Search results for: 3T3-L1 preadipocyte cell

Authors: Ahmed M. Haddad, Hadeel S. El-Shaal, Gadallah M. Abu-Elreesh

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Some of filamentous fungi can be used for biodiesel production as they are able to accumulate high amounts of intracellular lipids when grown at stress conditions. Aspergillus fumigatus sp. was isolated from Nile delta soil in Egypt. The fungus was primarily screened for its capacity to accumulate lipids using Nile red staining assay. The fungus could accumulate more than 20% of its biomass as lipids when grown at optimized minimal medium. After lipid extraction, we could use fungal cell debris to remove some heavy metals from contaminated waste water. The fungal cell debris could remove Cd, Cr, and Zn with absorption efficiency of 73%, 83.43%, and 69.39% respectively. In conclusion, the Aspergillus fumigatus isolate may be considered as a promising biodiesel producer, and its biomass waste can be further used for bioremediation of wastewater contaminated with heavy metals.

Keywords: biodiesel, bioremediation, fungi, heavy metals, lipids, oleaginous

Procedia PDF Downloads 207

Authors: Moizza Tahir, Uzma Bashir, Aisha Akhtar, Zainab Ansari, Sameen Ansari, Muhammad Ali Tahir

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Cancer burden has globally increased. Among cutaneous cancers basal cell carcinoma constitute vast majority of skin cancer. There is need for appropriate diagnostic, therapeutic and prognostic significance evaluation for skin cancers Present study report intraoperative frozen section (FS) histopathological clearance for excision of BCC in a tertiary care center and find the frequency of involvement of surgical margin with reference to anatomical site, with size and surgical technique. It was prospective open label interventional study conducted at Dermatology department of tertiary care hospital Rawalpindi Pakistan in lais on with histopathology department from January 2023 to April 2024. Total of thirty-six (n = 36) patients between age 45-80 years with basal cell carcinoma of 10-20mm on face were included following inclusion exclusion criteria by purposive sampling technique. Informed consent was taken. Surgical excision was performed and intraoperative frozen section histopathology clearance of tumor margin was taken from histopathologist on telephone. Surgical reconstruction was done. Final Histopathology report was reexamined on day 10th for margin and depth clearance. Descriptive statistics were calculated for age, gender, sun exposure, reconstructive technique, anatomical site, and tumor free margin report on frozen section analysis. Chi square test was employed for statistical significance of involvement of surgical margin with reference to anatomical site, size and decision on reconstructive surgical technique, p value of <0.05 was considered significant. Total of 36 patients of BCC were enrolled, males 12 (33.3%) and females were 24 (66.6%). Age ranged from 45 year to 80 year mean of 58.36 ±SD7.8. Size of BCC ranged from 10mm to 35mm mean of 25mm ±SD 0.63. Morphology was nodular 18 (50%), superficial spreading 11(30.6%), morphoeic 1 (2.8%) and ulcerative in 6(16.7%) cases. Intraoperative frozen section for histopathological margin clearance with 2-3 mm safety margin and surgical technique has p-value0.51, for anatomical site p value 0.24 and size p-0.84. Intraoperative frozen section (FS) histopathological clearance for BCC face with 2-3mm safety margin with reference to reconstructive technique, anatomical site and size of BCC were insignificant.

Keywords: basal cell carcinoma, tumor free amrgin, basal cell carcinoma and frozen section, safety margin

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Authors: Z. Kolacinski, L. Szymanski. G. Raniszewski, D. Koza, L. Pietrzak

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Magnetic Bio-Nano-Fluid (BNF) can be composed of a buffer fluid such as plasma and magnetic nanoparticles such as iron, nickel, cobalt and their oxides. However iron is one of the best elements for magnetization by electromagnetic radiation. It can be used as a tool for medical diagnosis and treatment. Radio frequency (RF) radiation is able to heat iron nanoparticles due to magnetic hysteresis. Electromagnetic heating of iron nanoparticles and ferro-fluids BNF can be successfully used for non-invasive thermal ablation of cancer cells. Moreover iron atoms can be carried by carbon nanotubes (CNTs) if iron is used as catalyst for CNTs synthesis. Then CNTs became the iron containers and they screen the iron content against oxidation. We will present a method of CNTs addressing to the required cells. For thermal ablation of cancer cells we use radio frequencies for which the interaction with human body should be limited to minimum. Generally, the application of RF energy fields for medical treatment is justified by deep tissue penetration. The highly iron doped CNTs as the carriers creating magnetic fluid will be presented. An excessive catalyst injection method using electrical furnace and microwave plasma reactor will be presented. This way it is possible to grow the Fe filled CNTs on a moving surface in continuous synthesis process. This also allows producing uniform carpet of the Fe filled CNTs carriers. For the experimental work targeted to cell ablation we used RF generator to measure the increase in temperature for some samples like: solution of Fe2O3 in BNF which can be plasma-like buffer, solutions of pure iron of different concentrations in plasma-like buffer and in buffer used for a cell culture, solutions of carbon nanotubes (MWCNTs) of different concentrations in plasma-like buffer and in buffer used for a cell culture. Then the targeted therapies which can be effective if the carriers are able to distinguish the difference between cancerous and healthy cell’s physiology are considered. We have developed an approach based on ligand-receptor or antibody-antigen interactions for the case of colon cancer.

Keywords: cancer treatment, carbon nano tubes, drag delivery, hyperthermia, iron

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Authors: Nazanin Amanat, Alison Tayler, Steve Whyard

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While chemical insecticides effectively control many insect pests, they also harm many non-target species. Double-stranded RNA (dsRNA) pesticides, in contrast, can be designed to target unique gene sequences and thus act in a species-specific manner. DsRNA insecticides do not, however, work equally well for all insects, and for some species that are considered refractory to dsRNA, a primary factor affecting efficacy is the relative ease by which dsRNA can enter a target cell’s cytoplasm. In this study, we are examining how different structured dsRNAs (linear, hairpin, and paperclip) can enter mosquito and lepidopteran cells, as they represent dsRNA-sensitive and refractory species, respectively. To determine how the dsRNAs enter the cells, we are using chemical inhibitors and RNA interference (RNAi)-mediated knockdown of key proteins associated with different endocytosis processes. Understanding how different dsRNAs enter cells will ultimately help in the design of molecules that overcome refractoriness to RNAi or develop resistance to dsRNA-based insecticides. To date, we have conducted chemical inhibitor experiments on both cell lines and have evidence that linear dsRNAs enter the cells using clathrin-mediated endocytosis, while the paperclip dsRNAs (pcRNAs) can enter both species’ cells in a clathrin-independent manner to induce RNAi. An alternative uptake mechanism for the pcRNAs has been tentatively identified, and the outcomes of our RNAi-mediated knockdown experiments, which should provide corroborative evidence of our initial findings, will be discussed.

Keywords: dsRNA, RNAi, uptake, insecticides, dipteran, lepidopteran

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Authors: Jordan Chamarande, Lisiane Cunat, Corentine Alauzet, Catherine Cailliez-Grimal

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Gut microbiota (GM) is now considered a new organ mainly due to the microorganism’s specific biochemical interaction with its host. Although mechanisms underlying host-microbiota interactions are not fully described, it is now well-defined that cell surface molecules and structures of the GM play a key role in such relation. The study of surface structures of GM members is also fundamental for their role in the establishment of species in the versatile and competitive environment of the digestive tract and as a potential virulence factor. Among these structures are capsular polysaccharides (CPS), fimbriae, pili and lipopolysaccharides (LPS), all well-described for their central role in microorganism colonization and communication with host epithelium. The health-promoting Parabacteroides distasonis, which is part of the core microbiome, has recently received a lot of attention, showing beneficial properties for its host and as a new potential biotherapeutic product. However, to the best of the authors’ knowledge, the cell surface molecules and structures of P. distasonis that allow its maintain within the GM are not identified. Moreover, although P. distasonis is strongly recognized as intestinal commensal species with benefits for its host, it has also been recognized as an opportunistic pathogen. In this study, we reported gene clusters potentially involved in the synthesis of the capsule, fimbriae-like and pili-like cell surface structures in 26 P. distasonis genomes and applied the new RfbA-Typing classification in order to better understand and characterize the beneficial/pathogenic behaviour related to P. distasonis strains. In context, 2 different types of fimbriae, 3 of pilus and up to 14 capsular polysaccharide loci, have been identified over the 26 genomes studied. Moreover, the addition of data to the rfbA-Type classification modified the outcome by rearranging rfbA genes and adding a fifth group to the classification. In conclusion, the strain variability in terms of external proteinaceous structure could explain the inter-strain differences previously observed in P. distasonis adhesion capacities and its potential pathogenicity.

Keywords: gut microbiota, Parabacteroides distasonis, capsular polysaccharide, fimbriae, pilus, O-antigen, pathogenicity, probiotic, comparative genomics

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Authors: Daniel Aberdam

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Haploinsufficiency of PAX6 in humans is the main cause of congenital aniridia, a rare eye disease characterized by reduced visual acuity. Patients have also progressive disorders including cataract, glaucoma and corneal abnormalities making their condition very challenging to manage. Aniridia-related keratopathy (ARK), caused by a combination of factors including limbal stem-cell deficiency, impaired healing response, abnormal differentiation, and infiltration of conjunctival cells onto the corneal surface, affects up to 95% of patients. It usually begins in the first decade of life resulting in recurrent corneal erosions, sub-epithelial fibrosis with corneal decompensation and opacification. Unfortunately, current treatment options for aniridia patients are currently limited. Although animal models partially recapitulate this disease, there is no in vitro cellular model of AKT needed for drug/therapeutic tools screening and validation. We used genome editing (CRISPR/Cas9 technology) to introduce a nonsense mutation found in patients into one allele of the PAX6 gene into limbal stem cells. Resulting mutated clones, expressing half of the amount of PAX6 protein and thus representative of haploinsufficiency were further characterized. Sequencing analysis showed that no off-target mutations were induced. The mutated cells displayed reduced cell proliferation and cell migration but enhanced cell adhesion. Known PAX6 targets expression was also reduced. Remarkably, addition of soluble recombinant PAX6 protein into the culture medium was sufficient to activate endogenous PAX6 gene and, as a consequence, rescue the phenotype. It strongly suggests that our in vitro model recapitulates well the epithelial defect and becomes a powerful tool to identify drugs that could rescue the corneal defect in patients. Furthermore, we demonstrate that the homeotic transcription factor Pax6 is able to be uptake naturally by recipient cells to function into the nucleus.

Keywords: Pax6, crispr/cas9, limbal stem cells, aniridia, gene therapy

Procedia PDF Downloads 188

Authors: Albina V. Moskvicheva, Gevorg G. Kazarian, Anna R. Valeeva, Marina A. Efimova, Malik N. Mukminov, Eduard A. Shuralev, Rustam Kh. Ravilov, Kamil S. Khaertynov

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Improving the early diagnosis of tuberculosis and solving a number of problems associated with the differential diagnosis of Mycobacterium bovis infection, nonspecific tuberculin reactions caused by sensitization of the body by non-tuberculosis mycobacteria, is urgent. The filtrates and extracts of M. bovis cell surface components are promising antigens with diagnostic potential. The purpose of this study was to isolate and characterize antigenic proteins and determine the dominant M. bovis antigens recognized by the humoral immune system. The mycobacterial cells were homogenized on FastPrep-24. Gel-filtration chromatography was used to fractionate the lysates of cell surface component extracts and proteins isolated from M. bovis culture supernatant. The separated fractions were analyzed using two-dimensional gel electrophoresis followed by determination of antigen serological activity using immunoblot with specific hyperimmune rabbit blood serum. As a result of electrophoretic separation of components by molecular weight, 23 antigen fractions were obtained. Analysis of densitograms showed that the fractions contained two zones of antigens with pronounced serological activity, corresponding to molecular weights of 28 and 21 kDa. The high serological activity of the 28 kDa antigen was established by immunoblot using hyperimmune blood sera. Separated and characterized by M. bovis specific antigen with a molecular weight of 28 kDa was added to the collection of specific marker antigens for M. bovis.

Keywords: antigen, gel-filtration chromatography, immunoblot, Mycobacterium bovis

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Authors: Somit Dutta, Pallab Kar, Arnab Kumar Chakraborty, Arnab Sen, Tapas Kumar Chaudhuri

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In the present study three species of Clerodendrum; Clerodendrum indicum, Volkameria inermis and Clerodendrum colebrookianum were used to investigate the possible activity against oxidative stress. A detailed in-vivo and in-vitro antioxidant profiling, directly associated with inflammation-related carcinogenesis, has been executed with a motive to evaluate the free radical scavenging activity of Clerodendrum extract. Measurement of cell viability and ROS generation in HEK-293 (Human Embryonic Kidney Cell Line) cells was also estimated. The immune cell proliferative properties (MTT) and in-vitro assay for evaluation of their antioxidant activities including hydroxyl radical, nitric oxide, singlet oxygen, peroxinitrate and hydrogen peroxide, etc. were investigated. GC-MS and FTIR analyses have been performed to identify the active biological compounds. These active biological compounds were further studied to assess their potential medicinal properties, aided by molecular docking and interaction analysis between the active compounds and different proteins related to oxidative stress leading to progression of carcinogenesis. The research article clearly demonstrates the role of ROS in various phases of carcinogenesis. Therefore, the antioxidant and free radical scavenging capacity of all the Clerodendrum species might prove beneficial for the immune system. It might be concluded that this plant species offers great promise for cancer prevention and therapy due to the presence of several bioactive compounds and potent antioxidant capacity of C. colebrookianum.

Keywords: antioxidant, cancer, oxidative stress, reactive oxygen species (ROS)

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Authors: Yingjeng James Li, Lung-Yu Sung, Huan-Jyun Ciou

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Durability of Membrane Electrode Assembly for Proton Exchange Membrane Fuel Cells was evaluated in both steady state and accelerated decay modes. Steady state mode was carried out at constant current of 800mA / cm2 for 2500 hours using air as cathode feed and pure hydrogen as anode feed. The degradation of the cell voltage was 0.015V after such 2500 hrs operation. The degradation rate was therefore calculated to be 6uV / hr. Accelerated mode was carried out by switching the voltage of the single cell between OCV and 0.2V. The durations held at OCV and 0.2V were 20 and 40 seconds, respectively, meaning one minute per cycle. No obvious change in performance of the MEA was observed after 10000 cycles of such operation.

Keywords: durability, lifetime, membrane electrode assembly, proton exchange membrane fuel cells

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Authors: Kanyarat Sikhao, Nichakorn Khondee

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Adjuvant is commonly mixed with agricultural spray solution during foliar application to improve the performance of microbial-based biological control, including better spreading, absorption, and penetration on a plant leaf. This research aims to replace chemical surfactants in adjuvant by biosurfactants for reducing a negative impact on antagonistic microorganisms and crops. Biosurfactant was produced from Brevibacterium casei NK8 and used as a cell-free broth solution containing a biosurfactant concentration of 3.7 g/L. The studies of microemulsion formation and phase behavior were applied to obtain the suitable composition of biosurfactant-based adjuvant, consisting of cell-free broth (70-80%), coconut oil-based fatty alcohol C12-14 (3) ethoxylate (1-7%), and sodium chloride (8-30%). The suitable formula, achieving Winsor Type III microemulsion (bicontinuous), was 80% of cell-free broth, 7% of fatty alcohol C12-14 (3) ethoxylate, and 8% sodium chloride. This formula reduced the contact angle of water on parafilm from 70 to 31 degrees. The non-phytotoxicity against plant seed of Oryza sativa and Brassica rapa subsp. pekinensis were obtained from biosurfactant-based adjuvant (germination index equal and above 80%), while sodium dodecyl sulfate and tween80 showed phytotoxic effects to these plant seeds. The survival of Bacillus subtilis in biosurfactant-based adjuvant was higher than sodium dodecyl sulfate and tween80. The mixing of biosurfactant and plant-based surfactant could be considered as a viable, safer, and acceptable alternative to chemical adjuvant for sustainable organic farming.

Keywords: biosurfactant, microemulsion, bio-adjuvant, antagonistic microorganisms

Procedia PDF Downloads 124

Authors: Mayuva Youngsabanant, Chanikarn Srinark, Supanyika Sengsai, Soratorn Kerdkriangkrai, Nongnuch Gumlungpat, Mayuree Pumipaiboon

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The follicular fluid proteins of healthy medium size follicles (4-6 mm in diameters) and large size follicles (7-8 mm in diameter) of large white pig ovaries were collected by using sterile technique. They were used for testing the effect on primary in vitro cell culture growth of porcine oviductal epithelial cells (pOEC). Porcine oviductal epithelial cells of luteal phase was culture in M199 and added with 10% fetal calf serum 2.2 mg/mL, NaHCO₃, 0.25 mM pyruvate, 15 µg/mL and 50 µg/mL, gentamycin sulfate at high humidified atmosphere with 5% CO₂ in 95% air atmosphere at 37°C for 96 h before testing. The optimized concentration of pFF of two follicle sizes (at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins) in culture medium was observed for 24 h using MTT assay. Results were analyzed with a one-way ANOVA in SPSS statistic. Moreover, pOEC was also studied in morphological characteristic on long-term culture. The results of long-term study revealed that pOEC showed 70-80 percentage of healthy morphology on epithelial-like character and contained 30 percentage of an elongated shape (fibroblast-like morphology) at 4 weeks of culture time. MTT assay reviewed an increase in the percentage of viability of pOEC in 2 treated of follicular fluid groups. Two treatment concentration groups were higher than control group (p < 0.05) but not in positive control group. Interestingly, at 200 µg protein of 2 treated follicular fluid groups were reached the highest cell viability which is higher than a positive control and it is significantly different form control group (P < 0.05). These cells are developed and had fibroblast elongate shape which is longer than the cells in control group and positive control group. This report implies that pFF of medium follicle size at 200 µg proteins and large follicle size at 200 and 500 µg proteins could be optimized concentration for using as a supplement in culture medium to promote cell growth and development instead of growth hormone from fetal calf serum. It could be applied in cell biotechnology researches. Acknowledgements: The project was funded by a grant from Silpakorn University Research and Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: in vitro, porcine follicular fluid protein (pFF), porcine oviductal epithelial cells (pOEC), MTT

Procedia PDF Downloads 124

Authors: Ayda Youssef, Mohammed Ali Khalaf, Tarek Rafaat

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Background: Langerhans cell histiocytosis (LCH) is a rare multi-systemic disease that shows an abnormal proliferation of these kinds of cells associated with a granular infiltration that affects different structures of the human body, including the lung, liver, spleen, lymph nodes, brain, mucocutaneous, soft tissue (head and neck), and salivary glands. Evaluation of the extent of disease is one of the major predictors of patient outcome. Objectives: To recognize the pathogenesis of Langerhans cell histiocytosis (LCH), describe the radiologic criteria that are suggestive of LCH in different organs rather than the bones and to illustrate the appropriate differential diagnoses for LCH in each of the common extra-osseous sites. Material and methods: A retrospective study was done on 150 biopsy-proven LCH patients from 2007 to 2012. All patients underwent imaging studies, mostly US, CT, and MRI. These patients were reviewed to assess the extra-skeletal manifestations of LCH. Results: In 150 patients with biopsy-proven LCH, There were 33 patients with liver affection, 5 patients with splenic lesions, 55 patients with enlarged lymph nodes, 9 patient with CNS disease and 11 patients with lung involvement. Conclusions: Because of the frequent LCH children and evaluation of the extent of disease is one of the major predictors of patient outcome. Radiologist need to be familiar with its presentation in different organs and regions of body outside the commonest site of affection (bones). A high-index suspicion should be raised a biopsy is recommended in the presence of radiological suspicion. Chemotherapy is the preferred therapeutic modality.

Keywords: langerhans cell histiocytosis, extra-skeletal, pediatrics, radiology

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Authors: Jingjing Huang, Nengwei Li, Guanghua Wei, Jiabin You, Chao Wang, Junliang Zhang

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In this work, molecular dynamics (MD) simulation is applied to analyze the mass transport process in the cathode of proton exchange membrane fuel cell (PEMFC), of which all types of molecules situated in the cathode is considered. a reasonable and effective MD simulation process is provided, and models were built and compared using both Materials Studio and LAMMPS. The mass transport is one of the key issues in the study of proton exchange membrane fuel cells (PEMFCs). In this report, molecular dynamics (MD) simulation is applied to analyze the influence of Nafion ionomer distribution and Pt nano-particle size on mass transport process in the cathode. It is indicated by the diffusion coefficients calculation that a larger quantity of Nafion, as well as a higher equivalent weight (EW) value, will hinder the transport of oxygen. In addition, medium-sized Pt nano-particles (1.5~2nm) are more advantageous in terms of proton transport compared with other particle sizes (0.94~2.55nm) when the center-to-center distance between two Pt nano-particles is around 5 nm. Then mass transport channels are found to be formed between the hydrophobic backbone and the hydrophilic side chains of Nafion ionomer according to the radial distribution function (RDF) curves. And the morphology of these channels affected by the Pt size is believed to influence the transport of hydronium ions and, consequently the performance of PEMFC.

Keywords: cathode catalytic layer, mass transport, molecular dynamics, proton exchange membrane fuel cell

Procedia PDF Downloads 199

Authors: Achitphol Chookaew, Paramee Thongsukhsai, Patamarerk Engsontia, Narongwit Nakwan, Pritsana Raugrut

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Lung cancer is one of the most common malignancies and primary cause of death due to cancer worldwide. Non-small cell lung cancer (NSCLC) is the main subtype in which majority of patients present with advanced-stage disease. Herein, we analyzed differentially expressed genes to find potential biomarkers for lung cancer diagnosis as well as prognostic markers. We used transcriptome data from our 2 NSCLC patients and public data (GSE81089) composing of 8 NSCLC and 10 normal lung tissues. Differentially expressed genes (DEGs) between NSCLC and normal tissue and between early-stage and late-stage NSCLC were analyzed by the DESeq2. Pairwise correlation was used to find the DEGs with false discovery rate (FDR) adjusted p-value £ 0.05 and |log2 fold change| ³ 4 for NSCLC versus normal and FDR adjusted p-value £ 0.05 with |log2 fold change| ³ 2 for early versus late-stage NSCLC. Bioinformatic tools were used for functional and pathway analysis. Moreover, the top ten genes in each comparison group were verified the expression and survival analysis via GEPIA. We found 150 up-regulated and 45 down-regulated genes in NSCLC compared to normal tissues. Many immnunoglobulin-related genes e.g., IGHV4-4, IGHV5-10-1, IGHV4-31, IGHV4-61, and IGHV1-69D were significantly up-regulated. 22 genes were up-regulated, and five genes were down-regulated in late-stage compared to early-stage NSCLC. The top five DEGs genes were KRT6B, SPRR1A, KRT13, KRT6A and KRT5. Keratin 6B (KRT6B) was the most significantly increased gene in the late-stage NSCLC. From GEPIA analysis, we concluded that IGHV4-31 and IGKV1-9 might be used as diagnostic biomarkers, while KRT6B and KRT6A might be used as prognostic biomarkers. However, further clinical validation is needed.

Keywords: differentially expressed genes, early and late-stages, gene ontology, non-small cell lung cancer transcriptomics

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Authors: Reham H. Soliman, Noha Noufal, Howayda AbdelAal

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Calcium/calmodulin-dependent serine protein kinase (CASK) belongs to the membrane-associated guanylate kinase (MAGUK) family and has been proposed as a mediator of cell-cell adhesion and proliferation, which can contribute to tumorogenesis. CASK has been linked as a good prognostic factor with some tumor subtypes, while considered as a poor prognostic marker in others. To our knowledge, no sufficient evidence of CASK role in colorectal cancer is available. The aim of this study is to evaluate the expression of Calcium/calmodulin-dependent serine protein kinase (CASK) in non-mucinous colorectal adenocarcinoma and adenomatous polyps as precursor lesions and assess its prognostic significance. The study included 42 cases of conventional colorectal adenocarcinoma and 15 biopsies of adenomatous polyps with variable degrees of dysplasia. They were reviewed for clinicopathological prognostic factors and stained by CASK; mouse, monoclonal antibody using heat-induced antigen retrieval immunohistochemical techniques. The results showed that CASK protein was significantly overexpressed (p <0.05) in CRC compared with adenoma samples. The CASK protein was overexpressed in the majority of CRC samples with 85.7% of cases showing moderate to strong expression, while 46.7% of adenomas were positive. CASK overexpression was significantly correlated with both TNM stage and grade of differentiation (p <0.05). There was a significantly higher expression in tumor samples with early stages (I/II) rather than advanced stage (III/IV) and with low grade (59.5%) rather than high grade (40.5%). Another interesting finding was found among the adenomas group, where the stronger intensity of staining was observed in samples with high grade dysplasia (33.3%) than those of lower grades (13.3%). In conclusion, this study shows that there is significant overexpression of CASK protein in CRC as well as in adenomas with high grade dysplasia. This indicates that CASK is involved in the process of carcinogenesis and functions as a potential trigger of the adenoma-carcinoma cascade. CASK was significantly overexpressed in early stage and low-grade tumors rather than tumors with advanced stage and higher histological grades. This suggests that CASK protein is a good prognostic factor. We suggest that CASK affects CRC in two different ways derived from its physiology. CASK as part of MAGUK family can stimulate proliferation and through its cell membrane localization and as a mediator of cell-cell adhesion might contribute in tumor confinement and localization.

Keywords: CASK, colorectal cancer, overexpression, prognosis

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Authors: Eva Filová, Jana Daňková, Věra Sovková, Matej Daniel

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Titanium alloys are biocompatible metals that are widely used in clinical practice as load bearing implants. The chemical modification may influence cell adhesion, proliferation, and differentiation as well as stiffness of the material. The aim of the study was to evaluate the adhesion, growth and differentiation of pig mesenchymal stem cells on the novel beta titanium alloy Ti36Nb6Ta compared to standard medical titanium alloy Ti6Al4V. Discs of Ti36Nb6Ta and Ti6Al4V alloy were sterilized by ethanol, put in 48-well plates, and seeded by pig mesenchymal stem cells at the density of 60×103/cm2 and cultured in Minimum essential medium (Sigma) supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cell viability was evaluated using MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay;Promega), cell proliferation using Quant-iT™ ds DNA Assay Kit (Life Technologies). Cells were stained immunohistochemically using monoclonal antibody beta-actin, and secondary antibody conjugated with AlexaFluor®488 and subsequently the spread area of cells was measured. Cell differentiation was evaluated by alkaline phosphatase assay using p-nitrophenyl phosphate (pNPP) as a substrate; the reaction was stopped by NaOH, and the absorbance was measured at 405 nm. Osteocalcin, specific bone marker was stained immunohistochemically and subsequently visualized using confocal microscopy; the fluorescence intensity was analyzed and quantified. Moreover, gene expression of osteogenic markers osteocalcin and type I collagen was evaluated by real-time reverse transcription-PCR (qRT-PCR). For statistical evaluation, One-way ANOVA followed by Student-Newman-Keuls Method was used. For qRT-PCR, the nonparametric Kruskal-Wallis Test and Dunn's Multiple Comparison Test were used. The absorbance in MTS assay was significantly higher on titanium alloy Ti6Al4V compared to beta titanium alloy Ti36Nb6Ta on days 7 and 14. Mesenchymal stem cells were well spread on both alloys, but no difference in spread area was found. No differences in alkaline phosphatase assay, fluorescence intensity of osteocalcin as well as the expression of type I collagen, and osteocalcin genes were observed. Higher expression of type I collagen compared to osteocalcin was observed for cells on both alloys. Both beta titanium alloy Ti36Nb6Ta and titanium alloy Ti6Al4V Ti36Nb6Ta supported mesenchymal stem cellsˈ adhesion, proliferation and osteogenic differentiation. Novel beta titanium alloys Ti36Nb6Ta is a promising material for bone implantation. The project was supported by the Czech Science Foundation: grant No. 16-14758S, the Grant Agency of the Charles University, grant No. 1246314 and by the Ministry of Education, Youth and Sports NPU I: LO1309.

Keywords: beta titanium, cell growth, mesenchymal stem cells, titanium alloy, implant

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Authors: Anushaa A.

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In this work, silver nanoparticles (AgNP) were manufactured directly without harmful chemicals utilising methanol extract (SNLME) Solanum nigrume leaves. We are using nigrum leaf extract from Solanum, which converts silver nitrate to silver ions, for synthesization purposes. An examination of the AgNP produced was performed using ultraviolet (UV-VIS) spectroscopy, infrared spectroscopy (FTIR) transformed from Fourier and scanning electrons (SEM). Biological activity was also tested. UV-VIS has proven that biosynthesized AgNP exists (420-450 nm). The FTIR spectrum has been utilised to confirm the presence of different functional groups within the biomolecules, which are a nanoparticular capping agent and the spectroscopic and crystal nature of AgNP. The viability of the silver nanoparticles was evaluated using zeta potential calculations. Negative zeta potential of -33.4 mV demonstrated the stability of silver-nanoparticles. The morphology of AgNP was examined using a scanning electron microscope. Greenly generated AgNP showed significant anti-Staphylococcus aureus, Candida, and Escherichia coli action. The green AgNP demonstration indicated that the IC50 for the human teratocarcinoma cell line was 29.24 μg/ml during 24 hours of therapy (PA1 Ovarian cell line). The dose-dependent effects were reported in both antibacterial and cytotoxicity assays and as an effective agent. Finally, the findings of this research showed that silver nanoparticles generated might serve as a viable therapeutic agent to combat microorganisms killing and curing cancer.

Keywords: antimicrobial activity, PA1 ovarian cancer cell line, silver nanoparticles, Solanum nigrum

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Authors: Yi-Chiang Huang, Hsu-Feng Lee, Yu-Chao Tseng, Wen-Yao Huang

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Proton exchange membranes as a key component in fuel cells have been widely studying over the past few decades. As proton exchange, membranes should have some main characteristics, such as good mechanical properties, low oxidative stability and high proton conductivity. In this work, trifluoromethyl groups had been introduced on polymer backbone and phenyl side chain which can provide densely located sulfonic acid group substitution and also promotes solubility, thermal and oxidative stability. Herein, a series of novel sulfonated aromatic hydrocarbon polyelectrolytes was synthesized by polycondensation of 4,4''''-difluoro-3,3''''- bis(trifluoromethyl)-2'',3''-bis(3-(trifluoromethyl)phenyl)-1,1':4',1'':4'',1''':4''',1''''-quinquephenyl with 2'',3''',5'',6''-tetraphenyl-[1,1':4',1'': 4'',1''':4''',1''''-quinquephenyl]-4,4''''-diol and post-sulfonated was through chlorosulfonic acid to given sulfonated polymers (SFC3-X) possessing ion exchange capacities ranging from 1.93, 1.91 and 2.53 mmol/g. ¹H NMR and FT-IR spectroscopy were applied to confirm the structure and composition of sulfonated polymers. The membranes exhibited considerably dimension stability (10-27.8% in length change; 24-56.5% in thickness change) and excellent oxidative stability (weight remain higher than 97%). The mechanical properties of membranes demonstrated good tensile strength on account of the high rigidity multi-phenylated backbone. Young's modulus were ranged 0.65-0.77GPa which is much larger than that of Nafion 211 (0.10GPa). Proton conductivities of membranes ranged from 130 to 240 mS/cm at 80 °C under fully humidified which were comparable or higher than that of Nafion 211 (150 mS/cm). The morphology of membranes was investigated by transmission electron microscopy which demonstrated a clear hydrophilic/hydrophobic phase separation with spherical ionic clusters in the size range of 5-20 nm. The SFC3-1.97 single fuel cell performance demonstrates the maximum power density at 1.08W/cm², and Nafion 211 was 1.24W/cm² as a reference in this work. The result indicated that SFC3-X are good candidates for proton exchange membranes in fuel cell applications. Fuel cell of other membranes is under testing.

Keywords: fuel cells, polyelectrolyte, proton exchange membrane, sulfonated polymers

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Authors: Asma Alhuqail, Nagwa Aref

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Programmed cell death resembles a real nature active defense in Datura metel against TMV after three days of virus infection. Physiological plant response was assessed for asymptomatic healthy and symptomatic infected detached leaves. The results indicated H2O2 and Chlorophyll-a as the most potential parameters. Chlorophyll-a was considered the only significant predictor variant for the H2O2 dependent variant with a P value of 0.001 and R-square of 0.900. The plant immune response was measured within three days of virus infection using the cutoff value of H2O2 (61.095 lmol/100 mg) and (63.201 units) for the tail moment in the Comet Assay. Their percentage changes were 255.12% and 522.40% respectively which reflects the stress of virus infection in the plant. Moreover, H2O2 showed 100% specificity and sensitivity in the symptomatic infected group using the receiver-operating characteristic (ROC). All tested parameters in the symptomatic infected group had significant correlations with twenty-five positive and thirty-one negative correlations where the P value was <0.05 and 0.01. Chlorophyll-a parameter had a crucial role of highly significant correlation between total protein and salicylic acid. Contrarily, this correlation with tail moment unit was (r = _0.930, P <0.01) where the P value was < 0.01. The strongest significant negative correlation was between Chlorophyll-a and H2O2 at P < 0.01, while moderate negative significant correlation was seen for Chlorophyll-b where the P value < 0.05. The present study discloses the secret of the three days of rapid transient production of activated oxygen species (AOS) that was enough for having potential quantitative physiological parameters for defensive plant response toward the virus.

Keywords: programmed cell death, plant–adaptive immune response, hydrogen peroxide (H2O2), physiological parameters

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Authors: Meihua Fang, Yipan Guo, Tao Fei, Pengyu Tian

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Space plasma caused spacecraft surface charging is the major space environment hazard. Particle in cell (PIC) method can be used to simulate the interaction between space plasma and spacecraft. It was proved that surface charging level of spacecraft in Jupiter’s orbits was high for its’ electron-heavy plasma environment. In this paper, Jovian plasma environment is modeled and surface charging analysis is carried out by PIC based software Spacecraft Plasma Interaction System (SPIS). The results show that the spacecraft charging potentials exceed 1000V at 2Rj, 15Rj and 25Rj polar orbits in the dark side at worst case plasma model. Furthermore, the simulation results indicate that the large Jovian magnetic field increases the surface charging level for secondary electron gyration.

Keywords: Jupiter, PIC, space plasma, surface charging

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Authors: Ali Golestani, Vahid Naseri, Hossein Taheri

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Recently, some of studies examined the effect of exercise on neurotrophic factors influencing the growth, protection, plasticity and function in central and peripheral nerve cells. The aim of this study was to investigate the effects of continuous and interval aerobic exercises with moderate intensity on serum levels of glial cell line-derived neurotrophic factor (GDNF) and aerobic capacity in obese children. 21 obese students with an average age of 13.6 ± 0.5 height 171 ± 5 and BMI 32 ± 1.2 were divided randomly to control, continuous aerobic and interval aerobic groups. Training protocol included continuous or interval aerobic exercises with moderate intensity 50-65%MHR, three times per week for 10 weeks. 48 hours before and after executing of protocol, blood samples were taken from the participants and their GDNF serum levels were measured by ELISA. Aerobic power was estimated using Shuttle-run test. T-test results indicated a small increase in their GDNF serum levels, which was not statistically significant (p =0.11). In addition, the results of ANOVA did not show any significant difference between continuous and interval aerobic training on the serum levels of their GDNF but their aerobic capacity significantly increased (p =0.012). Although continuous and interval aerobic exercise improves aerobic power in obese children, they had no significant effect on their serum levels of GDNF.

Keywords: aerobic power, continuous aerobic training, glial cell line-derived neurotrophic factor (GDNF), interval aerobic training, obese children

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Authors: Suharni Mohamad Suharni Mohamad, Nurul Izzati Hamzan Nurul Izzati Hamzan, Norhayu Abdul Rahman Norhayu Abdul Rahman, Siti Suraiya Md Noor Siti Suraiya Md Noor

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Human papillomavirus (HPV) is an important risk factor for development of oral cancer. HPV16 is the most common type found in HPV-positive squamous cell carcinoma. In the present study, we established a real-time loop-mediated isothermal amplification (real-time LAMP) for detection of HPV16. A set of six primers was specially designed to recognize eight distinct sequences of HPV16-E6. Detection and quantification was achieved by real-time monitoring using a real-time turbidimeter based on threshold time required for turbidity in the LAMP reaction. LAMP reagents (MgSO4, dNTPs, Bst polymerase concentrations) and various incubation times and temperatures were optimized. The sensitivity was determined using 10-fold serial dilutions of HPV16 standard strain. The specificity of was evaluated using other HPV genotypes. The optimized method was established with specifically designed primers by real-time detection in approximately 30 min at 65°C. The limit of detection of HPV16 using the LAMP assay was 10 pg/ml that could be detected in 30 min. The LAMP assay was 10 times more sensitive than the conventional PCR in detecting HPV16. No cross-reactivity with other HPV genotypes was observed. This quantitative real-time LAMP assay may improve diagnostic potential for the detection and quantification of HPV16 in clinical samples and epidemiological studies due to its rapidity, simplicity, high sensitivity and specificity. This assay will be further evaluated with HPV DNAs of saliva from patients with oral squamous cell carcinoma. Acknowledgement: This study was financially supported by the ScienceFund Grant, Ministry of Science, Technology and Innovation (305/PPSG/6113219).

Keywords: Oral Squamous Cell Carcinoma (OSCC), Human Papillomavirus 16 (HPV16), Loop-Mediated Isothermal Amplification (LAMP), rapid detection

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Authors: Esra Sengul, R. G. Aktas, M. E. Sitar, H. Isan

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Hepatocellular carcinoma (HCC) is a hepatocellular tumor commonly found on the surface of the chronic liver. HepG2 is the most commonly used cell type in HCC studies. The main proteins remaining in the blood serum after separation of plasma fibrinogen are albumin and globulin. The fact that the albumin showed hepatocellular damage and reflect the synthesis capacity of the liver was the main reason for our use. Alpha-Fetoprotein (AFP) is an albumin-like structural embryonic globulin found in the embryonic cortex, cord blood, and fetal liver. It has been used as a marker in the follow-up of tumor growth in various malign tumors and in the efficacy of surgical-medical treatments, so it is a good protein to look at with albumins. We have seen the morphological changes of dimethyl sulfoxide (DMSO) on HepG2 and decided to investigate its biochemical effects. We examined the effects of DMSO, which is used in cell cultures, on albumin, AFP and total protein at low doses. Material Method: Cell Culture: Medium was prepared in cell culture using Dulbecco's Modified Eagle Media (DMEM), Fetal Bovine Serum Dulbecco's (FBS), Phosphate Buffered Saline and trypsin maintained at -20 ° C. Fixation of Cells: HepG2 cells, which have been appropriately developed at the end of the first week, were fixed with acetone. We stored our cells in PBS at + 4 ° C until the fixation was completed. Area Calculation: The areas of the cells are calculated in the ImageJ (IJ). Microscope examination: The examination was performed with a Zeiss Inverted Microscope. Daytime photographs were taken at 40x, 100x 200x and 400x. Biochemical Tests: Protein (Total): Serum sample was analyzed by a spectrophotometric method in autoanalyzer. Albumin: Serum sample was analyzed by a spectrophotometric method in autoanalyzer. Alpha-fetoprotein: Serum sample was analyzed by ECLIA method. Results: When liver cancer cells were cultured in medium with 1% DMSO for 4 weeks, a significant difference was observed when compared with the control group. As a result, we have seen that DMSO can be used as an important agent in the treatment of liver cancer. Cell areas were reduced in the DMSO group compared to the control group and the confluency ratio increased. The ability to form spheroids was also significantly higher in the DMSO group. Alpha-fetoprotein was lower than the values of an ordinary liver cancer patient and the total protein amount increased to the reference range of the normal individual. Because the albumin sample was below the specimen value, the numerical results could not be obtained on biochemical examinations. We interpret all these results as making DMSO a caretaking aid. Since each one was not enough alone we used 3 parameters and the results were positive when we refer to the values of a normal healthy individual in parallel. We hope to extend the study further by adding new parameters and genetic analyzes, by increasing the number of samples, and by using DMSO as an adjunct agent in the treatment of liver cancer.

Keywords: hepatocellular carcinoma, HepG2, dimethyl sulfoxide, cell culture, ELISA

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Authors: Zakaria Baka, Halima Alem

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Cancer is a major worldwide health problem that has caused around ten million deaths in 2020. In addition, efforts to develop new anti-cancer drugs still face a high failure rate. This is partly due to the lack of preclinical models that recapitulate in-vivo drug responses. Indeed conventional cell culture approach (known as 2D cell culture) is far from reproducing the complex, dynamic and three-dimensional environment of tumors. To set up more in-vivo-like cancer models, 3D bioprinting seems to be a promising technology due to its ability to achieve 3D scaffolds containing different cell types with controlled distribution and precise architecture. Moreover, the introduction of microfluidic technology makes it possible to simulate in-vivo dynamic conditions through the so-called “cancer-on-chip” platforms. Whereas several cancer types have been modeled through the cancer-on-chip approach, such as lung cancer and breast cancer, only a few works describing ovarian cancer models have been described. The aim of this work is to combine 3D bioprinting and microfluidic technics with setting up a 3D dynamic model of ovarian cancer. In the first phase, alginate-gelatin hydrogel containing SKOV3 cells was used to achieve tumor-like structures through an extrusion-based bioprinter. The desired form of the tumor-like mass was first designed on 3D CAD software. The hydrogel composition was then optimized for ensuring good and reproducible printability. Cell viability in the bioprinted structures was assessed using Live/Dead assay and WST1 assay. In the second phase, these bioprinted structures will be included in a microfluidic device that allows simultaneous testing of different drug concentrations. This microfluidic dispositive was first designed through computational fluid dynamics (CFD) simulations for fixing its precise dimensions. It was then be manufactured through a molding method based on a 3D printed template. To confirm the results of CFD simulations, doxorubicin (DOX) solutions were perfused through the dispositive and DOX concentration in each culture chamber was determined. Once completely characterized, this model will be used to assess the efficacy of anti-cancer nanoparticles developed in the Jean Lamour institute.

Keywords: 3D bioprinting, ovarian cancer, cancer-on-chip models, microfluidic techniques

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Authors: Shamill Amedot Udonwa, Phyllis S. Y. Chong, Lim S. L. Julia, Wee-Joo Chng

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In this paper, we investigated how PRL-3 expression in H929 and U266 cells affects the efficacy of drug treatment. H929 and U266 cells were treated with Bortezomib (BTZ) of different concentrations, and it was observed that H929 cells were resistant to BTZ, while U266 cells were not viable. Investigations into how BTZ targets these cells were conducted, and it was observed that BTZ affects the PARP-Caspase3 pathway as well as PRL-3-Leo1 pathways. These pathways regulate cell proliferation and cell cycle, respectively. Hence, we are able to show the mechanism of how BTZ affects cells and also the role PRL-3 plays on downstream oncogenes such as cyclin-D1 and c-MYC. More importantly, this investigation into PRL-3 in BTZ resistance will be highly applicable in the future as the first clinical trials of PRL-3 antibody (PRL3-zumab) are ongoing at the National University Hospital, Singapore (NUHS). This would mean that understanding the mechanism of resistance through PRL-3, which has yet to be studied, will demonstrate the potential of PRL-3 in developing novel strategies to improve the treatment of MM.

Keywords: drug resistance, hematology, multiple myeloma, oncogene

Procedia PDF Downloads 125

Authors: Orkide Donma, M. Metin Donma, Burcin Nalbantoglu, Birol Topcu, Feti Tulubas, Murat Aydin, Tuba Gokkus, Ahmet Gurel

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Increasing prevalence of childhood obesity has increased the interest in early and late indicators of gaining weight. Cell blood counts may be indicators of proinflammatory states. The aim was to evaluate associations of hematological parameters, including Hematocrit (HTC), hemoglobin, blood cell counts, and their indices with the degree of obesity in pediatric population. A total of 249; -139 morbidly obese (MO), 82 healthy Normal Weight (NW) and 28 Overweight (OW) children were included into the scope of the study. WHO BMI-for age percentiles were used to form age- and sex-matched groups. Informed consent forms and the Ethics Committee approval were obtained. Anthropometric measurements were performed. Hematological parameters were determined. Statistical analyses were performed using SPSS. The degree for statistical significance was p≤0.05. Significant differences (p=0.000) between waist-to-hip ratios and head-to-neck ratios (hnrs) of MO and NW children were detected. A significant difference between hnrs of OW and MO children (p=0.000) was observed. Red cell Distribution Width (RDW) was higher in OW children than NW group (p=0.030). Such finding couldn’t be detected between MO and NW groups. Increased RDW was prominent in OW children. The decrease in Mean Corpuscular Hemoglobin Concentration (MCHC) values in MO children was sharper than the values in OW children (p=0.006 vs p=0.042) compared to those in NW group. Statistically higher HTC levels were observed between MO-NW (p=0.014), but none between OW-NW. Though the cause-effect relationship between obesity and erythrocyte indices still needs further investigation, alterations in RDW, HTC, MCHC during obesity may be of significance in the early life.

Keywords: anthropometry, children, erythrocytes, obesity

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Authors: Daniel Canales, Fabián Alvarez, Pablo Varela, Marcela Saavedra, Claudio García, Paula Zapata

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Calcium oxide nanoparticles (n-CaO) ca. 8 nm were obtained from eggshell waste. The n-CaO was incorporated into Poly(lactic acid) PLA matrix in 10 and 20 wt.% of filler content by electrospinning process to obtain PLA/n-CaO nanocomposite fibers as a potential use in scaffold for bone tissue regeneration. The fibers morphology and diameter were homogeneity, the PLA had a diameter of 2.2 ± 0.8 µm and, with the nanoparticles incorporation (20wt.%), reached ca. 2.9 ± 0.9 µm. The PLA/n-CaO nanocomposites fibers showed in vitro bioactivity, capable of inducing the precipitation of hydroxyapatite (HA) layer in the fiber surface after 7 days in Simulated Body Solution (SBF). The biocidal and biological properties of PLA/n-Cao with 20 wt.% were evaluated, showing a 30% reduction in bacterial viability against S. aureus and 11% for E. coli after 6 hours of bacterial suspensions exposure. Furthermore, the fibers did not show a cytotoxic effect on the bone marrow ST-2 cell line, permitting the cell adhesion and proliferation in Roswell Park Memorial Institute medium (RPMI). The PLA/n-CaO with 20 wt.% of nanoparticles showed a higher capacity to promote the osteogenic differentiation, significantly increasing the alkaline phosphatase (ALP) expression after 7 days compared to PLA and cell control. The in vivo analysis corroborated the biocompatibility of scaffolds prepared, the presence of n-CaO in PLA reduced the formation of fibrous encapsulation of the material improve the healing process.

Keywords: electrospun scaffolds, PLA based nanocomposites, calcium oxide nanoparticles, bioactive materials, tissue engineering

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Authors: P. Annapurna, Cecil Ross, Sudhir Krishna, Sweta Srivastava

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Irradiation plays a pivotal role in cervical cancer treatment, however some tumors exhibit resistance to therapy while some exhibit relapse, due to better repair and enhanced resistance mechanisms operational in their cells. The present study aims to understand the signaling mechanism operational in resistance phenotype and in the present study we report the role of Rho GTPase associated protein kinase (ROCK) signaling in cervical carcinoma radio-resistance. ROCK signaling has been implicated in several tumor progressions and is important for DNA repair. Irradiation of spheroid cultures of SiHa cervical carcinoma derived cell line at 6Gy resulted in generation of resistant cells in vitro which had better clonogenic abilities and formed larger and more colonies, in soft agar colony formation assay, as compared to the non-irradiated cells. These cells also exhibited an enhanced motility phenotype. Cell cycle profiling showed the cells to be blocked in G2M phase with enhanced pCDC2 levels indicating onset of possible DNA repair mechanism. Notably, 3 days post-irradiation, irradiated cells showed increased ROCK2 translocation to the nucleus with enhanced protein expression as compared to the non-irradiated cells. Radio-sensitization of the resistant cells was enhanced using Y27632, an inhibitor to ROCK signaling. The treatment of resistant cells with Y27632 resulted in increased cell death upon further irradiation. This observation has been confirmed using inhibitory antibodies to ROCK1/2. Result show that both ROCK1/2 have a functional contribution in radiation resistance of cervical cancer cells derived from cell lines. Interestingly enrichment of stem like cells (Hoechst negative cells) was also observed upon irradiation and these cells were markedly sensitive to Y27632 treatment. Our results thus suggest the role of ROCK signaling in radio-resistance in cervical carcinoma. Further studies with human biopsies, mice models and mechanistic of ROCK signaling in the context of radio-resistance will clarify the role of this molecule further and allow for therapeutics development.

Keywords: cervical carcinoma, radio-resistance, ROCK signaling, cancer treatment

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Authors: Harika Atmaca, Emir Bozkurt, Latife Merve Oktay, Selim Uzunoglu, Ruchan Uslu, Burçak Karaca

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Microarrays have been developed for highly parallel enzyme-linked immunosorbent assay (ELISA) applications. The most common protein arrays are produced by using multiple monoclonal antibodies, since they are robust molecules which can be easily handled and immobilized by standard procedures without loss of activity. Protein expression profiling with protein array technology allows simultaneous analysis of the protein expression pattern of a large number of proteins. Trabectedin, a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate, Ecteinascidia turbinata, has been shown to have antitumor effects. Here, we used a novel proteomic approach to explore the mechanism of action of trabectedin in prostate cancer cell line PC-3 by apoptosis antibody microarray. XTT cell proliferation kit and Cell Death Detection Elisa Plus Kit (Roche) was used for measuring cytotoxicity and apoptosis. Human Apoptosis Protein Array (R&D Systems) which consists of 35 apoptosis related proteins was used to assess the omic protein expression pattern. Trabectedin induced cytotoxicity and apoptosis in prostate cancer cells in a time and concentration-dependent manner. The expression levels of the death receptor pathway molecules, TRAIL-R1/DR4, TRAIL R2/DR5, TNF R1/TNFRSF1A, FADD were significantly increased by 4.0-, 21.0-, 4.20- and 11.5-fold by trabectedin treatment in PC-3 cells. Moreover, mitochondrial pathway related pro-apoptotic proteins Bax, Bad, Cytochrome c, and Cleaved Caspase-3 expressions were induced by 2.68-, 2.07-, 2.8-, and 4.5-fold and the expression levels of anti-apoptotic proteins Bcl-2 and Bcl-XL were reduced by 3.5- and 5.2-fold in PC-3 cells. Proteomic (antibody microarray) analysis suggests that the mechanism of action of trabectedin may be exerted via the induction of both intrinsic and extrinsic apoptotic pathways. The antibody microarray platform can be utilised to explore the molecular mechanism of action of novel anticancer agents.

Keywords: trabectedin, prostate cancer, omic protein expression profile, apoptosis

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Authors: Dinakaran Venkatachalam, Paul Chambers, Kavitha Kongara, Preet Singh

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The objective of this study is to formulate different topical preparations containing articaine and to investigate their permeation through goat skin. Initially, articaine and its hydrochloride salt were compared for in vitro permeation using Franz cell model. Goat skin samples were collected after euthanizing male goat kids purchased from the dairy goat farmers. Subcutaneous fat was removed and the skin was mounted on the donor chamber (orifice area 1.00 cm²) and drugs were applied onto the epidermis. Phosphate buffer saline (pH 7.4) was used to maintain sink condition in the receptor chamber (8 ml) of the Franz cell. Samples (0.4 ml) were collected at various intervals over 24 hours after each sampling equal volume of PBS was replaced in the receptor chamber. Articaine in the collected samples were quantified using LC/MS. The results suggested that articaine free base permeates better than its hydrochloride salt through goat skin. This study results support the fact that local anesthetics in its base form are lipophilic and thus penetrates faster through cell membranes than their salts. Later, articaine free base was formulated either using ethanol and octyl salicylate or dimethyl sulfoxide (DMSO) as penetration enhancers and was compared for in vitro permeation. The transdermal flux of articaine in the formulation containing DMSO was approximately 3.8 times higher than that of the formulation containing ethanol and octyl salicylate. Further studies to evaluate the local anesthetic efficacy of the topical formulation containing articaine for dermal anesthesia in animals have been planned.

Keywords: articaine, dermal anesthesia, local anesthetic, transdermal

Procedia PDF Downloads 219