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Background and Objective Coronavirus disease 2019 is a novel disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 virus. It was first detected in December 2019 and has since been declared a pandemic causing millions of deaths worldwide. Therefore, there is an urgent need to develop effective therapeutics against coronavirus disease 2019. A critical step in the crosstalk between the virus and the host cell is the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to the peptidase domain of the angiotensin-converting enzyme 2 (ACE2) receptor present on the surface of host cells. Methods An in silico approach was employed to design a 13-amino acid peptide inhibitor (13AApi) against the RBD of the SARS-CoV-2 spike protein. Its binding specificity for RBD was confirmed by molecular docking using pyDockWEB, ClusPro 2.0, and HDOCK web servers. The stability of 13AApi and the SARS-CoV-2 spike protein complex was determined by molecular dynamics simulation using the GROMACS program while the physicochemical and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of 13AApi were determined using the ExPASy tool and pkCSM server. Finally, in vitro validation of the inhibitory activity of 13AApi against the spike protein was performed by an enzyme-linked immunosorbent assay. Results In silico analyses indicated that the 13AApi could bind to the RBD of the SARS-CoV-2 spike protein at the ACE2 binding site with high affinity. In vitro experiments validated the in silico findings, showing that 13AApi could significantly block the RBD of the SARS-CoV-2 spike protein. Conclusions Blockage of binding of the SARS-CoV-2 spike protein with ACE2 in the presence of the 13AApi may prevent virus entry into host cells. Therefore, the 13AApi can be utilized as a promising therapeutic agent to combat coronavirus disease 2019. Supplementary Information The online version contains supplementary material available at 10.1007/s40268-021-00357-0.

Lipoxygenases (LOXs) are a family of enzymes that can oxygenate polyunsaturated fatty acids. As a member of the family, 15-lipoxygenase-1 (15-LOX-1) specifically metabolizes arachidonic acid and linoleic acid. 15-LOX-1 can affect physiological and pathophysiological events via regulation of the protein-lipid interactome, alterations in intracellular redox state and production of lipid metabolites that are involved in the induction and resolution of inflammation. Although several studies have shown that 15-LOX-1 has an antitumorigenic role in many different cancer models, including breast cancer, the role of the protein in cancer drug resistance has not been established yet. In this study, we, for the first time, aimed to show the potential role of 15-LOX-1 in acquired doxorubicin (DOX) resistance in MCF7 and HeLa cancer cell lines. Our results show that ALOX15 was transcriptionally downregulated in DOX-resistant cells compared with their drug-sensitive counterparts. Moreover, overexpression of ALOX15 in the drug-resistant cells resulted in resensitization of those cells to DOX in a cell-dependent manner. 15-LOX-1 expression could induce apoptosis by activating PPAR gamma and enhance the accumulation of DOX in drug-resistant MCF7 cells by altering cellular motility properties, and membrane dynamics. However, HeLa DOX cells did not show any of these effects but were susceptible to cell death when treated with 13(S)-HODE. These results underline the role and importance of 15-LOX-1 in cancer drug resistance, and points to novel mechanisms as a therapeutic approach to overcome cancer drug resistance.

Bacterial endotoxin-induced sepsis causes 30–40% of the deaths in the intensive care unit (ICU) globally, for which there is no pharmacotherapy. Lipopolysaccharide (LPS), a bacterial endotoxin, stimulates the Toll-like receptor (TLR)-4 signalling pathways to upregulate the expression of various inflammatory mediators. Here, we show that the TIRAP and c-Jun protein signalling complex forms in macrophages in response to LPS stimulation, which increases the AP1 transcriptional activity, thereby amplifying the expression of inflammatory mediators. Using a computer-aided molecular docking platform, we identified gefitinib as a putative inhibitor of the TIRAP-c-Jun signalling complex. Further, we demonstrated the ability of gefitinib to inhibit the interaction of TIRAP-c-Jun with in vitro experiments and with a mouse model of sepsis. Importantly, pre-treatment with gefitinib increased the survival of the mice that received a lethal dose of LPS compared to that of the controls. These findings verify the ability of gefitinib to directly disrupt the interaction of TIRAP and c-Jun, thereby inhibiting a major inflammatory response that is often observed in patients experiencing sepsis.

We report the surface modification of chitosan/poly(ethylene glycol) (chitosan/PEG) hydrogel materials via layer-by-layer (LbL) technique using stimuli-responsive polymers. Water-soluble complexes of Tannic Acid (TA) and a broad-spectrum antibiotic, Ciprofloxacin (Cipro) were prepared and co-assembled at the surface of Chitosan/PEG hydrogels with poly(N-vinyl caprolactam) (PVCL). Compared to the bare hydrogels, the surface spreading and proliferation of human fibroblasts were significantly enhanced on precast hydrogels coated with TA-Cipro/PVCL multilayers. LbL coating also provided enhanced Cipro release from the hydrogel surface at 37 °C compared to bare hydrogels. TA-Cipro/PVCL coated hydrogels showed antibacterial activity through chitosan and temperature-induced release of Cipro from multilayers. Chitosan and Cipro showed a coordinated antibacterial effect on Eschericia coli and Bacillus cereus, reducing their minimum inhibitory concentration (MIC). This effect was more pronounced on B. cereus. LbL modification of chitosan-based hydrogels using stimuli responsive polymers can be advantageous for bringing multiple functionalities to these materials without sacrificing their intrinsic properties such as antibacterial activity and biocompatibility. Such LbL-coated hydrogels hold promise in wound treatment as they may promote fibroblast proliferation and skin tissue regeneration.

Recently, there has been a resurgence of interest in metabolic rewiring of tumors to identify clinically relevant genes. However, most of these studies have had either focused on individual tumors, or are too general, providing a broad outlook on overall changes. In this study, we have first curated an extensive list of genes encoding metabolic enzymes and metabolite transporters relevant to carbohydrate, fatty acid and amino acid oxidation and biosynthesis. Next, we have used publicly available transcriptomic data for 20 different tumor types from The Cancer Genome Atlas Network (TCGA) and focused on differential expression of these genes between tumor and adjacent normal tissue. Our study revealed major transcriptional alterations in genes that are involved in central metabolism. Most tumors exhibit upregulation in carbohydrate and amino acid transporters, increased glycolysis and pentose phosphate pathway, and decreased fatty acid and amino acid oxidation. On the other hand, the expression of genes of the tricarboxylic acid cycle, anaplerotic reactions and electron transport chain differed between tumors. Although most transcriptomic alterations were conserved across many tumor types suggesting the initiation of common regulatory programs, expression changes unique to specific tumors were also identified, which can provide gene expression fingerprints as potential biomarkers or drug targets. Our study also emphasizes the value of transcriptomic data in the deeper understanding of metabolic changes in diseases.

Deregulation of metabolic pathways has increasingly been appreciated as a major driver of cancer in recent years. The principal cancer-associated alterations in metabolism include abnormal uptake of glucose and amino acids and the preferential use of metabolic pathways for the production of biomass and nicotinamide adenine dinucleotide phosphate (NADPH). Aldo-keto reductases (AKRs) are NADPH dependent cytosolic enzymes that can catalyze the reduction of carbonyl groups to primary and secondary alcohols using electrons from NADPH. Aldose reductase, also known as AKR1B1, catalyzes the conversion of excess glucose to sorbitol and has been studied extensively for its role in a number of diabetic pathologies. In recent years, however, high expression of the AKR1B and AKR1C family of enzymes has been strongly associated with worse outcomes in different cancer types. This review provides an overview of the catalysis-dependent and independent data emerging on the molecular mechanisms of the functions of AKRBs in different tumor models with an emphasis of the role of these enzymes in chemoresistance, inflammation, oxidative stress and epithelial-to-mesenchymal transition.

Proteinase-activated receptor 2 (PAR-2) is a G protein-coupled receptor activated by both trypsin and a specific agonist peptide, SLIGKV-NH2. It has been linked to various pathologies, including pain and inflammation. Several peptide and peptidomimetic agonizts for PAR-2 have been developed exhibiting high potency and efficacy. However, the number of PAR-2 antagonists is smaller. We screened the Food and Drug Administration library of approved compounds to retrieve novel antagonists for repositioning in the PAR-2 structure. The most efficacious compound bicalutamide bound to the PAR-2 binding groove near the extracellular domain as observed in the in silico studies. Further, it showed reduced Ca2+ release in trypsin activated cells in a dose-dependent manner. Hence, bicalutamide is a novel and potent PAR-2 antagonist which could be therapeutically useful in blocking multiple pathways diverging from PAR-2 signaling. Further, the novel scaffold of bicalutamide represents a new molecular structure for PAR-2 antagonism and can serve as a basis for further drug development.

We report the preparation of ultrathin coatings of zwitterionic block copolymer micelles and a comparison of their protein adsorption, adhesiveness, and antibacterial properties. Zwitterionic block copolymer micelles were obtained through pH-induced self-assembly of poly[3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate- b-2-(diisopropylamino)ethyl methacrylate] (βPDMA- b-PDPA) at pH 7.5. βPDMA- b-PDPA micelles with zwitterionic βPDMA-corona and pH-responsive PDPA-core were then used as building blocks to prepare layer-by-layer (LbL) assembled multilayer films together with hyaluronic acid (HA), tannic acid (TA), or poly(sodium 4-styrenesulfonate) (PSS). Protein adsorption tests showed that 3-layer βPDMA- b-PDPA micelles/HA films were the most effective to reduce the adhesion of BSA, lysozyme, ferritin, and casein. In contrast, βPDMA- b-PDPA micelles/TA films were the most attractive surfaces for protein adsorption. Bacterial antiadhesive tests against a model Gram-negative bacterium, Escherichia coli, and a model Gram-positive bacterium, Staphylococcus aureus, were in good agreement with the protein adsorption properties of the films. The differences in the antiadhesive properties between these three different film systems are discussed within the context of chemical nature and the functional chemical groups of the polyanions, layer number, and surface morphology of the films. Multilayers were found to lose their antiadhesiveness in the long term. However, by taking advantage of the pH-responsive hydrophobic micellar cores, we show that an antibacterial agent could be loaded into the micelles and multilayers could exhibit antibacterial activity in the long term especially at moderately acidic conditions. In contrast to antiadhesive properties, no significant differences were recorded in the antibacterial properties between the different film types.

Orthopedic implants have shown major success in clinics after successful orthopedic surgeries. However, they are also associated with several drawbacks such as local and systemic immune reaction in the body and improper integration of the bone with the implants. Osteoconductivity is a property of orthopedic implants that promotes bone cell adhesion and bone tissue integration on implant surfaces. This study presents use of a biodegradable cationic polyester, poly(4-hydroxy-L-proline ester) (PHPE) together with Tannic Acid (TA) as building blocks in construction of layer-by-layer (LbL) films to impart osteoconductive properties to multilayer films. Water-soluble complexes of PHPE and TA (PHPE-TA) were prepared at pH 4 and then LbL deposited at the surface without using a polymer counterpart. Multilayers were then cross-linked using NaIO4 to enhance their stability under physiological conditions. Potential of multilayers as an osteoconductive coating were assessed by (i) osteoblast-like cell adhesion; (ii) determination of collagen deposition by cells and (iii) the determination of extracellular matrix (ECM) mineralization and the results were compared to control substrates, i.e. cell culture plate well surface and collagen-coated substrates. PHPE-TA multilayers were found to be adhesive for SaOS-2 osteoblast-like cells and promoted collagen-rich nodule formation and mineralization of the ECM without causing cytotoxicity. PHPE-TA multilayers promoted higher alkaline phosphatase (ALP) activity and mineralization of the ECM, compared to collagen-coated surfaces. Importantly, PHPE-TA multilayers exhibited osteoconductive behavior without need for incorporation of bioactive natural polymers like collagen and hyaluronic acid or calcium phosphate ceramics into the multilayer films. Such LbL films are promising to modify the surfaces of orthopedic implants to impart osteoconductive properties to a surface.

Intestinal epithelial cells are derived from stem cells at the crypts that undergo differentiation into transit‐amplifying cells, which in turn form terminally differentiated enterocytes as these cells reach the villus. Extensive alterations in both transcriptional and translational programs occur during differentiation, which can induce the activation of cellular stress responses such as ER stress‐related unfolded protein response (UPR) and autophagy, particularly in the cells that are already committed to becoming absorptive cells. Using an epithelial cell model of enterocyte differentiation, we report a mechanistic study connecting enterocyte differentiation to UPR and autophagy. We report that differentiated colon epithelial cells showed increased cytosolic Ca2+ levels and activation of all three pathways of UPR: inositol‐requiring enzyme 1 (IRE1), protein kinase RNA‐like ER kinase, and activating transcription factor 6 (ATF6) compared to the undifferentiated cells. Enhanced UPR in the differentiated cells was accompanied by the induction of autophagy as evidenced by increased ratio of light chain 3 II/I, upregulation of Beclin‐1, and downregulation of p62. We show for the first time that mechanistically, the upregulation of hepatocyte nuclear factor 4α (HNF4α) during differentiation led to increased promoter binding and transcriptional upregulation of two major proteins of UPR: X‐box binding protein‐1 and ATF6, implicating HNF4α as a key regulator of UPR response during differentiation. Integrating wet‐lab with in silico analyses, the present study links differentiation to cellular stress responses, and highlights the importance of transcription factor signaling and cross‐talk between the cellular events in the regulation of intestinal cell differentiation. TÜBİTAK. Grant Numbers: 114S937, 113S985 Bilecik Şeyh Edebali University Scientific Research Fund. Grant Number: 2018‐01.BŞEÜ.12‐01

This review focuses on exosomes derived from various cancer cells. The review discusses the possibility of differentiating macrophages in alternatively activated anti-inflammatory pro-tumorigenic M2 macrophage phenotypes and classically activated pro-inflammatory, anti-tumorigenic M1 macrophage phenotypes in the tumor microenvironment (TME). The review is divided into two main parts, as follows: (1) role of exosomes in alternatively activating M2-like macrophages-breast cancer-derived exosomes, hepatocellular carcinoma (HCC) cell-derived exosomes, lung cancer-derived exosomes, prostate cancer-derived exosomes, Oral squamous cell carcinoma (OSCC)—derived exosomes, epithelial ovarian cancer (EOC)—derived exosomes, Glioblastoma (GBM) cell-derived exosomes, and colorectal cancer-derived exosomes, (2) role of exosomes in classically activating M1-like macrophages, oral squamous cell carcinoma-derived exosomes, breast cancer-derived exosomes, Pancreatic-cancer derived modified exosomes, and colorectal cancer-derived exosomes, and (3) exosomes and antibody-dependent cellular cytotoxicity (ADCC). This review addresses the following subjects: (1) crosstalk between cancer-derived exosomes and recipient macrophages, (2) the role of cancer-derived exosome payload(s) in modulating macrophage fate of differentiation, and (3) intracellular signaling mechanisms in macrophages regarding the exosome’s payload(s) upon its uptake and regulation of the TME. Under the electron microscope, nanoscale exosomes appear as specialized membranous vesicles that emerge from the endocytic cellular compartments. Exosomes harbor proteins, growth factors, cytokines, lipids, miRNA, mRNA, and DNAs. Exosomes are released by many cell types, including reticulocytes, dendritic cells, B-lymphocytes, platelets, mast cells, and tumor cells. It is becoming clear that exosomes can impinge upon signal transduction pathways, serve as a mediator of signaling crosstalk, thereby regulating cell-to-cell wireless communications. Based on the vesicular cargo, the molecular constituents, the exosomes have the potential to change the fate of macrophage phenotypes, either M1, classically activated macrophages, or M2, alternatively activated macrophages. In this review, we discuss and describe the ability of tumor-derived exosomes in the mechanism of macrophage activation and polarization.

New aziridine 2-phosphonic acids were prepared by monohydrolysis of the aziridine 2-phosphonates that were obtained by the modified Gabriel-Cromwell reaction of vinyl phosphonate or alpha-tosylvinyl phosphonate with a primary amine or a chiral amine. The cellular cytotoxicity of these compounds was tested against the HCT-116 colorectal cancer cell lines and the CCD-18Co normal colon fibroblast lines using the MTT assay. Three of the synthesized phosphonic acid derivatives 2e (ethyl hydrogen {(2S)-1-[(1S)-1-(naphthalen-2-yl)ethyl]aziridin-2-yl}phosphonate), 2h (ethyl hydrogen (1-benzylaziridin-2-yl)phosphonate), and 2i (ethyl hydrogen (1-cyclohexylaziridin-2-yl)phosphonate) showed higher cytotoxicity than the reference cancer treatment agent etoposide. Cell death was through a robust induction of apoptosis even more effectively than etoposide, a well-known apoptosis inducing agent.

Protein katlanması veolgunlaşması Endoplazmik Retikulum (ER)’da gerçekleşir. ER fonksiyonundakibozulmalar, yanlış katlanmış proteinlerin birikmesine neden olur ki bu durum“ER stresi” olarak bilinir. Katlanmamış Protein Yanıtı (Unfolded ProteinResponse-UPR), ER stresi durumunda hücresel homeostazın sağlanması için ER’deprotein katlanmasının yeniden ve doğrulukla gerçekleştirilmesi amacı iledüzenlenmiş bir hücre sinyalizasyon programıdır. Bu çalışmada ER stresinin,kolon kanseri hücrelerinde VEGF (Vascular Endothelial Growth Factor) ifadesinive VEGF’in hücre dışı salınımını arttırdığı gösterilmiştir. ER stresi modülatörlerikanser hücrelerinde ER stresine bağlı apoptozisi tetikleme potansiyelleri ileumut verici tedavi seçenekleri olarak sunulsa da, bu çalışma sonucu elde edilenveriler, ER stresini tetikleyici yaklaşımların, artmış otokrin/intrakrin VEGFsinyali ile tedaviye karşı dirence neden olabileceğini, hatta anjiyogenezidestekleyebileceğini öne sürmektedir., Endoplasmic Reticulum (ER) is an important sitefor protein folding and maturation. Accumulation of unfolded or misfoldedproteins in the ER leads to “ER stress”. The unfolded protein response (UPR) isa finely regulated cell-signaling program to re-establish ER folding capacityfor building up cellular homeostasis. Although ER stress modulators have emergedas promising treatment options with their potential to trigger ERstress-mediated apoptosis in cancer cells, here we show that ER stressupregulates VEGF (Vascular Endothelial Growth Factor) expression and secretionin colon cancer cells, which may result in resistance against the treatmentthrough enhanced autocrine/intracrine VEGF signaling and through supportingfurther angiogenesis.

The role of mitochondrial dysfunction in the pathogenesis of inflammatory bowel diseases (IBD) is still being investigated. This study evaluated the therapeutic effect of curcumin (Cur), a polyphenolic electrophile in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis and mitochondrial dysfunction, in mice. Colitis was induced by rectal instillation to mice of 30 mg kg−1 TNBS, alone or followed by daily intraperitoneal injections of Cur 25 mg kg−1. Animals were euthanized at days 3, 7, and 14, post TNBS challenge. Colon mitochondria of control mice were treated with 5 µM Cur, and TNBS (50, 100 µM)-toxicity was evaluated by measuring swelling, respiration, and aconitase and fumarase activities. Redox status was evaluated in colon mucosa and in mitochondria. In vitro, a short-term Cur treatment controlled the dose and time dependent mitochondrial toxicity induced by TNBS, by collapsing the generation of superoxide anion and hydroperoxy lipids, rebalancing nitric oxide synthase and aconitase activities, and recoupling mitochondria. In vivo, a daily low-dose Cur abolished mice mortality which reached 27% in model group. Cur improved in a time dependent manner mucosal redox homeostasis, cell apoptosis, mucin depleted crypts and crypt abscesses by controlling prooxidant activity of myeloperoxidase and NO synthase associated to phagocytes influx, quenching hydroperoxy lipids, and reboosting GSH levels. Cur, by quenching intra and extra mitochondrial ROS generation, rebalancing aconitase/fumarase and MDA/GSH ratios, and recoupling mitochondria, may support mithormesis priming and remitting in IBD.

Cemtirestat, 3-mercapto-5H-[1,2,4]-triazino[5,6-b]indole-5-acetic acid was recently designed and patented as a highly selective and efficient aldose reductase inhibitor endowed with antioxidant activity. The aim of the present study was to assess the general toxicity of cemtirestat using in silico predictions, in vitro and in vivo assays. ProTox-II toxicity prediction software gave 17 “Inactive” outputs, a mild hepatotoxicity score (0.52 probability) along with a predicted LD50 of 1000 mg/kg. Five different cell lines were used including the immortalized mouse microglia BV-2, the primary human fibroblasts VH10, the insulinoma pancreatic β-cells INS-1E, the human colon cancer cells HCT116 and the human immortalized epithelial endometrial cell lines HIEEC. In contrast to the clinically used epalrestat, cemtirestat showed remarkably low cytotoxicity in several different cell culture viability tests such as MTT proliferation assay, neutral red uptake, BrdU incorporation, WST-1 proliferation assay and propidium iodide staining followed by flow cytometry. In a yeast spotting assay, the presence of cemtirestat in incubation of Saccaromyces cerevisiae at concentrations as high as 1000 µM did not affect cell growth rate significantly. In the 120-day repeated oral toxicity study in male Wistar rats with daily cemtirestat dose of 6.4 mg/kg, no significant behavioral alterations or toxicological manifestations were observed in clinical and pathological examinations or in hematological parameters. In summary, these results suggest that cemtirestat is a safe drug that can proceed beyond preclinical studies.

Cancer cells grown under the stress of nutrient restriction can rewire endo-lysosomal activation and initiate autophagy as a survival mechanism. Colorectal cancer (CRC) cells are known to be particularly susceptible to the restriction of specific nutrients such as glucose and glutamine; nonetheless this susceptibility can vary considerably between cell lines. Caco-2, T84 and LoVo CRC cell lines were cultured for 2h - 48h in a pre-optimized starvation medium containing low serum, glucose and L-glutamine and pathways associated with autophagy, as well as lysosomal trafficking, biogenesis and degradation were examined in these cells. With nutrient restriction, all three cell lines showed induction of autophagy via an increase in LC3-II and p-AMPK. However, autophagic flux was inhibited in starved T84 and LoVo cells reflected by an increase in the cargo protein p62; starved Caco-2 cells showed normal autophagic flux. We therefore hypothesized a recalibration of nutrient sensing signals via the endo-lysosomal pathway in T84 and LoVo cells under nutrient restricted conditions. Nutrient restricted LoVo cells showed a surprising increase in mTORC1 activity and a decrease in the mRNA expression of MCOLN1, a cation-permeable channel on lysosomes and a transcriptional target of the master transcription regulator of lysosomal biogenesis Transcription factor EB (TFEB). Nutrient restricted T84 and Caco-2 cells showed a decrease in mTORC1 activity and a modest increase in MCOLN1 expression. A remarkable increase in the protein expression of early (Rab5) and late (Rab7) endosomal markers was observed in LoVo and T84 but not in Caco-2 cells. Additionally, treatment of both unstarved and starved T84 and LoVo cells with the v-ATPase inhibitor Bafilomycin A1 (Baf) led to an accumulation of Rab5 and Rab7; this accumulation was reversed in unstarved but not in starved cells upon withdrawal of Baf. The mRNA expression of Rab5 and Rab7 either decreased or remained unchanged in the starved T84 cells, implicating either post-transcriptional regulation via miRNAs or enhanced protein synthesis. Small RNA sequencing and subsequent confirmation with RT-qPCR of nutrient restricted cells indicated the differential expression of a network of miRNAs targeting protein involved in endo-lysosomal signalling. Additionally, the activation of the mTORC1 pathway is strongly suggestive of enhanced protein synthesis in response to the stress of starvation. Overall, our data suggests that nutrient restriction can lead to a highly rapid deregulation in endo-lysosomal signalling via post-transcriptional mechanisms. Future studies will indicate whether such deregulation provides a survival advantage to cancer cells and whether perturbation of nutrient sensing pathways can be used as a therapeutic option. Citation Format: Aliye E. Gulec, Hepsen H. Husnugil, Goksu Oral, Ilir Sheraj, Sreeparna Banerjee. Investigation of the effects of nutrient restriction on endolysosomal signaling in colorectal cancer (CRC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1983.

Detection of Diabetic Retinopathy and Age related Macular Degeneration is a challenge for the ophthalmologists as the abnormalities are merely visible at the early stage. Early detection of these diseases can prevent permanent vision loss. Handling a large amount of retinal images and detection of abnormalities due to these diseases is laborious as well as time consuming. In this research work, an algorithm is developed for identifying the abnormal objects in retina, if any with a machine learning technique using Naive Bayes classification is proposed. A training set is generated with features of abnormalities present in retinal image and the type of disease the retina is suffering from. The Naive Bayes classifier helps to predict the disease for each retinal image after gathering the knowledge from training set. The correctness of prediction is calculated to measure the efficiency of the classifier. The system achieves 97.014% of accuracy on an average.

Blindness is one of the most serious complications among diabetic patients due to diabetic retinopathy. Primary cause of diabetic retinopathy is abnormality of retinal blood vessels which becomes fragile and often raptured, due to which lipid deposits gets accumulated in the intra retinal space and are visible as yellowish colour in varying shapes sizes and locations. This paper presents a method which uses 2D Otsu thresholding to detect the presence of exudates in digital retinal fundus image. The proposed method uses various techniques, like Hough transform, image thresholding etc. and exhibits a sensitivity of 97.60%, specificity of 98.15% and accuracy of 97.85%.

In this study, celecoxib (CLX), a highly hydrophobic nonsteroidal anti-inflammatory drug with relatively low bioavailability, was used as a model drug to determine loading in different solvents and release properties from silica particles. Hydrothermal synthesis method was used to synthesize SBA-15 particles, which were functionalized by post-synthesis grafting method with (3-Aminopropyl) triethoxysilane (APTES). Additionally, boron doped SBA-15 samples were prepared to generate borosilicate samples. After functionalization, drug loading was carried out in three different solvents: ethanol, methanol and hexane and their effect on drug loading and release properties were examined. Particle morphology and solvent effect on drug loading capacity of silica particles, as well as the effect of pH on drug release were analyzed. For this purpose, SBA-15 particles were characterized by using X-ray Diffraction (XRD), Small-Angle X-ray Spectrometry (SAXS), N 2 adsorption - desorption, Fourier Transform Infra-red Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), Ultra-Violet Spectrometry (UV-VIS) and Thermogravimetric Analysis (TGA). Herein we demonstrate a highly improved release rate of CLX by using SBA-15 silica particles as drug carriers compared to the commercial drug, Celebrex. We have observed slow release from SBA-15-A (APTES functionalized SBA-15) particles, while there was burst release from SBA-15-B (boron doped SBA-15) particles and all silica samples prepared in hexane. We also observed greater loss of viability with silica-CLX complexes compared to empty silica particles on colon cancer cell lines HCT-116 and HT-29 in vitro . Our results demonstrate the use of silica supports as potential drug delivery carriers for poorly water soluble drugs.

Prostanoids play an important role in a variety of physiological and pathophysiological processes including inflammation and cancer. The rate-limiting step in the prostanoid biosynthesis pathway is catalyzed by cyclooxygenases (COXs). Aberrant expression of the inducible isoform COX-2 plays a significant role in colon cancer initiation and progression. In this study, we have hypothesized that COX-2 specific inhibitors such as Valdecoxib (VLX), being highly hydrophobic, may alter biophysical properties of cellular lipids. In this study, COX-2 expressing (HT29) and COX-2 non-expressing (SW620) colon cancer cell lines were treated with VLX and examined using attenuated total reflection infrared spectroscopy. The results revealed that VLX treatment decreased lipid fluidity in the cells irrespective of COX-2 expression status and affected order parameters of the lipids in both cell lines. Cluster analysis also indicated that the spectral differences between the two cell lines are profound and could be successfully differentiated. Valdecoxib treatment could enhance the composition, order and dynamics of the lipids of colon cancer cells independently of its COX-2 inhibitory mechanism. Valdecoxib has therapeutic effects upon colon cancer, therefore it can be used as an adjuvant and/or chemopreventive agent for colon cancer.

We have previously shown that ethanolic extract from bark (EEB) of Salix aegyptiaca (Musk Willow) can inhibit proliferation and motility and induce apoptosis in colon cancer cells. Tandem mass spectrometry revealed EEB to be rich in catechin, catechol, and salicin. The present study investigated the chemopreventive effect of HPLC-fingerprinted EEB on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) formation in mice. DMH (20 mg/kg body weight) was weekly injected subcutaneously to mice for the first 2 weeks. EEB (100 and 400 mg/kg body weight) was provided orally from the 7th to 14th week, after which colon tissues were evaluated histologically and biochemically. DMH treatment induced high number of ACF; EEB significantly reduced the number and multiplicity of ACF, along with a restoration in goblet cells and mucin accumulation. EEB supplementation improved the markers of inflammation (myeloperoxidase and neutrophil infiltration) and oxidative stress. More importantly, EEB amplified apoptosis of neoplastic cells in the colon mucosa of DMH-treated mice. It also lowered levels of markers for early transformation events such as EGFR, nuclear β-catenin, and COX-2 in colon cancer cell lines HT-29 and HCT-116. The innocuity of EEB (up to 1600 mg/kg) to mice reinforces its potential as a chemopreventive agent.

Quercetin (Qc) shows strong antitumor effects but has limited clinical application due to poor water solubility and bioavailability. In a screening of novel semi-synthetic derivatives of Qc, 3,7-dihydroxy-2-[4-(2-chloro-1,4-naphthoquinone-3-yloxy)-3-hydroxyphenyl]-5-hydroxychromen-4-one (CHNQ) could ameliorate acetic acid induced acute colitis in vivo more efficiently than Qc. Since inflammation contributes to colorectal cancer (CRC), we have hypothesized that CHNQ may have anti-cancer effects. Using CRC cell lines HCT-116 and HT-29, we report that CHNQ was three-fold more cytotoxic than Qc along with a robust induction of apoptosis. As expected from naphthoquinones such as CHNQ, a strong induction of oxidative stress was observed. This was accompanied by reactive oxygen species (ROS) induced autophagy marked by a dramatic increase in the lipidation of LC3, decreased activation of Akt/PKB, acidic vesicle accumulation and puncta formation in HCT-116 cells treated with CHNQ. Interestingly, an incomplete autophagy was observed in HT-29 cells where CHNQ treatment led to LC3 lipidation, but not the formation of acidic vacuoles. CHNQ-induced cytotoxicity, ROS formation and autophagy were also detected in vivo in Saccharomyces cerevisiae strain RDKY3615 (WinstonS288C background). Overall, we propose that CHNQ can induce cancer cell death through the induction of oxidative stress, and may be examined further as a potential chemotherapeutic drug.

In this study, luminescent and magnetic core/shell Gd1-x-yCexTbyBO3@SiO2 nanoparticles were synthesized and used to design a drug delivery system for Celecoxib (CLX). CLX was chosen as the model drug because it is a nonsteroidal anti-inflammatory drug that is highly hydrophobic with relatively low bioavailability. The core was synthesized by Pechini sol-gel method and silica coating was carried out by a Modified Stober method. Drug loading was carried out in ethanol with high efficiency and an improved drug dissolution was obtained in phosphate-buffered saline (PBS) at pH 7.4. Dissolution rate of CLX adsorbed onto the drug carrier at different pH conditions were examined and compared. The obtained core particles had a diameter of about 240 nm and core/shell particles had a diameter of about 300 nm. The core/shell particles possessed high luminescence intensity and soft magnetic properties. Drug loading was carried out in ethanol with 12.7% loading efficiency and a substantial decrease of about 8% in photoluminescence intensity was observed in drug loaded samples, which made the quantification of the released drug possible by comparing the photoluminescence intensities. The solubility of the drug was enhanced when compared to crystalline CLX, a sustained release profile was achieved and increasing luminescence intensity was observed during drug release. 55% of the loaded drug was released after 72 h in PBS at pH 7.4 while 20% drug release was observed in a pH 5.5 solution. Drug release at pH 5.5 followed a first order release kinetic model while the release at pH 7.4 followed a Higuchi's kinetic model with Fickian diffusion mechanism. An in vitro loss in cell viability with CLX loaded carrier was observed for the colon cancer cell line HCT-116. These results suggest that the designed drug carrier has promising applications for poorly soluble drugs with improved bioavailability, in addition to its application as a bioimaging agent in fluorescence microscopy and magnetic resonance imaging.

Multilayers of poly(2-alkyl-2-oxazoline)s (PAOX) with varying side chains were constructed through layer-by-layer (LbL) self-assembly using Tannic Acid (TA). Surface morphology, stability, wettability and stimuli-responsive drug release properties of the films were correlated with antiadhesive and antibacterial properties of the multilayers. Thickness, roughness, wettability and stability of multilayers were affected by the chemical nature of PAOX side chain, phase behaviour of PAOX and the binding strength between the layers. PAOX-multilayers released remarkably different amounts of Ciprofloxacin (CIP) especially at acidic pH and 37 °C. Antibacterial activity of the films against S. aureus was in good agreement with the amounts of CIP released from multilayers. Despite the differences in the released CIP amount, PAOX-multilayers showed similar antibacterial activity against E. coli under all conditions. Multilayers with greater wettability exhibited greater resistance against Bovine Serum Albumin (BSA) adsorption and were more effective to reduce adherence of E. coli. None of the PAOX-films exhibited antiadhesiveness against S. aureus.

Tunçer, Sinem (Bilecik, Author) Colloidal platinum (Pt) is widely consumed due to its health promoting benefits. However, the exact biological effects of these nanoparticles have not been studied in detail, particularly in the gut. In the present study we observed that colloidal Pt was not cytotoxic towards three different epithelial colon cancer cell lines. Co-treatment of the colon cancer cell line Caco-2 with the oxidative stress inducing agent hydrogen peroxide (H2O2) and colloidal Pt resulted in a significant decrease in H2O2 induced oxidative stress. Colloidal Pt by itself did not induce any oxidative stress. Additionally, both overnight pretreatment of Caco-2 cells with colloidal Pt followed by 1 h treatment with H2O2, or co-treatment of cells for 1 h with colloidal Pt and H2O2 resulted in a significant recovery of cell death. Of note, the same protective effects of colloidal Pt were not observed when the oxidative stress was induced in the presence of 2, 2-azobis (2-amidinopropane) dihydrochloride, indicating that the source of free radicals may define the outcome of anti-oxidant activity of colloidal Pt. Colloidal Pt was also able to cross a model intestinal barrier formed in vitro with differentiated Caco-2 cells easily. Overall, our data indicate that colloidal Pt was not toxic towards intestinal epithelial cells, reduced H2O2 induced oxidative stress, protected from oxidative stress related death of intestinal epithelial cells and could pass a model gut barrier easily. Colloidal Pt can therefore be consumed orally for its anti-oxidant and other health promoting benefits. Science Academy of Turkey (BAGEP) Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) - 2218 YÖK 100/2000 WOS:000463067400064 PMID:30963117 1-s2.0-S2405844018363989 Q2

Dimethyl sulfoxide (DMSO) is a small molecule with polar, aprotic and amphiphilic properties. It serves as a solvent for many polar and nonpolar molecules and continues to be one of the most used solvents (vehicle) in medical applications and scientific research. To better understand the cellular effects of DMSO within the concentration range commonly used as a vehicle (0.1–1.5%, v/v) for cellular treatments, we applied Attenuated Total Reflectance (ATR) Fourier Transform Infrared (FT-IR) spectroscopy to DMSO treated and untreated epithelial colon cancer cells. Both unsupervised (Principal Component Analysis-PCA) and supervised (Linear Discriminant Analysis-LDA) pattern recognition/modelling algorithms applied to the IR data revealed total segregation and prominent differences between DMSO treated and untreated cells at whole, lipid and nucleic acid regions. Several of these data were supported by other independent techniques. Further IR data analyses of macromolecular profile indicated comprehensive alterations especially in proteins and nucleic acids. Protein secondary structure analysis showed predominance of β-sheet over α-helix in DMSO treated cells. We also observed for the first time, a reduction in nucleic acid level upon DMSO treatment accompanied by the formation of Z-DNA. Molecular docking and binding free energy studies indicated a stabilization of Z-DNA in the presence of DMSO. This alternate DNA form may be related with the specific actions of DMSO on gene expression, differentiation, and epigenetic alterations. Using analytical tools combined with molecular and cellular biology techniques, our data indicate that even at very low concentrations, DMSO induces a number of changes in all macromolecules, which may affect experimental outcomes where DMSO is used as a solvent.

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by a scavenger receptor in macrophages initiates necrosis core formation that characterizes atherosclerosis. We report that NOS1-derived nitric oxide (NO) facilitates low-density lipoprotein (LDL) uptake by macrophages independent of the inflammatory response. LDL uptake could be dramatically suppressed by NOS1 specific inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM). Importantly, the notion that NOS1 can mediate uptake of lipoproteins suggests that the foam cell formation is regulated by NOS1-derived NO-mediated mechanism. This is a novel study involving NOS1 as a critical player of foam cell formation and reveals much about the key molecular proteins involved in atherosclerosis. Targeting NOS1 would be a useful strategy in reducing LDL uptake by macrophages and hence dampening the atherosclerosis progression.

The oxygenation of polyunsaturated fatty acids such as arachidonic and linoleic acid through lipoxygenases (LOXs) and cyclooxygenases (COXs) leads to the production of bioactive lipids that are important both in the induction of acute inflammation and its resolution. Amongst the several isoforms of LOX that are expressed in mammals, 15-LOX-1 was shown to be important both in the context of inflammation, being expressed in cells of the immune system, and in epithelial cells where the enzyme has been shown to crosstalk with a number of important signalling pathways. This review looks into the latest developments in understanding the role of 15-LOX-1 in different disease states with emphasis on the emerging role of the enzyme in the tumour microenvironment as well as a newly re-discovered form of cell death called ferroptosis. We also discuss future perspectives on the feasibility of use of this protein as a target for therapeutic interventions.

Background Osteoarthritis (OA), is a leading cause of joint dysfunction and the disease is characterised by progressive destruction of the articular cartilage.1 At the molecular level, degeneration of the cartilage is attributed to multifactorial events including oxidative stress, mitochondrial dysfunction, apoptosis and associated changes in inflammatory and catabolic gene expression.2 Recent studies have revealed the essential role of plant-derived antioxidants in preventing pathophysiological events observed in joint diseases by modulating redox signalling.3 Objectives Here, we studied whether the polyphenolic compounds verbascoside and hydroxytyrosol exert chondroprotective effects by suppressing oxidative stress pathways and IL-1, IL-1β-converting enzyme (ICE) expression in human OA chondrocytes. Methods Chondrocytes were isolated from the joint cartilages of OA patients (grade 3–4) during total knee arthroplasty with the approval of Kecioren Training and Research Hospital, Clinical Research Ethics Committee (B.10.4.ISM.4.06.68.49), as described previously.4 The alterations in cell counts, viability (MTT, NRU), proliferation (RTCA-iCELLigence System), reactive oxygen species (ROS) generation, stress-related signalling proteins and inflammatory progenitors ICE/caspase-1 (ELISA) were determined. Results Cell viability was increased at lower concentrations of hydroxytyrosol in OA chondrocytes (P1, 30th day). On the other hand, verbascoside treated cells did not show any difference in the activity of mitochondrial oxidoreductases by the MTT assay. However, both polyphenols significantly increased proliferation and reduced intracellular ROS generation in OA chondrocytes at lower concentrations. Although verbascoside has no effect on ICE/caspase-1, treatment with hydroxytrosol downregulated ICE/caspase-1, indicating a potential anti-inflammatory effect. Both polyphenols modulated the activation of stress activated signalling pathways via p38 and JNK proteins. Conclusions At low concentrations the antioxidants hydroxytyrosol and verbascoside may have potential chondroprotective effects in OA. References [1] Lepetsos P, Papavassiliou AG. ROS/oxidative stress signaling in osteoarthritis. Biochim Biophys Acta2016Apr;1862(4):576–591. [2] Khan NM, Ahmad I, Haqqi TM. Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis. Free Radic Biol Med. 2018Jan 12:S0891–5849(18)30023–6. [3] Chin KY, Pang KL. Therapeutic Effects of Olive and Its Derivatives on Osteoarthritis: From Bench to Bedside. Nutrients2017Sep 26;9(10):E1060. [4] Naranda J, Gradisnik L, Gorenjak M, Vogrin M, Maver U. Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA). Peer J. 2017Mar 21;5:e3079. Acknowledgements Acknowledgement: This study is supported by TUBITAK (The Scientific and Technological Research Council of Turkey) Project No: 315 S012. This study is supported by TUBITAK (The Scientific and Technological Research Council of Turkey) Project No: 315 S012. Disclosure of Interest None declared

Retinopathy is a common disease among those who are suffering from Diabetes for a long period. Several abnormalities are related to Diabetic Retinopathy. Cotton Wool Spot is one among them. It causes from nerve fiber layer breaking from occlusion of pre-capillary arterioles. It occurs in retina as whitish spots causing blindness in some cases. Early detection of CWS can prevent severe damage of retina which may lead to permanent vision loss. In this paper an algorithm is developed which can detect these spots automatically from a retinal image affected by Diabetic Retinopathy. The automatic detection can help the doctors for accurate detection of Cotton Wool Spots and also for the longitudinal study of a retinal image damaged by Diabetic Retinopathy.

The most common abnormalities in retina images occur due to age related macular degeneration and diabetic retinopathy. In this paper a decision support system is proposed to classify these abnormalities. The process involves the combination of contextual information with images obtained from a database. Decisions trees are proposed for this purpose as they can combine contextual information with images. Images are pre- processed and segmented to obtain the regions of interest for the individual manifestations in each of these diseases. Matching of candidate segmented images with prototype segmented images is performed along with the matching of the associated contextual information.

Diabetic retinopathy is a retinal abnormality found among diabetes patients which may lead to blindness. Hard exudates (HE) is one of the most significant symptoms of DR, which are lipoprotein and cellular debris leaked out of the damaged blood vessels of retina. Detection of HE during early stages helps the patients from loss of vision. This paper proposes a Tabu search-based dynamic thresholding approach to detect HE in retinal images. The proposed method uses Hough transform, median filtering, image thresholding, etc., with a goal to detect HE. The proposed method proves the effectiveness of Tabu search algorithm-based image thresholding technique for the detection of diabetic retinopathy. The proposed method is tested using DIARETDB0 and DIARETDB1 image database and it exhibits a sensitivity of 97.45% and specificity of 96.85%.

Diabetic retinopathy is considered to be one of the major causes of blindness among diabetes mellitus patients. Due to diabetic retinopathy blood vessels of retina gets damaged and fat, lipoprotein substances gets leaked out of the damaged blood vessels and are deposited in the intra retinal space. These substances are viewed as yellowish or whitish in color and are termed as exudates. They are the most important visible sign of the presence of diabetic retinopathy. Exudates are of two types, (a) hard exudates and (b) soft exudates. If the disease is not detected in early stages then it may lead to complete loss of vision to the diabetes patients. Detection of exudates is extremely difficult to detect by visual inspection due to small inner diameter of retina and inadequate lighting conditions. An efficient image analysis program can detect the presence effectively. In this paper we have proposed an automatic method for detection of hard exudates. The proposed method exhibits a sensitivity of 97.60% and specificity of 93% and accuracy of 95.70%.

Short chain fatty acids (SCFAs) are produced by commensal bacteria in the gut and are known to reduce inflammation through transcriptional inhibition of cytokines and inflammatory proteins such as cyclooxygenase-2 (COX-2). Butyrate is a SCFA that was reported to alter the mRNA stability of inflammatory genes by increasiing the expression of the RNA binding protein (RBP) Tristetraprolin (TTP). We have hypothesized that butyrate may regulate gene expression post-transcriptionally through global effects on the expression or cytoplasmic trans location of RBPs. Using bioinformatics analyses of publicly available microarray data as well as colon cancer cell lines treated with sodium butyrate, we have observed that butyrate treatment led to a general reduction in expression of several (but not all) RBPs and inhibition in the cytosolic translocation of HuR, a well-known stabilizing RBP. This was reflected in reduced NanoLuc reporter activity of several different AU-rich element (ARE) sequences in the presence of butyrate; this suppression was retained even when HuR was overexpressed. Mechanistically, we have shown that reduced activity of HuR was related to decreased phosphorylation of p38 and MK2 and enhanced phosphorylation of Chk2. As a proof of concept, we show butyrate-mediated inhibition in binding of HuR to the 3'UTR of COX-2 mRNA resulting in reduced mRNA and protein levels of the inflammatory gene. Overall, our data suggest that butyrate can reduce the expression of inflammatory genes not only by transcriptional regulation, but also by post-transcriptional regulation via inhibition of mRNA stabilizing proteins.

Biodegradable polymers have been broadly used as agents that can complex with and deliver osteoinductive agents, but osteoinductivity of the polymers themselves has been rarely studied. Here we report the osteoinductivity of poly(4-hydroxy-L-proline ester) (PHPE), a biodegradable cationic polymer with cell penetrating properties. Under physiological conditions, PHPE degrades into trans-4-hydroxy-L-proline (trans-Hyp), a non-coded amino acid with essential functions in collagen fibril formation and fibril stability. Treatment of SaOS-2 osteoblast-like cells and hFOB 1.19 primary osteoblast cells with PHPE promoted earlier collagen nodule formation and mineralization of the extracellular matrix compared to untreated cells, even when mineralization activators were absent in the growth medium. Our results indicate that PHPE is a potential osteoinductive agent in vitro that can favor bone regeneration. Moreover, this osteoinductive property could be partly attributed to the degradation product trans-Hyp, which could recapitulate some, but not all of the osteogenic activity. The primary findings of this study can be summarized as follows: treatment of cells with PHPE led to (1) the induction of COL1A1 expression, collagen synthesis and secretion in osteoblast-like cells, (2) mineralization of the ECM in both SaOS-2 and hFOB 1.19 primary osteoblasts, and (3) induction of BMP2 gene and protein expression in osteoblast-like cells, which can promote mineralization of the ECM and regeneration of the bone tissue. Our results suggest that PHPE is a non-cytotoxic polymer and can be potentially used to overcome collagenopathies such as osteogenesis imperfecta.

Nanosized liposomal drug delivery systems are well suited for selective drug delivery at tumor sites. Celecoxib (CLX) is a highly hydrophobic cyclooxygenase-2 inhibitor that can reduce the incidence of colorectal polyps; however, the adverse cardiovascular effects limit its applicability. Here, we report a liposomal formulation of CLX using 1,2-Distearoyl-sn-glycero-3-phosphocholine, cholesterol, and polyethylene glycol. Encapsulation efficiency of the drug was greater than 70%; the release was slow and sustained with only 12%-20% of CLX released in the first 12h. Flow cytometry and confocal microscopy studies using the colon cancer cell lines HCT-116 and SW620 showed significantly higher cellular association and internalization of the liposomes after incubation for 6h when compared with 30min. The liposomes did not colocalize with transferrin, but had a punctuate appearance, indicating vesicular localization. Cell proliferation was inhibited by 95% and 78%, respectively, in SW620 and HT29 cells after incubation with 600M liposomal CLX for 72h. Moreover, cellular motility, as shown by a scratch wound healing assay, was also significantly (p=0.006) inhibited when SW620 cells were incubated with 400M liposomal CLX. This is the first report of the successful encapsulation of CLX in a long-circulating liposomal formulation that could be effective against colorectal cancer. (c) 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3666-3677, 2013

The Caco-2 colorectal cancer cell line is widely used as a model for intestinal differentiation and barrier function. These cells, upon reaching confluency, spontaneously differentiate into enterocyte-like cells, synthesize intestinal enzymes, and form domes. Caco-2 cells also undergo autophagy in the course of differentiation. The criteria to establish the induction of autophagy in cells are already well established. Here, we describe the protocol for the spontaneous differentiation of Caco-2 cells and the detection of autophagy using Western blot, flow cytometry, and immunofluorescence.

The aim of this study was to investigate the effects of Raloxifene (Ral) on degeneration-related changes in osteoarthritis (OA)-like chondrocytes using two- and three-dimensional models. Five-azacytidine (Aza-C) was used to induce OA-like alterations in rat articular chondrocytes and the model was verified at molecular and macrolevels. Chondrocytes were treated with Ral (1, 5 and 10 mu M) for 10 days. Caspase-3 activity, gene expressions of aggrecan, collagen II, alkaline phosphatase (ALP), collagen X, matrix metalloproteinases (MMP-13, MMP-3 and MMP-2), and MMP-13, MMP-3 and MMP-2 protein expressions were studied in two-dimensional model. Matrix deposition and mechanical properties of agarose-chondrocyte discs were evaluated in three-dimensional model. One mu M Ral reduced expression of OA-related genes, decreased apoptosis, and MMP-13 and MMP-3 protein expressions. It also increased aggrecan and collagen II gene expressions relative to untreated OA-like chondrocytes. In three-dimensional model, 1 mu M Ral treatment resulted in increased collagen deposition and improved mechanical properties, although a significant increase for sGAG was not observed. In summation, 1 mu M Ral improved matrix-related activities, whereas dose increment reversed these effects except ALP gene expression and sGAG deposition. These results provide evidence that low-dose Ral has the potential to cease or reduce the matrix degeneration in OA.

Intestinal epithelial differentiation entails the formation of highly specialized cells with specific absorptive, secretory, digestive and immune functions. Cell-cell and cell-microenvironment interactions appear to be crucial in determining the outcome of the differentiation process. Using the Caco-2 cell line, which undergoes spontaneous re-differentiation when grown past confluency, we observed a loss of VCAM-1 (vascular cell adhesion molecule 1) mRNA expression, while ICAM-1 (intercellular cell adhesion molecule 1) mRNA expression was seen to increase over the course of differentiation. Protein kinase Cθ (PKCθ) acted downstream of protein kinase Cα (PKCα) to inactivate inhibitor of κB (IκB) and activate nuclear factor κB (NF-κB) in undifferentiated cells, and this pathway was inhibited in the differentiated cells. The increase in ICAM-1 mRNA expression in the differentiated cells was due to increased promoter recruitment of C/EBPβ, which transcriptionally up-regulated ICAM-1 mRNA. However, protein expression of ICAM-1 was found to decrease over the course of differentiation due to degradation in the proteasome and lysosome. Immunohistochemistry using tumor samples from colon cancer patients indicated that non-transformed matched normal cells (well-differentiatied) showed no ICAM-1 expression, but the poorly differentiated tumor cells showed higher expression. Functionally, a decrease in adhesion to human umbilical vein endothelial cells was observed in the differentiated Caco-2 cells. Thus, regulation of ICAM-1 and VCAM-1, although both NF-κB target genes, appears to be different over the course of epithelial differentiation in Caco-2 cells.

CLX (celecoxib), a selective COX-2 (cyclo-oxygenase-2) inhibitor, has numerous pleiotropic effects on the body that may be independent of its COX-2 inhibitory activity. The cancer chemopreventive ability of CLX, particularly in CRC (colorectal cancer), has been shown in epidemiological studies. Here we have, for the first time, examined the biophysical effects of CLX on the cellular membranes of COX-2 expressing (HT29) and COX-2 non-expressing (SW620) cell lines using ATR-FTIR (attenuated total reflectance–Fourier transform IR) spectroscopy and SL-ESR (spin label–ESR) spectroscopy. Our results show that CLX treatment decreased lipid fluidity in the cancer cell lines irrespective of COX-2 expression status. As metastatic cells have higher membrane fluidity, we examined the effect of CLX on the metastatic potential of these cells. The CLX treatment efficiently decreased the proliferation, anchorage-independent growth, ability to close a scratch wound and migration and invasion of the CRC cell lines through Matrigel. We propose that one of the ways by which CLX exerts its anti-tumorigenic effects is via alterations in cellular membrane fluidity which has a notable impact on the cells' metastatic potential.

Sodium butyrate (NaBt) is a histone deacetylase inhibitor (HDACi) produced in the colon by commensal microbiota-mediated fermentation of dietary fibers. It is regarded to have tumor suppressive and anti-inflammatory effects. NaBt can regulate gene expression through chromatin remodeling and altered transcription. However, it has not been addressed adequately whether inflammatory genes may also be regulated by NaBt in a post transcriptional manner via AU rich elements (ARE) in their 3'UTR. Overexpression of Cyclooxygenase-2 (COX-2) is observed in chronic inflammatory diseases and colorectal cancer (CRC). The aim of this study was to explore whether NaBt regulates COX-2 expression via post-transcriptional mechanisms. Pre-confluent Caco-2 and HT-29 cells were treated with different concentrations (1-5mM) of NaBt for short (3-6h) and long (24-48h) time points. Gene expression was determined by qPCR or Western blot. mRNA stability was determined with an Actinomycin D (ActD) chase assay. 3'UTR activity was determined in an RPSM30 vector containing the ARE rich and corresponding mutated regions of the 3'UTR from COX-2. NaBt strongly reduced COX-2 mRNA and protein expression with both short and long-term treatments. This reduction was not through NF-κB, since short-term treatment of cells with NaBt could not alter NF-κB activity in a luciferase reporter assay. An Act D chase assay indicated that in the absence of new mRNA synthesis, NaBt treatment induced stability of COX-2 mRNA in Caco-2 cells while it enhanced COX-2 mRNA decay in HT-29 cells. However, we did not observe any change in the 3'UTR activity of COX-2 in the presence of NaBt alone, indicating that the presence of a stress factor (such as ActD) was necessary for mRNA stabilization. Treatment of Caco-2 cells with NaBt and ActD together resulted in enhanced activation of the stress-related protein kinase MAPKAPK2 (MK2) compared to ActD alone, which can induce mRNA stabilization through nucleo-cytoplasmic shuttling of the ARE binding protein HuR. This was also reflected in increased COX-2 mRNA and protein levels. In HT-29 cells, simultaneous (NaBt+ActD) treatment resulted in a decrease in p-MK2, but an increase in p-Chk2 compared to ActD alone. p-Chk2 can preserve Cdk1 in a hyperphosphorylated state where it may promote retention of HuR in the nucleus, facilitating COX2 mRNA degradation and lower protein levels. No change in p-Chk2 was observed in Caco-2 cells in a similar experimental set up. These findings suggest that in the presence of a stress-inducing agent like ActD, NaBt can alter mRNA stability of COX-2 in a contextual manner, dependent on upstream signaling. Thus, while the rate of COX-2 mRNA degradation in HT-29 was enhanced, it slows down the same in Caco-2 cells. Future studies will indicate how NaBt can affect the dichotomy in Chk2/MK2 activation in the presence of stress. Citation Format: Sreeparna Banerjee, Shabnam Enayat, Sinem Tuncer, Doğukan H. Ulgen, Aydan Torun. NaBt can regulate the expression of COX-2 post-transcriptionally in the presence of chemically induced stress in colon epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4438.

Leaves, bark and catkins of Salix aegyptiaca L. were extracted into solvents of increasing polarity from cyclohexane (non-polar), butanol, ethanol and water (polar) and analysed for their antioxidant capacity, total phenol and flavonoids. The highest antioxidant activity (19 μg/ml IC50 for inhibition of DPPH radical activity), total phenolic content (212 mg gallic acid equivalents/g of dried extract) and total flavonoid (479 mg catechin equivalents/g of dried extract) was observed in the ethanolic extract of bark. HPLC identification of phenolic compounds from the extracts indicated the presence of gallic acid, caffeic acid, vanillin and p-coumaric acid, myricetin, catechin, epigallocatechin gallate, rutin, quercetin as well as salicin. Our data indicates the presence of high amounts of phenols and flavonoids in different parts of S. aegyptiaca species and propose that extracts from this plant may be utilised as a source of health promoting antioxidants.

Selenium-doped hydroxyapatite (HA) was biomimetically coated on Ti6Al4V plates with the aim of combining the anticancer and antibacterial properties of selenium with the biocompatibility and bioactivity of HA. For the coating process, the composition of 1.5 × SBF (solution with ion concentrations at 1.5 times that of simulated body fluid, SBF) was modified to include 0.15 mM selenate (SeO42−) ion. The selenium-doped HA coating was characterized by several methods, such as scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The cytotoxicity of selenium on osteoblast and osteosarcoma cells was determined. The coating was shown to inhibit the growth of Staphylococcus epidermidis.

The polyunsaturated fatty acid (PUFA) in fish render them potentially susceptible to enzymatic oxidation. Lipoxygenase (LOX) activity from mackerel (Scomber scombrus) was characterized with respect to pH stability and activity, temperature stability, buffer and substrate prefeences and inhibition patterns. The partially puriped enzyme was stable over the pH range 4.0-1 I. 0 and had a pH-activity optimum pH of 5.6. Mackerel muscle LOX (&OX) was stable ot 0-50C but lost activity at higher temperatures. Enzymatic assays using diflereru buffers revealed that MES-NaOH and Bistris-HCl were the suitable b@ers for reaction. Substrate preference was for linoleic acid compared to linolenic, arachidonic, docosahexaenoic or oleic acid. K,,, values were 0.69 and 0.57 mM and V, was 0.058 and 0.019 pmol min" for linoleic and linolmic acid, respectively. &OX was inhibited by classical LOX inhibitors such as BHA, BHT, NDGA, esculetin and also by the heme protein inhibitor KCN. Values for the IC,, (concentration for halfmaximclr inhibition) was between 0.02 pM and 41 pM. Normal phase high pegormance liquid chromatography (HPLC) analyses revealed that both 13- and 9-hydroperoxides were formed during mmLOX catalyzed oxidation with linoleic acid substme although a higher proportion of the 13-isomer was formed. With enzymes from different mackerel tissues (skin, gills and muscle), &OX had the highest hydroperoxide forming ability.

We report on preparation of substrates with dual function coatings, i.e. bacterial anti-adhesive and antibacterial agent releasing polymer films of zwitterionic block copolymer micelles (BCMs). BCMs were obtained by pH-induced self-assembly of poly[3-dimethyl (methacryloyloxyethyl) ammonium propane sulfonate- b -2-(diisopropylamino)ethyl methacrylate] (βPDMA- b -PDPA), resulting in BCMs with zwitterionic βPDMA-coronae and pH-responsive PDPA-core. These zwitterionic BCMs were then used as building blocks to construct mono- and multi-layer films. We found that the number of layers in the film was critical for the anti-adhesive property and 3-layer films were the most anti-adhesive against a model Gram-positive bacterium, Staphylococcus aureus . Antibacterial activity could be introduced to the films by loading Triclosan into βPDMA- b -PDPA micelles. Triclosan containing films were effective against Triclosan-sensitive Staphylococcus aureus specifically at moderately acidic conditions due to pH-induced disintegration of the micellar core blocks and release of Triclosan from the surface. Three-layer films also exhibited anti-adhesive property at physiological pH against a model Gram-negative bacterium, Escherichia coli . At moderately acidic pH, the coatings showed a contact antibacterial effect against an isolate of Escherichia coli with low sensitivity to Triclosan only when micellar cores were loaded with Triclosan. Such dual function films can be promising to combat biofouling at the non-homogeneous and/or defective parts of an anti-adhesive coating. Moreover, considering the moderately acidic conditions around an infection site, these multilayers can be advantageous due to their property of pH-induced antibacterial agent release. Statement of Significance This study presents preparation of substrates with dual function ultra-thin coatings of zwitterionic block copolymer micelles which show bacterial anti-adhesive properties against a Gram-positive and a Gram-negative bacterium. Such coatings are also capable of releasing antibacterial compounds in response to pH changes. Films were prepared by self-assembly of polymers at the surface. Our findings showed that zwitterionic micellar coronae introduced bacterial anti-adhesive property to the films, whereas pH-responsive micellar cores enabled release of an antibacterial agent from the surface at acidic pH. Considering the moderately acidic conditions around an infection site, such multilayers can be promising for the coating of implants/medical devices.

The high polyunsaturated fatty acid content of oily fish such as mackerel (Scomber scombrus) makes it particularly susceptible to oxidative degradation. We have shown previously the presence of lipoxygenase (LOX), a lipid oxygenase, in mackerel muscle. In the current study, commercially available green tea polyphenols were shown to effectively inhibit the LOX activity of mackerel muscle. EGCG (epigallocatechin gallate) was the strongest inhibitor tested with an IC50 (concentration for half maximal inhibition) value of 0.13 nM. All the tea catechins showed a mixed non-competitive type inhibition. In addition, antioxidants such as BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), esculetin, caffeic acid, ascorbic acid, and ethylene diamine tetraacetic acid (EDTA) were effective to varying degrees (IC50 values between 0.02 and >50 μM) in the inhibition of mackerel muscle LOX. Nordihydroguaiaretic acid (NDGA), a classical LOX inhibitor and potassium cyanide (KCN), a heme protein inhibitor were assayed for their inhibitory activities for comparison. Post harvest spoilage of fish account for loss of as much as 10% of the world’s catches of cultured fish. This data indicates that the green tea polyphenols, nature’s very potent antioxidants, may be used as an effective and natural means of reducing post harvest spoilage in fish.