell count in G1 phase significantly increased, accompanied with inhibition of cell growth. In western blot analysis, VER-155008 reduced expression of p-Akt, while the expression of Akt, p-JNK, JNK, LC3A, LAMP2A and HSP70 were not altered. In macroautophagy analysis by the use of mesothelioma cells, which were transfected with the plasmid and stably expressed GFP-LC3-RFP-LC3ΔG, 20 µM VER-155008, 5.0 µM gefitinib and deprivation of fetal bovine serum (FBS) induced macroautophagy. In addition, VER-155008 enhanced FBS-deprivation-induced macroautophagy. From these results, functional inhibition of HSP70 by VER-155008 suppressed cell growth in pleural mesothelioma cells, accompanied with enhancement of macroautophagy. Citation Format: Kosuke Sakai, Maya Inoue, Hiroaki Nishimura, Shintaro Mikami, Yoshiki Kuwabara, Akitoshi Kojima, Maiko Toda, Yumiko Ogawa, Satoshi Kikuchi, Yusuke Hirata, Yuriko Mikami, Hiroyuki Kyoyama, Gaku Moriyama, Akihiko Gemma, Kazutsugu Uematsu. Evaluation of anti-tumor effects of VER-155008, a functional inhibitor of HSP70, through macroautophagy in mesothelioma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1830.

Abstract 4087: ALDH1A1 inhibitor induces cell cycle arrest and reduces cell proliferation of cisplatin-resistant ovarian cancer cells

Abstract Introduction: Ovarian cancer is one of the most common malignancies among women that causes severe mortality in USA and globally. Despite advances in cancer treatments in the last few decades, there is no substantial improvement in the survival rates of patients with advanced ovarian cancers. This is mainly attributed to drug resistance and tumor recurrence. Most patients develop resistance to platinum compounds, which are often used as the first-line therapy. Several studies have shown that aldehyde dehydrogenase (ALDH1A1) plays an important role in cancer stemness and confers resistance to cell death induced by platinum compounds used to treat ovarian cancers. Therefore, in this study, we determined the effect of a small-molecule inhibitor of ALDH1A1 (A37) in cisplatin-resistant ovarian cancer cells and assessed it's molecular mechanism of action. Experimental methods: The cisplatin-resistant ovarian cancer cells (A2780-CR) were exposed to various concentrations of ALDH1A1 inhibitor (A37) and then the cell proliferation by water-soluble tetrazolium salt (WST-1) assay, cell cycle analysis by flow cytometry and oxygen consumption rate (OCR) by Seahorse extracellular flux analysis (Seahorse XFe24 Analyzer) were performed. Furthermore, the protein expression of critical signaling molecules that modulate the proliferation of ovarian cancer cells was assessed by western blot analysis. Results: A37 treatment drastically decreased the proliferation of A2780-CR cells at 24 and 48 h. In addition, cell cycle analysis revealed a significant increase in G1 phase and dramatic decrease in S phase of the cell cycle upon A37 treatment. Interestingly, A37 suppressed the basal OCR, an indicator of mitochondrial respiration. Furthermore, A37 reduced the protein expression of mammalian target of rapamycin (mTOR), which plays a critical role in cancer cell cycle and proliferation. Conclusions: Our findings indicate that the ALDH1A1 inhibitor (A37) induces cell cycle arrest and suppresses the cell proliferation of cisplatin-resistant ovarian cancer cells, possibly through downregulation of mTOR signaling. Most importantly, A37 affects the cancer cell metabolism by reducing the mitochondrial respiration. Taken together, our study highlights the importance of potential therapeutic implication of ALDH1A1 inhibitor towards the treatment of chemoresistant-ovarian cancers. Citation Format: Ilangovan Ramachandran, Sivakumar Ramadoss, Selvendiran Karuppaiyah, Lauren Nathan, R. Ileng Kumaran, Gautam Chaudhuri. ALDH1A1 inhibitor induces cell cycle arrest and reduces cell proliferation of cisplatin-resistant ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4087.

Abstract 2863: Preclinical evaluation of astatine-211-conjugated anti-tissue factor antibody

Abstract It has been well-known that tissue factor (TF), the trigger protein of the extrinsic blood coagulation cascade, is overexpressed in various cancers including gastric cancer, pancreatic cancer and malignant gliomas. So far, we produced anti-TF monoclonal antibodies for targeting overexpressed TF in tumor site. Moreover, among them, we found that the clone 1084 possessed deep penetration property leading to more homogenous distribution in tumor than others. Currently, we are focusing on developing clone 1084 antibody armed with alpha emitters for cancer treatment, because alpha-particles are characterized by higher liner energy transfer (LET) and shorter range in tissue than other types of radiation, resulting in efficient deoxyribonucleic acid (DNA) double-strand breaks in cancer cells and minor effects on normal cells adjacent to tumor. In this study, we synthesized astatine-211, one of the most promising alpha-particle emitters, in the 209Bi(α,2n)211At reaction using the RIKEN Azimuthally Varying Field (AVF) cyclotron and labeled humanized clone 1084 with this radionuclide. First, we chemically attached a linker containing organic tin compound to the antibody and then labeled the liker-antibody complex with astatine-211 via a halogen-exchange reaction. Thin-layer chromatography (TLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies showed that we successfully labeled the antibody with astatine-211 and purified the radioactive antibody. We evaluated binding activity and in vitro cytocidal effect of the radioactive antibody by flow cytometry and water-soluble tetrazolium salt (WST-8) assay, respectively. The radioactive antibody specifically bound to TF expressed on cell membrane of gastric cancer cells and showed potent cell killing activity depending on TF expression of the cancer cells. Single photon emission computed tomography/computed tomography (SPECT/CT) study revealed that astatine-211 alone selectively accumulated in stomach and thyroid such as radioactive iodine. On the other hand, radioactive antibody showed similar pharmacokinetics to the naked 1084 antibody. Moreover, we confirmed in vivo anti-tumor effects of the radioactive antibody in a TF-overexpressing gastric cancer xenograft model. In conclusion, we successfully created astatine-211-conjugated anti-TF antibody and confirmed its anti-tumor effects. Citation Format: Hiroki Takashima, Yoshikatsu Koga, Kazunobu Onuki, Shino Manabe, Ryo Tsumura, Takahiro Anzai, Nozomi Iwata, Masahiro Yasunaga, Wang Yang, Takuya Yokokita, Yukiko Komori, Daiki Mori, Hiromitsu Haba, Hirofumi Fujii, Yasuhiro Matsumura. Preclinical evaluation of astatine-211-conjugated anti-tissue factor antibody [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2863.

Glyoxal and Methylglyoxal as E-cigarette Vapor Ingredients-Induced Pro-Inflammatory Cytokine and Mucins Expression in Human Nasal Epithelial Cells.

Glyoxal (GO), and methylglyoxal (MGO) are among the most toxic compounds emitted by electronic cigarette (E-cig) and regular tobacco cigarette smoke. Airway diseases presented mucus over production as their major pathophysiologic feature. However, the effects of GO and MGO on pro-inflammatory cytokines and mucin expression in human nasal epithelial cells, as well as the underlying signaling pathway, have not yet been studied. This study is to determine whether GO and MGO induce pro-inflammatory cytokines, and MUC5AC/5B expression via mitogen-activated protein kinase (MAPK)s and nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways. The effect of GO, and MGO on pro-inflammatory cytokines, mucins expression and the signalling pathway of GO and MGO were investigated using water-soluble tetrazolium salt-1, enzyme immunoassays, and immunoblot analysis with specific inhibitors and small interfering RNA. GO and MGO did not affect cell viability up to 2 mM in human nasal epithelial cells. GO and MGO increased production of pro-inflammatory such as interleukin (IL)-1β and IL-6) and MUC5AC/5B. Additionally, GO and MGO significantly activated extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and NF-κB. Whether ERK1/2, p38 MAPK, and NF-κB signaling pathway were involved in GO and MGO-induced production of pro-inflammatory cytokines (IL-1β and IL-6) and MUC5AC/5B, we used specific inhibitors and siRNA transfection. These significantly repressed GO- and MGO-induced expression of pro-inflammatory cytokines (IL-1β and IL-6) and MUC5AC/5B. GO and MGO induced pro-inflammatory cytokines and MUC5AC/5B expression via ERK1/2, p38 MAPK, and NF-κB in human nasal epithelial cells. These results suggested that GO and MGO may be involved in mucus hypersecretion-related airway diseases.

Improving the regenerative microenvironment during tendon healing by using nanostructured fibrin/agarose-based hydrogels in a rat Achilles tendon injury model.

Achilles tendon injuries are a frequent problem in orthopaedic surgery due to their limited healing capacity and the controversy surrounding surgical treatment. In recent years, tissue engineering research has focused on the development of biomaterials to improve this healing process. The aim of this study was to analyze the effect of tendon augmentation with a nanostructured fibrin-agarose hydrogel (NFAH) or genipin cross-linked nanostructured fibrin-agarose hydrogel (GP-NFAH), on the healing process of the Achilles tendon in rats. NFAH, GP-NFAH, and MatriDerm (control) scaffolds were generated (five in each group). A biomechanical and cell-biomaterial-interaction characterization of these biomaterials was then performed: Live/Dead Cell Viability Assay, water-soluble tetrazolium salt-1 (WST-1) assay, and DNA-released after 48 hours. Additionally, a complete section of the left Achilles tendon was made in 24 Wistar rats. Animals were separated into four treatment groups (six in each group): direct repair (Control), tendon repair with MatriDerm, or NFAH, or GP-NFAH. Animals were euthanized for further histological analyses after four or eight weeks post-surgery. The Achilles tendons were harvested and a histopathological analysis was performed. Tensile test revealed that NFAH and GP-NFAH had significantly higher overall biomechanical properties compared with MatriDerm. Moreover, biological studies confirmed a high cell viability in all biomaterials, especially in NFAH. In addition, in vivo evaluation of repaired tendons using biomaterials (NFAH, GP-NFAH, and MatriDerm) resulted in better organization of the collagen fibres and cell alignment without clinical complications than direct repair, with a better histological score in GP-NFAH. In this animal model we demonstrated that NFAH and GP-NFAH had the potential to improve tendon healing following a surgical repair. However, future studies are needed to determine the clinical usefulness of these engineered strategies. Cite this article: Bone Joint J 2020;102-B(8):1095-1106.

Molecular expression analysis and characterization of rockfish (Sebastes schlegelii) B cell activating factor.

B cell activating factor (BAFF) is recognized as a member of the TNF superfamily proteins that mediate the immune responses. In this study, BAFF from rockfish (Sebastes schlegelii) (SsBAFF) was characterized based on its functional aspects. The open reading frame of SsBAFF is 804bp in length and encodes a 267 long amino acid residue protein with predicted molecular weight of 29.48kDa. The deduced protein sequence comprises with transmembrane domain, furin cleavage site and TNF domain carrying Flap binding site that unique to TNF family. Recombinant SsBAFF (rSsBAFF) significantly enhanced rockfish lymphocytes proliferation and viability in a concentration dependent-manner according to the results from water soluble tetrazolium salt (WST-1) assay and flow cytometric assay. rSsBAFF also modulated the expression of genes involved in anti-inflammatory (IL-10 and NFκB-2) and anti-apoptotic (Bcl-2 and Bax) signal pathways. SsBAFF mRNA expression was detected ubiquitously in all analyzed rockfish tissues, with the highest levels in the spleen and head kidney. Further, the expression of SsBAFF in spleen were significantly induced following LPS, poly (I:C) and Streptococcus iniae challenges. These findings strongly suggest that SsBAFF might play an important role in rockfish immune system through regulating the inflammatory response and proliferation of immune cells.

In Vitro Evaluation of Lignin-Containing Nanocellulose.

The increasing importance of environmental sustainability has led to the development of new materials that are environmentally friendly, functional, and cost-effective. Lignin-containing cellulose nanomaterials are a common example of these. The advantages of lignocelluloses include their renewability, sustainability, and functionality combined with molecular rigidity and enhanced hydrophobicity. In order to valorize these beneficial traits from lignin-containing nanocellulose, various approaches have been examined in industrial applications. However, the safety of these materials has not been tested or validated in humans. In this study, we tested 21 wt% lignin-containing nanocellulose (L-MFC) in vitro using the human lung and kidney cell lines, H460 and HEK293 cells, respectively. The cytotoxicity of cellulose, L-MFC, and lignin was compared using the water-soluble tetrazolium salt assays. In addition, the gene expressions of HSP70 and HSP90 as cellular stress markers treated with cellulose, L-MFC, and lignin were quantified using real-time polymerase chain reaction (PCR) and Western blotting. Our data indicated little cytotoxicity for cellulose and significant cytotoxicity for lignin and a relatively low level of cytotoxicity for L-MFC, providing the lethal median concentration (LC50) values of L-MFC and lignin. The gene expression of HSP70 and HSP90 was little affected by moderate concentrations of L-MFC. Interestingly, the lignin contained in L-MFC influenced the cell viability and the gene expression of HSP70 and HSP90 less than the same amount of lignin alone. These results indicate that L-MFC displays cell-type-dependent sensitivity and suggest that L-MFC could serve as a new eco-friendly material that is relatively safe for humans.

Targeting CD146 using folic acid-conjugated nanoparticles and suppression of tumor growth in a mouse glioma model.

Glioma stem cells (GSCs) are responsible for tumor initiation, therapeutic resistance, and recurrence. CD146 is mainly expressed in dividing GSCs and regulates cell cycle progression. However, the evaluation of the efficacy of targeted therapy against CD146 in vivo remains to be investigated. In this study, the authors aimed to develop gene therapy targeting GSCs using chitosan oligosaccharide lactate (COL) nanoparticles (NPs) conjugated with folic acid-polyethylene glycol (FA-PEG-COL NPs) for in vitro and in vivo delivery of CD146 small-interfering RNA (siCD146) and to determine the effect of CD146 knockdown on tumor growth. To examine the uptake of NPs by tumor cells, immunofluorescence staining, flow cytometry, and in vivo imaging were performed. The knockdown effect of siCD146 was measured by western blot and water-soluble tetrazolium salt-8 assay in mouse glioma cells. The efficacy of siRNA therapy-targeted GSCs was evaluated by monitoring tumor growth through in vivo imaging and histological analysis. In vivo accumulation of the FA-PEG-COL NPs in subcutaneous and intracranial gliomas following NP administration via a mouse tail vein was observed. Additionally, in vitro delivery of siCD146 ionically cross-linked NPs, reduced CD146 levels, and suppressed growth in the glioma tumor sphere. Evaluation of the in vivo therapeutic effects of siCD146-cross-linked NPs in a mouse glioma model revealed significant suppression of intracranial tumor growth, with complete removal of the tumor observed in some mice on histological examination. Furthermore, delivery of siCD146 significantly reduced the Ki-67 index in residual tumor tissues relative to that in control mice. CD146 is a potential therapeutic target, and folic acid-conjugated NPs delivering siRNA may facilitate gene therapy in malignant gliomas.

Dentin Conditioning Protocol for Regenerative Endodontic Procedures

IntroductionThis study focused on the optimization of sodium hypochlorite–EDTA irrigation in terms of the viability and morphology of dental pulp stem cells (DPSCs) and the effects of an optimized EDTA protocol alone or prepared with nanobubble (NB) water on cell behavior. MethodsIn the first part, human dentin discs were conditioned with the following protocols:(1) Sodium hypochlorite followed by phosphate-buffered saline (PBS),(2) Irrigation protocol from group 1 followed by EDTA,(3) Irrigation protocol from group 2 followed by PBS,(4) Sodium hypochlorite followed by EDTA,(5) Irrigation protocol from group 4 followed by PBS. DPSC viability and morphology were determined. In the second part, dentin discs were conditioned with the (1) optimized protocol in the first part, (2) EDTA prepared using NB water, (3) ultrasonic-activated EDTA, or (4) ultrasonic-activated EDTA prepared using NB water. Transforming growth factor beta release and DPSC viability, morphology, and migration were determined using the enzyme-linked immunosorbent assay, the water-soluble tetrazolium salt-1 cell viability assay and live-dead assay, and the transwell migration assay, respectively. Data were analyzed using Kruskal-Wallis or one-way analysis of variance and post hoc tests. ResultsThe highest cell viability was observed in group 3 followed by group 5 (P < .05) in which PBS was used as a final rinse. Irrigation protocol from group 3 was used for the subsequent experiments. Ultrasonic-activated EDTA improved transforming growth factor beta release, viability, and migration of the cells compared with EDTA (P < .05). The preparation of EDTA with NBs did not change the biological properties of the EDTA-conditioned dentin (P > .05). ConclusionsRemoving the residual EDTA using PBS improved the cell viability on the dentin surface. Ultrasonic activation enhanced the growth factor release and biological properties, whereas the preparation of EDTA with NBs showed a similar effect to regular EDTA without compromising the cellular effect.

Devitalizing Effect of High Hydrostatic Pressure on Human Cells-Influence on Cell Death in Osteoblasts and Chondrocytes.

Chemical and physical processing of allografts is associated with a significant reduction in biomechanics. Therefore, treatment of tissue with high hydrostatic pressure (HHP) offers the possibility to devitalize tissue gently without changing biomechanical properties. To obtain an initial assessment of the effectiveness of HHP treatment, human osteoblasts and chondrocytes were treated with different HHPs (100–150 MPa, 250–300 MPa, 450–500 MPa). Devitalization efficiency was determined by analyzing the metabolic activity via WST-1(water-soluble tetrazolium salt) assay. The type of cell death was detected with an apoptosis/necrosis ELISA (enzyme-linked immune sorbent assay) and flow cytometry. Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) were carried out to detect the degree of cell destruction. After HHP treatment, the metabolic activities of both cell types decreased, whereas HHP of 250 MPa and higher resulted in metabolic inactivation. Further, the highest HHP range induced mostly necrosis while the lower HHP ranges induced apoptosis and necrosis equally. FESEM and TEM analyses of treated osteoblasts revealed pressure-dependent cell damage. In the present study, it could be proven that a pressure range of 250–300 MPa can be used for cell devitalization. However, in order to treat bone and cartilage tissue gently with HHP, the results of our cell experiments must be verified for tissue samples in future studies.

SERS biosensors for ultrasensitive detection of multiple biomarkers expressed in cancer cells

The design and fabrication of multifunctional surface-enhanced Raman scattering (SERS) nanotags are key issues in their application to biological imaging of cells and tissues. In this study, highly sensitive, reproducible and long-term stable SERS nanotags were developed for the identification of localized distribution of multiple protein biomarkers expressed on breast cancer cells. To enhance the surface electromagnetic fields of Raman reporter molecules, Ag-encapsulated Au (Ag-Au) hollow nanospheres were synthesized. Strong Raman signal enhancement effects could be achieved by positioning Raman reporter molecules in nanogaps between the Au hollow nanospheres and silver shell. In addition, the signal was also enhanced due to the localization of surface electromagnetic fields through the pinholes on the surface of Au hollow nanospheres. To maintain the long-term stability of the Au hollow-Ag core/shell nanospheres, their surface was coated with a polyethylene glycol (PEG) layer. The biocompatibility of PEGylated Ag-Au hollow nanospheres was investigated using the premix water soluble tetrazolium salt (WST-1) cell viability test. These SERS nanotags also enabled a high-resolution multiplexed live cell imaging. Our proposed SERS imaging technique using the new SERS nanotags provides a new platform for fast and accurate classification of different phenotypes of breast cancer cells.

Human CD200 Suppresses the HL-60 Mediated Xenocytotoxicity

BackgroundNeutrophils play an important role in xenogeneic rejection and represent a major obstacle in clinical application of xenografts. CD200 and its receptor CD200R are both type-1 membrane glycoproteins, which are members of the immunoglobulin superfamily (IgSF) and the ligation of CD200 with CD200R induces inhibitory NPXY signaling. The expression of CD200R appears in myeloid cells such as macrophages and granulocytes. Thus, we hypothesized that human CD200 expression on porcine cells might suppress the xenogeneic neutrophil-mediated cytotoxicity against porcine cells. MethodsTo prove our hypothesis, the suppressive effect of human CD200 in neutrophil-like human cell line 60 (HL-60)–mediated xenogeneic cytotoxicity against swine endothelial cells (SECs) was examined. Cytotoxicity was assessed with water-soluble tetrazolium salt 8 (WST-8) assay. ResultsHL-60 cells differentiated into CD66b+ CD200R+ neutrophil-like cells in the presence of dimethyl sulfoxide (DMSO). HL-60-mediated cytotoxicity against SECs was significantly suppressed by human CD200 on SECs. ConclusionsThe findings in this study indicate that human CD200 may suppress neutrophil-mediated xenogeneic rejection.

Proteome-wide identification of arginine methylation in colorectal cancer tissues from patients

BackgroundProtein arginine methylation reaction is catalyzed by protein arginine methyltransferase (PRMT) and the modification is implicated in various diseases including cancer. Currently, thousands of arginine methylation sites have been identified using high-resolution mass spectrometry-based proteomics technology. However, identification of arginine methylation using clinical samples at proteome level has not been reported yet. The objective of the present study was to identify, monomethyl-arginine (MMA) and asymmetric dimethyl-arginine (ADMA) sites in colorectal cancer (CRC) tissues at proteome level.MethodsPooled CRC tissue samples from 10 patients with stage II and III were digested by trypsin and these digests were further processed and lyophilized. Using monomethyl- or asymmetric dimethyl arginine (MMA or ADMA, respectively) motif kits, methylarginine-containing peptides were enriched and subsequently analyzed by high-resolution LC-MS/MS. DLD1 and HCT116 colon cancer cells were treated with type I PRMTs inhibitor (MS023) alone or combined with SN-38, and the effect of the drugs on CRC cell proliferation and apoptosis was measured by water-soluble tetrazolium salt (WST-1) assay and FACS analysis, respectively.ResultsIn the present study, 455 MMA sites of 272 proteins and 314 ADMA sites of 155 proteins were identified from CRC tissues acquired from patients. In addition, 216 methylation sites and 75 substrates for PRMTs were newly identified. These results reveal the significant presence of MMA and ADMA sites on nucleic acid binding proteins and protein complexes involved in transcription. To investigate the effect of protein arginine methylation in CRC proliferation and apoptosis, MS023 was treated to two CRC cell lines. After 48 h treatment with various concentrations of MS023, CRC cell proliferation was significantly suppressed, with concomitant apoptosis induction. Furthermore, MS023 treatment significantly enhanced the inhibitory effect of SN-38 on CRC cell proliferation.ConclusionThis work reports the first comprehensive analysis of arginine methylation with clinical sample and suggests that type I PRMTs are potential therapeutic targets for drug discovery in CRC.

Human CD31 on Swine Endothelial Cells Induces SHP-1 Phosphorylation in Macrophages

BackgroundInnate immunity by natural killer (NK) cells, macrophages, and neutrophils cause severe rejections in xenotransplantation. Therefore, the development of strategies for suppressing macrophages has considerable potential in practical applications of xenotransplantation. Recently, we found that human CD31 on swine endothelial cells (SECs) suppresses neutrophil-mediated xenogeneic rejection through homophilic binding. Since a significant amount of CD31 is expressed not only on neutrophils but also on macrophages, we studied the function of human CD31 in macrophage-mediated cytotoxicity. MethodsSECs and hCD31-transfected SECs (SEC/hCD31) were co-cultured with macrophages and cytotoxicity by macrophages was evaluated with water-soluble tetrazolium salt, or WST-8, assay. To confirm whether or not inhibitory signals are induced by hCD31 homophilic binding, the phosphorylation of the enzyme SHP-1 was investigated with Western blotting. ResultsNo suppression of cytotoxicity was induced in macrophages that had been co-cultured with SEC/CD31. However, phosphorylation of SHP-1 was induced in macrophages that had been co-cultured with SEC/hCD31. ConclusionsHuman CD31 on SEC may induce not only inhibitory signals but also activation signals via the binding to other receptors for hCD31.

Vitamin D regulates cell viability, migration and proliferation by suppressing galectin-3 (Gal-3) gene in ovarian cancer cells

Vitamin D deficiency is identified as a risk factor for the occurrence and recurrence of ovarian cancer. Galectin-3 (Gal-3) participates in many physiological and pathological processes. In present study, serum vitamin D level was detected using chemiluminescence enzyme immunoassay. Gal-3 expression was examined using real-time polymerase chain reaction (PCR), Western blot and immunocytochemistry analysis. SKOV3 cells viability was assessed by the water-soluble tetrazolium salt (WST-1) assay, the migration of SKOV3 cells was detected using transwell assay, and the proliferation of SKOV3 cells was measured by 3H-thymidine incorporation (3H-TdR). Our study demonstrated that vitamin D levels were lower in 40 ovarian cancer patients: vitamin D deficiency is closely related to the pathogenesis of ovarian cancer. Treatment with vitamin D reduced the migration and proliferation of ovarian cancer cells. Gal-3 was overexpressed in ovarian cancer, which could induce the viability, migration and proliferation ability of ovarian cancer cells, and these effects were abrogated by vitamin D downregulating the expression of Gal-3 gene. Therefore, our results support that vitamin D may suppress Gal-3-induced viability, migration and proliferation ability of ovarian cancer cells, which suggests that the use of vitamin D may have beneficial effects in preventing and treating ovarian cancer.

MON-513 Suppressing the Growth of Human Medullary Thyroid Cancer Cells Using FDA-Approved Drug

Medullary thyroid carcinoma (MTC) is a solid tumor of the parafollicular cells in the thyroid gland. MTC has worse prognosis, when compared with other differentiated thyroid cancers, and MTC patients with distant metastases have a low survival rate unless thyroidectomy is performed at an early stage. Furthermore, conventional treatments have only marginal benefits. Therefore, there is a need to develop novel therapeutics for MTC. Several drugs that are developed and tested in preclinical trials fail in clinical trials. Therefore, repurposing the already US Food and Drug Administration (FDA)-approved drugs towards the treatment of cancers may have potential benefits, like saving the lives of cancer patients and lowering the investment cost of drug development. Here, we explored a novel precision treatment for thyroid cancers by repurposing the FDA-approved small molecule anti-parasitic drug Nitazoxanide (NTZ). In our study, we examined the anticancer effects of NTZ on human MTC cells using the TT cell line. We treated the TT cells with different concentrations of NTZ and assessed the cell proliferation by water-soluble tetrazolium salt (WST-1) assay and oxygen consumption rate (OCR) by Seahorse extracellular flux analysis (Seahorse XFe24 Analyzer). Additionally, we determined the effects of NTZ on the protein expression of key signaling molecules that regulate MTC cell growth by western blot analysis. Our results indicated that NTZ significantly suppressed the growth of TT cells at 24 h treatment. Very importantly, NTZ reduced the basal OCR demonstrating the inhibition of mitochondrial respiration. Moreover, protein expression studies revealed that NTZ markedly reduced the key Hippo signaling pathway effector molecule TAZ and the oncogene c-myc. Interestingly, NTZ decreased the expression of epidermal growth factor receptor (EGFR) that plays an important role for RET activation in MTC. Importantly, NTZ increased the expression of p53 upregulated modulator of apoptosis (Puma). Taken together, our findings demonstrate for the first time that NTZ inhibits the growth of MTC cells and decreases the cancer cell metabolism. The mechanisms by which NTZ targets the MTC cells involve the suppression of key oncogenic proteins and upregulation of tumor suppressor molecule. Thus, our study highlights that repurposing this FDA-approved currently used drug may have a greater advantage of being tested in preclinical models of MTC, and therefore, for the rapid consideration of NTZ as a potential therapeutic drug to treat MTC patients in the near future.

The Effect of Composol Medium on miR-16 Expression during Platelet Storage up to Day 7 at Room Temperature.

Objective MicroRNAs (miRNAs) are short, noncoding RNAs that play vital roles in gene regulation. It has been shown that storage has an effect on platelet miRNAs. MiR-16 is highly expressed in platelets and it appears to target the genes involved in cell death. It has been shown that platelets could be stored in Composol for a longer period of time. The aim of the present study was to assess and compare the expression pattern of miR-16 in platelet concentrates (PCs) in plasma and Composol media. Materials and Methods In an experimental study, ten PC bags were collected and each bag was divided into two separate bags, one with the 70% Composol and the other with only plasma. Both bags were stored for 7 days at 22˚C and tested on days 1, 3, 5, and 7 of storage. For each sample, we performed quantitative real-time polymerase chain reaction (qRT-PCR). The water-soluble tetrazolium salt-1 (WST-1) test was used to assess platelet viability in all of the samples. Statistical analysis was done by SPSS and REST software. A P<0.05 was considered statistically significant. Results miR-16 was significantly elevated during the storage days, with fold changes of 3.47 (plasma) and 2.77 (Composol). The Composol group had significantly decreased miR-16 expression compared with the plasma group. Results of the WST-1 test showed less decrease in optical density (OD) in the Composol group (0.93 ± 0.4) during the storage days compared with the plasma group (0.75 ± 0.3). ConclusionOur finding supported results from previous studies that reported an increase in miR-16 expression during platelet storage. In addition, miR-16 down-regulation in Composol medium implied that Composol might be a good solution for long-term platelet storage because it has the potential to elevate the shelf-life of platelets stored at 22˚C.