Search results for: modified Krieger and Dougherty expression

Authors: Mahmoud M. Zakaria, Omnia F. Elmoursi, Mahmoud M. Gabr, Camelia A. AbdelMalak, Mohamed A. Ghoneim

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Many protocols were publicized for differentiation of human mesenchymal stem cells (MSCS) into insulin-producing cells (IPCs) in order to excrete insulin hormone ingoing to treat diabetes disease. Our aim is to evaluate relative gene expression for each independent protocol. Human bone marrow cells were derived from three volunteers that suffer diabetes disease. After expansion of mesenchymal stem cells, differentiation of these cells was done by three different protocols (the one-step protocol was used conophylline protein, the two steps protocol was depending on trichostatin-A, and the three-step protocol was started by beta-mercaptoethanol). Evaluation of gene expression was carried out by real-time PCR: Pancreatic endocrine genes, transcription factors, glucose transporter, precursor markers, pancreatic enzymes, proteolytic cleavage, extracellular matrix and cell surface protein. Quantitation of insulin secretion was detected by immunofluorescence technique in 24-well plate. Most of the genes studied were up-regulated in the in vitro differentiated cells, and also insulin production was observed in the three independent protocols. There were some slight increases in expression of endocrine mRNA of two-step protocol and its insulin production. So, the two-step protocol was showed a more efficient in expressing of pancreatic endocrine genes and its insulin production than the other two protocols.

Keywords: mesenchymal stem cells, insulin producing cells, conophylline protein, trichostatin-A, beta-mercaptoethanol, gene expression, immunofluorescence technique

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Authors: Mircea I. Pastrav, Cornelia Baera, Florea Dinu

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A different concept for designing and detailing of reinforced concrete precast frame structures is analyzed in this paper. The new detailing of the joints derives from the special hybrid moment frame joints. The special reinforcements of this alternative detailing, named modified special hybrid joint, are bondless with respect to both column and beams. Full scale tests were performed on a plan model, which represents a part of 5 story structure, cropped in the middle of the beams and columns spans. Theoretical approach was developed, based on testing results on twice repaired model, subjected to lateral seismic type loading. Discussion regarding the modified special hybrid joint behavior and further on widening research needed concludes the presentation.

Keywords: modified hybrid joint, repair, seismic loading type, acceptance criteria

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Authors: N. R. Putra, A. H. Abdul Aziz, A. S. Zaini, Z. Idham, F. Idrus, M. Z. Bin Zullyadini, M. A. Che Yunus

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The content of omega-3 in soybean oil is important in the development of infants and is an alternative for the omega-3 in fish oils. The investigation of extraction of soybean oil is needed to obtain the bioactive compound in the extract. Supercritical carbon dioxide extraction is modern and green technology to extract herbs and plants to obtain high quality extract due to high diffusivity and solubility of the solvent. The aim of this study was to obtain the optimum condition of soybean oil extraction by modified supercritical carbon dioxide. The soybean oil was extracted by using modified supercritical carbon dioxide (SC-CO₂) under the temperatures of 40, 60, 80 °C, pressures of 150, 250, 350 Bar, and constant flow-rate of 10 g/min as the parameters of extraction processes. An experimental design was performed in order to optimize three important parameters of SC-CO₂ extraction which are pressure (X₁), temperature (X₂) to achieve optimum yields of soybean oil. Box Behnken Design was applied for experimental design. From the optimization process, the optimum condition of extraction of soybean oil was obtained at pressure 338 Bar and temperature 80 °C with oil yield of 2.713 g. Effect of pressure is significant on the extraction of soybean oil by modified supercritical carbon dioxide. Increasing of pressure will increase the oil yield of soybean oil.

Keywords: soybean oil, SC-CO₂ extraction, yield, optimization

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Authors: Shan-Ying Wu, Sheng-Hui Lan, Xi-Zhang Lin, Ih-Jen Su, Ting-Fen Tsai, Chia-Jui Yen, Tsung-Hsueh Lu, Fu-Wen Liang, Huey-Jen Su, Chun-Li Su, Hsiao-Sheng Liu

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In hepatocelluar carcinoma (HCC), dysregulated expression of cyclin D1 and impaired autophagy has been reported separately. However, the relationship between them has not been explored. In this study, we demonstrated that autophagy was inversely correlated with cyclin D1 expression in 147 paired HCC patient specimens. HCC specimen with highly expression of cyclin D1 shows correlation with poor overall survival rate. Furthermore, induction of autophagy by amiodarone (antiarrhythmic drug) in Hep 3B cells, cyclin D1 was recruited into autophagosomes demonstrated by immune-gold labeling of cyclin D1 after extraction of autophagosomes. We further demonstrated that autophagy suppresses Hep 3B cell proliferation, and further analysis revealed that cell cycle was arrested at G1 phase. The interaction between LC3 (maker of autophagy) and cyclin D1 was increased after autophagy induction. In addition, ubiquitinated-cyclin D1 was also increased after autophagy induction, which is selectively degraded by autophagosome through binding with SQSTM1/p62 (an adaptor protein). In vivo study showed that amiodarone induced autophagy suppresses liver tumor formation in xenograft mouse and orthotopic rat model through decreasing cyclin D1 expression and inhibition of cell proliferation. Altogether, we reveal a novel mechanism that ubiquitinated cyclin D1 degraded by autophagic pathway by p62 and amiodarone is a promising drug for targeting cyclin D1 in liver cancer therapy.

Keywords: autophagy, cyclin D1, hepatocellular carcinoma, amiodarone

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Authors: Haveesha Buddhdev

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The most important right for a human being in a society is the freedom of expression as stated by Article 18 and 19 of the Universal Declaration of Human rights pledged by member states of United Nations. Will it be fair to expect him/her to be of sound mental health if this right is taken away? Religion as a primary social institution controls many rights, freedoms and duties of people in a society. It does so by imposing certain values and beliefs on people which would either enhance quality of life or curb their freedom adversely thus affecting individual mental health. This paper aims to study the positive and negative role that religion plays in influencing one’s freedom of expression. This paper will focus on reviewing existing studies on the positive and negative impacts of religion on mental health. It will also contain data collected by the researcher about the impacts of religion on freedom of expression which will be obtained by surveying a sample of 30 adolescents and young adults. The researcher will use a Likert scale for these purpose, with response options ranging from strongly disagree to strongly agree and quantify it accordingly. Descriptive statistics would be used to analyse the data. Such research would help to identify possible problems faced by adolescents and young adults when it comes to religio-cultural ethos and also facilitate further researches to study the role that religion plays in mental health.

Keywords: cultural Ethos, freedom of expression, adolescent mental health, social science

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Authors: Naseem Fatima, A. N. Srivastava, Tasleem Raza, Vijay Kumar

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Introduction: Carcinoma gallbladder is third most common gastrointestinal lethal disease with the highest incidence and mortality rate among women in Northern India. Scientists have found several risk factors that make a person more likely to develop gallbladder cancer; among these risk factors, deregulation of miRNAs has been demonstrated to be one of the most crucial factors. The changes in the expression of specific miRNA genes result in the control of inflammation, cell cycle regulation, stress response, proliferation, differentiation, apoptosis and invasion thus mediate the process in tumorgenesis. The aim of this study was to investigate the role of MiRNA-335 and may as a molecular marker in early detection of gallbladder cancer in suspected cases. Material and Methods: A total of 20 consecutive patients with gallbladder cancer aged between 30-75 years were registered for the study. Total RNA was extracted from tissue by using the mirVANA MiRNA isolation Kit according to the manufacturer’s protocol. The MiRNA- 335 and U6 snRNA-specific cDNA were reverse-transcribed from total RNA using Taqman microRNA reverse-transcription kit according to the manufacturer’s protocol. TaqMan MiRNA probes hsa-miR-335 and Taqman Master Mix without AmpEase UNG, Individual real-time PCR assays were performed in a 20 μL reaction volume on a Real-Time PCR system (Applied Biosystems StepOnePlus™) to detect MiRNA-335 expression in tissue. Relative quantification of target MiRNA expression was evaluated using the comparative cycle threshold (CT) method. The correlation was done in between cycle threshold (CT Value) of target MiRNA in gallbladder cancer with respect to non-cancerous Cholelithiasis gallbladder. Each sample was examined in triplicate. The Newman-Keuls Multiple Comparison Test was used to determine the expression of miR-335. Results: MiRNA335 was found to be significantly downregulated in the gallbladder cancer tissue (P<0.001), when compared with non-cancerous Cholelithiasis gallbladder cases. Out of 20 cases, 75% showed reduced expression of MiRNA335, were at last stage of disease with low overall survival rate and remaining 25% were showed up-regulated expression of MiRNA335 with high survival rate. Conclusion: The present study showed that reduced expression of MiRNA335 is associated with the advancement of the disease, and its deregulation may provide important clues to understanding it as a prognostic marker and opportunities for future research.

Keywords: carcinoma gallbladder, downregulation, MiRNA-335, RT-PCR assay

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Authors: Alina Beraudi, Simona Catalani, Dalila De Pasquale, Eva Bianconi, Umberto Santoro, Susanna Stea, Pietro Apostoli

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Recently the European Community, in line with the international scientific community such as with the Consensus Statement, has determined to stop the use of metal-on-metal big head stemmed hip prosthesis. Among the factors accounted as responsible for the high failure rates of these hip implants are the release and accumulation of metal ions. Many studies have correlated the presence of these ions, besides other factors, with the induction of oxidative stress response. In our study on 12 subjects, we observed the patient specific capability to eliminate metal ions after revision surgery. While for cobalt all the patients were able to completely excrete cobalt ions within 5-7 months after metal-on-metal bearing removal, for chromium ions it didn’t happen. If on the one hand the toxicokinetic differences between the two types of ions are confirmed by toxicological and occupational studies, on the other hand, this peculiar way of exposition represents a novel and important point of view. Thus, two different approaches were performed to better understand the subject specific capability to transport metal ions (albumin study) and to manage the response to them (heme-oxygenase-1 study): - a mutational screening of ALBUMIN gene was conducted in 30 MoM prosthetic patients resulting in the absence of nucleotidic changes compared with the ALB reference sequence. To this study was also added the analysis of expression of modified albumin protein; - a gene and protein expression study on 44 patients of heme-oxygenase-1, that is one of the most important antioxidant enzyme induced by metallic ions, was performed. This study resulted in no statistically significant differences in the expression of the gene and protein heme-oxygenase-1 between prosthetic and non-prosthetic patients, as well as between patients with high and low ions levels. Our results show that the protein studied (albumin and heme-oxygenase-1) seem to be not involved in determining chromium and cobalt ions level. On the other hand, achromium and cobalt elimination rates are different, but similar in all patients analyzed, suggesting that this process could be not patient-related. We support the importance of researching more about ions transport within the organism once released by hip prosthesis, about the chemical species involved, the districts where they are contained and the mechanisms of elimination, not excluding the existence of a subjective susceptibility to these metals ions.

Keywords: chromium, cobalt, hip prosthesis, individual susceptibility

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Authors: Gizem Buldum, Alexander Bismarck, Athanasios Mantalaris

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Cellulose is the most abundant biopolymer on earth and it is currently being utilised in a multitude of industrial applications. Over the last 30 years, attention has been paid to the bacterial cellulose (BC), since BC exhibits unique physical, chemical and mechanical properties when compared to plant-based cellulose, including high purity and biocompatibility. Although Acetobacter xylinum is the most efficient producer of BC, it’s long doubling time results in insufficient yields of the cellulose production. This limits widespread and continued use of BC. In this study, E. coli BL21 (DE3) or E. coli HMS cells are selected as host organisms for the expression of bacterial cellulose synthase operon (bcs) of A.xylinum. The expression system is created based on pET-Duet1 and pCDF plasmid vectors, which carry bcs operon. The results showed that all bcs genes were successfully transferred and expressed in E.coli strains. The expressions of bcs proteins were shown by SDS and Native page analyses. The functionality of the bcs operon was proved by congo red binding assay. The effect of culturing temperature and the inducer concentration (IPTG) on cell growth and plasmid stability were monitored. The percentage of plasmid harboring cells induced with 0.025 mM IPTG was obtained as 85% at 22˚C in the end of 10-hr culturing period. It was confirmed that the high output cellulose production machinery of A.xylinum can be transferred into other organisms.

Keywords: bacterial cellulose, biopolymer, recombinant expression system, production

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Authors: Garima Chaudhary

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The gender of an individual in forensic DNA analysis is normally accessed by using the STR multiplexes with the incorporated gender based marker amelogenin or in other words by presence or absence of Y-Chromosome, but it may not be true in all the cases. We hereby report an interesting case of a phenotypic female carrying a male karyotype (46XY). In the alleged murder case, the deceased female with XY genotype was noticed. The expression of 18 Y-linked genes was studied to measure the extent of expression. Expression at 4 loci was observed that might have caused the misinterpretation in forensic casework. This clinical situation of the deceased in this case was diagnosed as testicular feminization syndrome, which characterize a female phenotype with a male karyotype (46, XY). Most of these cases have SRY (testis determining factor). The genetic explanation of this phenomenon is not very clear. Here, we are discussing the impact of such situations of genetic discrepancy in forensic interpretation of results. In the presented murder case of a phenotypic female, sexual assault was also suspected. For confirmation vaginal swabs and micro slides were also sent to us for DNA examination. After DNA analysis using STR markers, Y-chromosome was detected in the samples which supporting the suspicion of sexual assault before murder. When the reference blood sample of the deceased was analyzed, it was found to be case of testicular feminization syndrome. Interesting inferences were made from the results obtained.

Keywords: DNA profiling, forensic case study, Y chromosome, females

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Authors: Precious Barnes, Abraham Mensah, Leonard Derkyi-Kwarteng, Benjamin Amoani, George Adjei, Ernest Adankwah, Faustina Pappoe, Kwabena Dankwah, Daniel Amoako-Sakyi, Samuel Victor Nuvor, Dorcas Obiri-Yeboah, Ewura Seidu Yahaya, Patrick Kafui Akakpo, Roland Osei Saahene

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Context: Breast cancer is a prevalent and aggressive type of cancer among African women, with high mortality rates in Ghana. Nuclear factor kappa B (NF-kB) is a transcription factor that has been associated with tumor progression in breast cancer. However, there is a lack of published data on NF-kB in breast cancer patients in Ghana or other African countries. Research Aim: The aim of this study was to assess the prognostic significance of NF-kB (p65) expression and its association with various clinicopathological features in breast cancer patients at the Cape Coast Teaching Hospital in Ghana. Methodology: A total of 90 formalin-fixed breast cancer tissues and 15 normal breast tissues were used in this study. The expression level of NF-kB (p65) was examined using immunohistochemical techniques. Correlation analysis between NF-kB (p65) expression and clinicopathological features was performed using SPSS version 25. Findings: The study found that NF-kB (p65) was expressed in 86.7% of breast cancer tissues. There was a significant relationship between NF-kB (p65) expression and tumor grade, proliferation index (Ki67), and molecular subtype. High-level expression of NF-kB (p65) was more common in tumor grade 3 compared to grade 1, and Ki67 > 20 had higher expression of NF-kB (p65) compared to Ki67 ≤ 20. Triple-negative breast cancer patients had the highest overexpression of NF-kB (p65) compared to other molecular subtypes. There was no significant association between NF-kB (p65) expression and other clinicopathological parameters. Theoretical Importance: This study provides important insights into the expression of NF-kB (p65) in breast cancer patients in Ghana, particularly in relation to tumor grade and proliferation index. The findings suggest that NF-kB (p65) could serve as a potential biological marker for cancer stage, progression, prognosis and as a therapeutic target. Data Collection and Analysis Procedures: Formalin-fixed breast cancer tissues and normal breast tissues were collected and analyzed using immunohistochemical techniques. Correlation analysis between NF-kB (p65) expression and clinicopathological features was performed using SPSS version 25. Question Addressed: This study addressed the question of the prognostic significance of NF-kB (p65) expression and its association with clinicopathological features in breast cancer patients in Ghana. Conclusion: This study, the first of its kind in Ghana, demonstrates that NF-kB (p65) is highly expressed among breast cancer patients at the Cape Coast Teaching Hospital, especially in triple-negative breast cancer patients. The expression of NF-kB (p65) is associated with tumor grade and proliferation index. NF-kB (p65) could potentially serve as a biological marker for cancer stage, progression, prognosis, and as a therapeutic target.

Keywords: breast cancer, Ki67, NF-kB (p65), tumor grade

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Authors: Yen-Ni Teng, Hsing-Yi Chen, Hsien-An Pan, Yung-Ming Lin, Hany A. Omar, Jui-Hsiang Hung

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Leucine-rich repeats and WD repeat domain containing 1 (LRWD1) is highly expressed in the testes of healthy males. On the other hand, LRWD1 is significantly down-regulated in the testicular tissues of patients with severe spermatogenic defects. In our study, the downregulation of LRWD1 expression by shRNA caused a significant reduction of cell growth and mitosis and a noteworthy increase in the cell microtubule atrophy rate. Here, we used EMBOSS CpG plot analysis to explore the promoter region of LRWD1 gene. We found that CpG islands are located between positions -253 to +5 nucleotides upstream from the LRWD1 transcription start site. Luciferase reporter assay revealed that the hypermethylation of the LRWD1 promoter reduced the transcription activity in cells. In addition, quantitative methylation-specific PCR and immunostaining showed that the methylation inhibitor, 5-Aza-2'-deoxycytidine, increased LRWD1 promoter activity, LRWD1 mRNA, protein expression and cell viability. Whereas, the methylation activator, S-adenosylmethionine, caused opposite effects. The overexpression of p53 and Nrf2 in NT2/D1 cells increased LRWD1 promoter activity while 5-fluorodeoxyuridine decreased it. In conclusion, this study highlights evidence that the methylation status of LRWD1 promoter is associated with LRWD1 expression. Since the expression level of LRWD1 plays an important role in spermatogenesis, the methylation status of LRWD1 may serve as a novel molecular diagnostic or therapeutic approach in male's infertility.

Keywords: LRWD1, DNA methylation, p53, Nrf2

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Authors: Z. Chekini, P. Afsharian, F. Ramezanali, A. A. Akhlaghi, R. Aflatoonian

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Macrophage migration inhibitory factor (MIF) is a key pro-inflammatory cytokine that involves in pathophysiological events of endometriosis. We aimed to evaluate the association between mRNA expression levels and polymorphisms of MIF in endometriosis. Seventy endometriosis patients and 70 volunteer fertile women were recruited. RFLP was applied to determine -173G/C polymorphism. ORF polymorphisms and -794(CATT)5-8 were detected by sequencing. Q-PCR was used for expression study of 14 ectopic tissues of patients. Homozygote of CATT5 was observed only in controls. The CATT5/G haplotype related to controls (p=0.094, OR=0.61). Expression level of MIF with -794(CATT)6,7/-173GC was significantly more than the other haplotypes (p=0.00). We identified four SNPs including: +254rs2096525 (p=0.843), +626rs33958703 (p=0.029), +656rs2070766 (p=0.703) and +509rs182012324 (p=1.00). In conclusion, increased repeat of CATT and presence of C allele in promoter of MIF were significantly associated with mRNA level in patients. It seems that +509rs182012324 and +626rs33958703 SNPs were significantly correlated with susceptibility to endometriosis.

Keywords: endometriosis, haplotype, macrophage migration inhibitory factor, polymorphism

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Authors: Emad Heydarnia, Aref Sepasi, Nika Asefi, Sara Khakshournia, Javad Mohammadnejad

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Background: Despite breakthrough therapeutics in breast cancer, it is one of the main causes of mortality among women worldwide. Thus, drug therapies for treating breast cancer have recently been developed by scientists. Metformin and Sorafenib are well-known therapeutic in breast cancer. In the present study, we combined Sorafenib and PCL-sorafenib with metformin to improve drug absorption and promote therapeutic efficiency. Methods: The MCF-7 cells were treated with Metformin, Sorafenib, or PCL-sorafenib. The growth inhibitory effect of these drugs and cell viability were assessed using MTT and flow cytometry assays, respectively. The expression of targeted genes involved in cell proliferation, signaling, and the cell cycle was measured by Real-time PCR. Results: The results showed that MCF-7 cells treated with Metformin/Sorafenib and PCL-sorafenib/Metformin co-treatment contributed to 50% viability compared to untreated group. Moreover, PI and Annexin V staining tests showed that the cells viability for Metformin/Sorafenib and PCL-sorafenib/Metformin was 38% and 17%, respectively. Furthermore, Sorafenib/Metformin and PCL-sorafenib/Metformin leads to p53 gene expression increase by which they can increase ROS, thereby decreasing GPX4 gene expression. In addition, they affected the expression of BCL2, and BAX genes and altered the cell cycle. Conclusion: Together, the combination of PCL-sorafenib/Metformin and Sorafenib/Metformin increased Sorafenib absorption at lower doses and also leads to apoptosis and oxidative stress increases in MCF-7 cells.

Keywords: breast cancer, metformin, nanotechnology, sorafenib

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Authors: So Youn Park, Yi Sle Lee, Ki Whan Hong, Chi Dae Kim

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The role of metabolism in the pathogenesis of osteoarthritis is an emerging field. Metabolic alterations may be a role in osteoarthritis (OA) pathogenesis, and these changes influence joint destruction via several cytokine. Especially, in OA patients, levels of IL-1β are elevated in the synovial fluid, synovial membrane, subchondral bone, and cartilage. The IL-1β is activated by NLRP3 inflammasomes, and NLRP3 inflammasomes are cytosolic complexes that drive the production of other inflammatory cytokines, including IL-1β. In this study, we examined that SIRT1 suppresses IL-1β through inhibiting NLRP3 inflammasomes and SIRT1 ameliorates osteoarthritis. OA fibroblasts were isolated from synovium of OA patients. IL-1β and NLRP3 were detected in synovium of OA patients by immunohistochemistry. Lipopolysaccharides (LPS) stimulated the expression of active IL-1β mRNA in OA fibroblasts and combination of LPS, and adenosine triphosphate increased more the expression of active IL-1β in OA fibroblasts. The level of IL-1β was measured by western blot and ELISA assay. NLRP3 inflammasomes complex were measured by western blot. SIRT1 did not inhibit expression of NLRP3 inflammasome. So caspase-1, apoptotic speck-like protein containing a caspase recruitment domain (ASC) and NLRP3 protein were expressed in OA fibroblasts. But SIRT1 suppressed activation of IL-1β by inhibiting activity of caspase-1 via NLRP3 inflammasome in OA fibroblasts under LPS plus ATP stimulation. These results suggest that SIRT1 is a modulator of NLRP3 inflammasomes in OA fibroblasts and ameliorate IL-1β, so expression of SIRT1 in OA fibroblast may be a potential strategy for OA inflammation treatment.

Keywords: osteoarthritis, inflammasome, SIRT1, IL-1beta

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Authors: Ghada Esheba, Fatimah Alturkistani, Arwa Obaid, Ahdab Bashehab, Moayad Alturkistani

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Introduction: Craniopharyngiomas (CPs) are rare epithelial tumors located mainly in the sellar/parasellar region. CPs have been classified histopathologically, genetically, clinically and prognostically into two distinctive subtypes: adamantinomatous and papillary variants. Aim: To examine the pattern of expression of both the β-catenin and epidermal growth factor receptor (EGFR) in surgically resected samples of adamantinomatous CP, and to asses for the possibility of using anti-EGFR in the management of ACP patients. Materials and methods: β-catenin and EGFR immunostaining was performed on paraffin-embedded tissue sections of 18 ACP cases. Result: 17 out of 18 cases (94%) of ACP exhibited strong nuclear/cytoplasmic expression of β-catenin, 15 (83%) of APC cases were positive for EGFR. Conclusion: Nuclear accumulation of β-catenin is a diagnostic hallmark of ACP. EGFR positivity in most cases of ACP could qualify the use of anti-EGFR therapy. 

Keywords: craniopharyngioma, adamantinomatous, papillary, epidermal growth factor receptor, B-catenin

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Authors: Nathalie Baeza-Kallee, Raphaël Bergès, Victoria Hein, Stéphanie Cabaret, Jeremy Garcia, Abigaëlle Gros, Emeline Tabouret, Aurélie Tchoghandjian, Carole Colin, Dominique Figarella-Branger

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Glioblastoma (GBM) contains cancer stem cells that are resistant to treatment. GBM cancer stem cell expresses glycolipids recognized by the A2B5 antibody. A2B5, induced by the enzyme ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyl transferase 3 (ST8Sia3), plays a crucial role in the proliferation, migration, clonogenicity, and tumorigenesis of GBM cancer stem cells. Our aim was to characterize the resulting effects of neuraminidase that remove A2B5 in order to target GBM cancer stem cells. To this end, we set up a GBM organotypic slice model; quantified A2B5 expression by flow cytometry in U87-MG, U87-ST8Sia3, and GBM cancer stem cell lines, treated or not by neuraminidase; performed RNAseq and DNA methylation profiling; and analyzed the ganglioside expression by liquid chromatography-mass spectrometry in these cell lines, treated or not with neuraminidase. Results demonstrated that neuraminidase decreased A2B5 expression, tumor size, and regrowth after surgical removal in the organotypic slice model but did not induce a distinct transcriptomic or epigenetic signature in GBM CSC lines. RNAseq analysis revealed that OLIG2, CHI3L1, TIMP3, TNFAIP2, and TNFAIP6 transcripts were significantly overexpressed in U87-ST8Sia3 compared to U87-MG. RT-qPCR confirmed these results and demonstrated that neuraminidase decreased gene expression in GBM cancer stem cell lines. Moreover, neuraminidase drastically reduced ganglioside expression in GBM cancer stem cell lines. Neuraminidase, by its pleiotropic action, is an attractive local treatment against GBM.

Keywords: cancer stem cell, ganglioside, glioblastoma, targeted treatment

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Authors: Belloufi Mohammed, Sellami Badreddine

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This paper deals with a new nonlinear modified three-term conjugate gradient algorithm for solving large-scale unstrained optimization problems. The search direction of the algorithms from this class has three terms and is computed as modifications of the classical conjugate gradient algorithms to satisfy both the descent and the conjugacy conditions. An example of three-term conjugate gradient algorithm from this class, as modifications of the classical and well known Hestenes and Stiefel or of the CG_DESCENT by Hager and Zhang conjugate gradient algorithms, satisfying both the descent and the conjugacy conditions is presented. Under mild conditions, we prove that the modified three-term conjugate gradient algorithm with Wolfe type line search is globally convergent. Preliminary numerical results show the proposed method is very promising.

Keywords: unconstrained optimization, three-term conjugate gradient, sufficient descent property, line search

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Authors: A. K. M. Kafi, S. N. Nina, Mashitah M. Yusoff

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In this work, we have described a new 3-dimensional (3D) network of crosslinked Horseradish Peroxidase/Carbon Nanotube (HRP/CNT) on a thiol-modified Au surface in order to build up the effective electrical wiring of the enzyme units with the electrode. This was achieved by the electropolymerization of aniline-functionalized carbon nanotubes (CNTs) and 4-aminothiophenol -modified-HRP on a 4-aminothiophenol monolayer-modified Au electrode. The synthesized 3D HRP/CNT networks were characterized with cyclic voltammetry and amperometry, resulting the establishment direct electron transfer between the redox active unit of HRP and the Au surface. Electrochemical measurements reveal that the immobilized HRP exhibits high biological activity and stability and a quasi-reversible redox peak of the redox center of HRP was observed at about −0.355 and −0.275 V vs. Ag/AgCl. The electron transfer rate constant, KS and electron transfer co-efficient were found to be 0.57 s-1 and 0.42, respectively. Based on the electrocatalytic process by direct electrochemistry of HRP, a biosensor for detecting H2O2 was developed. The developed biosensor exhibits excellent electrocatalytic activity for the reduction of H2O2. The proposed biosensor modified with HRP/CNT 3D network displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 1.0×10−7 to 1.2×10−4M with a detection limit of 2.2.0×10−8M at 3σ. Moreover, this biosensor exhibits very high sensitivity, good reproducibility and long-time stability. In summary, ease of fabrication, a low cost, fast response and high sensitivity are the main advantages of the new biosensor proposed in this study. These obvious advantages would really help for the real analytical applicability of the proposed biosensor.

Keywords: biosensor, nanomaterials, redox enzyme, thiol-modified Au surface

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Authors: Ho Seong Shin

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Introduction: A research in relation to wound healing also showed that platelet-rich plasma (PRP) was effective on normal tissue regeneration. Nonetheless, there is no evidence that when platelet-rich plasma was applied on diabetic wound, it normalize diabetic wound healing process. In this study, we have analyzed matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) expression to know the effect of PRP on diabetic wounds using Reverse transcription-polymerase chain reaction (RT-PCR) of MMP-2, MMP-9 mRNA. Materials and Methods: Platelet-rich plasma (PRP) was prepared from blood of 6 rats. The whole 120-mL was added immediately to an anticoagulant. Citrate phosphonate dextrose(CPD) buffer (0.15 mg CPDmL) in a ratio of 1 mL of CPD buffer to 5 mL of blood. The blood was then centrifuged at 220g for 20minutes. The supernatant was saved to produce fibrin glue. The participate containing PRP was used for second centrifugation at 480g for 20 minutes. The pellet from the second centrifugation was saved and diluted with supernatant until the platelet concentration became 900,000/μL. Twenty male, 4week-old OLETF rats were underwent operation; each rat had two wounds created on left and right sides. The each wound of left side was treated with PRP gel, the wound of right side was treated with physiologic saline gauze. Results: RT-PCR analysis; The levels of MMP-2 mRNA in PRP applied tissues were positively related to postwounding days, whereas MMP-2 mRNA expression in saline-applied tissues remained in 5day after treatment. MMP-9 mRNA was undetectable in saline-applied tissues for either tissue, except 3day after treatment. Following PRP-applied tissues, MMP-9 mRNA expression was detected, with maximal expression being seen at third day. The levels of MMP-9 mRNA in PRP applied tissues were reported high intensity of optical density related to saline applied tissues.

Keywords: diabetes, MMP-2, MMP-9, OLETF, PRP, wound healing MMP-9

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Authors: Jorge Olivares, Fernando Maass, Pablo Martin

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Fractional derivatives have been considered recently as a way to solve different problems in Engineering. In this way, second kind modified Bessel functions are considered here. The order α fractional differential equations of second kind Bessel functions, Kᵥ(x), are studied with simple initial conditions. The Laplace transform and Caputo definition of fractional derivatives are considered. Solutions have been found for ν=1/3, 1/2, 2/3, -1/3, -1/2 and (-2/3). In these cases, the solutions are the sum of two hypergeometric functions. The α fractional derivatives have been for α=1/3, 1/2 and 2/3, and the above values of ν. No convergence has been found for the integer values of ν Furthermore when α has been considered as a rational found m/p, no general solution has been found. Clearly, this case is more difficult to treat than those of first kind Bessel Function.

Keywords: Caputo, modified Bessel functions, hypergeometric, linear fractional differential equations, transform Laplace

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Authors: H. Elgubbi, A. Mlitan, A. Alzridy

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Ninhydrin is the most well known spray reagent for identification of amino acids. Spring with Ninhydrin as a non-specific reagent is well-known and widely used for its remarkable high sensitivity. Using Ninhydrin reagent alone to detect amino acid on thin layer chromatography (TLA) paper is not advisable due to its lower sensitivity. A new spray reagent, Stannus chloride solution (Sn CL2) has been used to detect amino acids on filtter paper (witman 14) and TLC paper, silica Gel, 60 F254 TLC Aluminium Sheet 20x20cm Merck- Germany. Also, modified TLC pre-staining method was used, which only consisted of 3 steps: spotting, separating and color. The improved method was rapid and inexpensive and the results obtained were clear and reliable. In addition, it is suitable for screening different amino acid.

Keywords: amino acid, ninhydrin, modified ninhydrin reagent, stannus chloride reagent, thin-layer chromatography (TLC), TLC pre-staining

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Authors: Khushbu Priyadarshi, Chandramani Pathak

Abstract:

Cancer cells can develop resistance to conventional therapies especially chemotherapeutic drugs. Resistance to chemotherapy is another challenge in cancer therapeutics. Therefore, it is important to address this issue. Gallic acid (GA) is a natural plant compound that exhibits various biological properties including anti-proliferative, anti-inflammatory, anti-oxidant and anti-bacterial. Despite of the wide spectrum biological properties GA has cytotoxic response and low bioavailability. To overcome this problem, GA was conjugated with the Polyamidoamine(PAMAM) dendrimer for improving the bioavailability and efficient delivery in drug-resistant HCT-116 Colon Cancer cells. Gallic acid was covalently linked to 4.0 G PAMAM dendrimer. PAMAM dendrimer is well established nanocarrier but has cytotoxicity due to presence of amphiphilic nature of amino group. In our study we have modified surface of PAMAM dendrimer with Gallic acid and examine their anti-proliferative effects in drug-resistant HCT-116 cells. Further, drug-resistant colon cancer cells were established and thereafter treated with different concentration of PAMAM-GA to examine their anti-proliferative potential. Our results show that PAMAM-GA conjugate induces apoptotic cell death in HCT-116 and drug-resistant cells observed by Annexin-PI staining. In addition, it also shows that multidrug-resistant drug transporter P-gp protein expression was downregulated with increasing the concentration of GA conjugate. After that we also observed the significant difference in Rh123 efflux and accumulation in drug sensitive and drug-resistant cancer cells. Thus, our study suggests that conjugation of anti-cancer agents with PAMAM could improve drug resistant property and cytotoxic response to treatment of cancer.

Keywords: drug resistance, gallic acid, PAMAM dendrimer, P-glycoprotein

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Authors: JiYoung Park, SueGoo Rhee, HyunAe Woo

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In liver regeneration, quiescent hepatocytes need to be primed to fully respond to growth factors such as hepatocyte growth factor. To understand the priming process, it is necessary to analyze patterns of gene expression that occur during liver regeneration after partial hepatectomy (PHx). Recently, tyrosine phosphorylation of signal transducer and activator of transcription 3 (pYSTAT3) has been shown to play an important role in initiating liver regeneration. In order to evaluate the role of pYSTAT3 on liver regeneration after PHx, we used an intrabody which can selectively inhibit pYSTAT3. In our previous studies, an intrabody had been shown that it bound specifically to the pYSTAT3. Adenovirus-mediated expression of the intrabody in HepG2 cells, as well as mouse liver, blocked both accumulation of pYSTAT3 in the nucleus and downstream target of pYSTAT3. In this study, PHx was performed on intrabody-expressing mice and the expression levels of liver regeneration-related genes were analyzed. We also measured liver/body weight ratios and the related cellular signaling pathways were analyzed. Acute phase response genes were reduced in an intrabody-expressing mice during liver regeneration than in control virus-injected mice. However, the time course of liver mass restoration in intrabody-expressing mice was similar to that observed in control virus-injected mice. We also observed that the expression levels of anti-apoptotic genes, such as Bcl2 and Bcl-xL were decreased in intrabody-expressing mice whereas the expression of cell cycle-related genes such as cyclin D1, and c-myc was increased. Liver regeneration after PHx was partially impaired by the selective inhibition of pYSTAT3 with a phosphorylation site-specific intrabody and these results indicated that pYSTAT3 might have limited role in liver mass recovery.

Keywords: STAT3, pYSTAT3, liver regeneration, intrabody

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Authors: Abdelkrim Khadir, Sina Kavalakatt, Preethi Cherian, Ali Tiss

Abstract:

Soluble epoxide hydrolase (sEH) is an emerging therapeutic target in several chronic states that have inflammation as a common underlying cause such as immunometabolic diseases. Indeed, sEH is known to play a pro-inflammatory role by metabolizing anti-inflammatory, epoxyeicosatrienoic acids (EETs) to pro-inflammatory diols. Recently, it was shown sEH to be linked to diet and microbiota interaction in rat models of obesity. Nevertheless, the functional contribution of sEH and its anti-inflammatory substrates EETs in obesity remain poorly understood. In the current study, we compared the expression pattern of sEH between lean and obese nondiabetic human subjects using subcutaneous adipose tissue (SAT) and peripheral blood mononuclear cells (PBMCs). Using RT-PCR, western blot and immunofluorescence confocal microscopy, we show here that the level of sEH mRNA and protein to be significantly increased in obese subjects with concomitant increase in endoplasmic reticulum (ER) stress components (GRP78 and ATF6α) and inflammatory markers (TNF-α, IL-6) when compared to lean controls. The observation that sEH was overexpressed in obese subjects’ prompt us to investigate whether physical exercise could reduce its expression. In this study, we report here 3-months supervised physical exercise significantly attenuated the expression of sEH in both the SAT and PBMCs, with a parallel decrease in the expression of ER stress markers along with attenuated inflammatory response. On the other hand, homocysteine, a sulfur containing amino acid deriving from the essential amino acid methionine was shown to be directly associated with insulin resistance. When 3T3-L1 preadipocytes cells were treated with homocysteine our results show increased sEH levels along with ER stress markers. Collectively, our data suggest that sEH upregulation is strongly linked to ER stress in adiposity and that physical exercise modulates its expression. This gives further evidence that exercise might be useful as a strategy for managing obesity and preventing its associated complications.

Keywords: obesity, adipose tissue, epoxide hydrolase, ER stress

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Authors: M. Błoch, P. Karpiński, P. Gasperowicz, R. Płoski, A. Lebioda, P. Skiba, A. Rozensztrauch, D. Patkowski, R. Śmigiel

Abstract:

Introduction: The cause of the isolated form of esophageal atresia has been yet unknown. Objectives: The primary objective of this study was to indicate epigenetic factors which may play an important role in the etiopathogenesis of esophageal atresia. Methods: We recruited a group of 6 pairs of twins, among whom one of the twins developed EA. The selection of such a group for testing allows for excluding external factors (e.g., infections, drugs, toxins) as the cause of the birth defect. The analyzes were performed with the use of genetic material isolated from the whole blood and esophagus tissue of a patient with EA. The reduced representation bisulphite sequencing (RRBS) technique was used to study the change in the genomic imprinting -a change in the expression of genes, which may be the epigenetic cause of EA. Results: In the course of the analyzes, significant hypomethylation and hypermethylation regions were identified. 65 genes with probably increased expression and 65 with decreased expression were selected. These genes have not been marked in literature as possibly pathogenic in esophageal atresia. However, their participation in the pathogenesis of esophageal atresia cannot be clearly excluded. Conclusion: We suggest a role of hypomethylation or hypermethylation of selected genes as one of the possible epigenetic factors in EA pathogenesis. The use of the RRBS technique in the search for the cause of EA is pioneer research; therefore, it seems necessary to extend the research group to new patients with EA. Acknowledgment: The work was supported by the National Science Centre, Poland, under research project 2016/21/N/NZ5/01927.

Keywords: esophageal atresia, epigenetics, embryonic development, surgery, genes expression, twins

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Authors: Haibo Zhao, Thomas Andre, Katherine Avery, Alper Kiziltas, Deborah Mielewski

Abstract:

Silica aerogels were fabricated through a modified rapid supercritical extraction (RSCE) process. The silica aerogels were made using a tetramethyl orthosilicate precursor and then placed in a hot press and brought to the supercritical point of the solvent, ethanol. In order to control the pressure release without a pressure controller, a needle valve was used. The resulting aerogels were then characterized for their physical and chemical properties and compared to silica aerogels created using similar methods. The aerogels fabricated using this modified RSCE method were found to have similar properties to those in other papers using the unmodified RSCE method. Silica aerogel infused glass blanket composite, graphene reinforced silica aerogel composite were also successfully fabricated by this new method. The modified RSCE process and system is a prototype for better gas outflow control with a lower cost of equipment setup. Potentially, this process could be evolved to a continuous low-cost high-volume production process to meet automotive requirements.

Keywords: aerogel, automotive, rapid supercritical extraction process, low cost production

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Authors: Saeed Ghali, Hoda El-Faramawy, Mamdouh Eissa, Michael Mishreky

Abstract:

Scale-resistant austenitic stainless steel, X45CrNiW 18-9, has been developed, and modified steels produced through partial and total nickel replacement by nitrogen. These modified steels were produced in a 10 kg induction furnace under different nitrogen pressures and were cast into ingots. The produced modified stainless steels were forged, followed by air cooling. The phases of modified stainless steels have been investigated using the Schaeffler diagram, dilatometer, and microstructure observations. Both partial and total replacement of nickel using 0.33-0.50% nitrogen are effective in producing fully austenitic stainless steels. The nitrogen contents were determined and compared with those calculated using the Institute of Metal Science (IMS) equation. The results showed great deviations between the actual nitrogen contents and predicted values through IMS equation. So, an equation has been derived based on chemical composition, pressure, and temperature at 1600oC. [N%] = 0.0078 + 0.0406*X, where X is a function of chemical composition and nitrogen pressure. The derived equation has been used to calculate the nitrogen content of different steels using published data. The results reveal the difficulty of deriving a general equation for the prediction of nitrogen content covering different steel compositions. So, it is necessary to use a narrow composition range.

Keywords: solubility, nitrogen, stainless steel, Schaeffler

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Authors: A. Y. Tsidulko, T. M. Pankova, E. V. Grigorieva

Abstract:

High-grade gliomas are the most frequent and most aggressive brain tumors which are characterized by active invasion of tumor cells into the surrounding brain tissue, where the extracellular matrix (ECM) plays a crucial role. Disruption of ECM can be involved in anticancer drugs effectiveness, side-effects and also in tumor relapses. The anti-inflammatory agent dexamethasone is a common drug used during high-grade glioma treatment for alleviating cerebral edema. Although dexamethasone is widely used in the clinic, its effects on normal brain tissue ECM remain poorly investigated. It is known that proteoglycans (PGs) are a major component of the extracellular matrix in the central nervous system. In our work, we studied the effects of dexamethasone on the ECM proteoglycans (syndecan-1, glypican-1, perlecan, versican, brevican, NG2, decorin, biglican, lumican) using RT-PCR in the experimental animal model. It was shown that proteoglycans in rat brain have age-specific expression patterns. In early post-natal rat brain (8 days old rat pups) overall PGs expression was quite high and mainly expressed PGs were biglycan, decorin, and syndecan-1. The overall transcriptional activity of PGs in adult rat brain is 1.5-fold decreased compared to post-natal brain. The expression pattern was changed as well with biglycan, decorin, syndecan-1, glypican-1 and brevican becoming almost equally expressed. PGs expression patterns create a specific tissue microenvironment that differs in developing and adult brain. Dexamethasone regimen close to the one used in the clinic during high-grade glioma treatment significantly affects proteoglycans expression. It was shown that overall PGs transcription activity is 1.5-2-folds increased after dexamethasone treatment. The most up-regulated PGs were biglycan, decorin, and lumican. The PGs expression pattern in adult brain changed after treatment becoming quite close to the expression pattern in developing brain. It is known that microenvironment in developing tissues promotes cells proliferation while in adult tissues proliferation is usually suppressed. The changes occurring in the adult brain after dexamethasone treatment may lead to re-activation of cell proliferation due to signals from changed microenvironment. Taken together obtained data show that dexamethasone treatment significantly affects the normal brain ECM, creating the appropriate microenvironment for tumor cells proliferation and thus can reduce the effectiveness of anticancer treatment and promote tumor relapses. This work has been supported by a Russian Science Foundation (RSF Grant 16-15-10243)

Keywords: dexamthasone, extracellular matrix, glioma, proteoglycan

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Authors: Petra Borilova Linhartova, Michaela Ruckova, Sabina Sevcikova, Natalie Mlcuchova, Jan Bohm, Katerina Zukalova, Monika Vlachova, Jiri Dolina, Lumir Kunovsky, Radek Kroupa, Zdenek Pavlovsky, Zdenek Danek, Tereza Deissova, Lydie Izakovicova Holla, Ondrej Slaby, Zdenek Kala

Abstract:

Esophageal adenocarcinoma (EAC) is a highly aggressive malignancy that frequently develops from Barrett's esophagus (BE), a premalignant pathologic change occurring in the lower end of the esophagus. Specific microRNAs (miRNAs), small non-coding RNAs that function as posttranscriptional regulators of gene expression, were repeatedly proved to play key roles in the pathogenesis of these diseases. This pilot study aimed to analyze four selected miRNAs in esophageal tissues from healthy controls (HC) and patients with reflux esophagitis (RE)/BE/EAC, as well as to compare expression at the site of Barrett's mucosa/adenocarcinoma and healthy esophageal tissue outside the area of the main pathology in patients with BE/EAC. In this pilot study, 22 individuals (3 HC, 8 RE, 5 BE, 6 EAC) were included and endoscopically examined. RNA was isolated from the fresh-frozen esophageal tissue (stored in the RNAlater™ Stabilization Solution −70°C) using the AllPrep DNA/RNA/miRNA Universal Kit. Subsequent RT-qPCR analysis was performed using selected TaqMan MicroRNA Assays for miR-21, miR-34a, miR-196a, miR-196b, and endogenous control (RNU44). While the expression of miR-21 in the esophageal tissue with the main pathology was decreased in BE and EAC patients in comparison to the group of HC and RE patients (p=0.01), the expression of miR-196a was increased in the BE and EAC patients (p<0.01). Correlations between those miRNAs expression in tissue and severity of diagnosis were observed (p<0.05). In addition, miR-196a was significantly more expressed at the site with the main pathology than in paired adjacent esophageal tissue in BE and EAC patients (p<0.01). In conclusion, our pilot results showed that miR-196a, which regulates the proliferation, invasion, and migration (and was previously associated with esophageal squamous cell carcinoma and marked as a potential therapeutic target), could be a diagnostic tissue biomarker for BE and EAC as well.

Keywords: microRNA, barrett´s esophagus, esophageal adenocarcinoma, biomarker

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Authors: Zhang Zhenzhen

Abstract:

Valproate (VPA) is commonly used in the treatment of bipolar disorder and epilepsy. The mechanism is complicated, including its ability to inhibit histone deacetylases (HDACs). Here, we show that VPA attenuated VEGF gene expression and the morphological changes in choroidal neovascularization (CNV) induced by photocoagulation in retina. C57BL/6 mice were injected subcutaneously at 300mg/kg twice daily with VPA before insult. Vascular endothelial growth factor (VEGF)-A and VEGF-B were examined in the eyes of VPA-treated mice and in human retinal pigment epithelial cell lines (ARPE-19) exposed to VPA. In addition, CNV was induced by photocoagulation in mice injected with VPA, and the volume of CNV was compared by fluorescence-labeled choroidal flat mount. Morphological changes were analyzed on stained histological sections. Western blot analysis was used to determine protein levels of VEGF-A and VEGF-B, and acetylation of histone H3 in each group. VPA injected intraperitoneally attenuated the VEGF-A and VEGF-B expression in the retina, accompanied by the hyperacetylation of retina tissue, indicating that VPA acts directly on retina tissues through acetylation to reduce the expression of VEGF. VPA also attenuated the VEGF-A mRNA expression in the retinal pigment epithelium showed by immunohistochemistry. Moreover, the administration of VPA significantly attenuated photocoagulation-induced CNV in mice. These results demonstrate that VPA attenuated VEGF production in retina associated with choroidal neovascularization possibly via the HDAC inhibition.

Keywords: retina, acetylation, chorodial neovascularization, vascular endothelial growth factor

Procedia PDF Downloads 167